CA2759405A1 - Method - Google Patents
Method Download PDFInfo
- Publication number
- CA2759405A1 CA2759405A1 CA2759405A CA2759405A CA2759405A1 CA 2759405 A1 CA2759405 A1 CA 2759405A1 CA 2759405 A CA2759405 A CA 2759405A CA 2759405 A CA2759405 A CA 2759405A CA 2759405 A1 CA2759405 A1 CA 2759405A1
- Authority
- CA
- Canada
- Prior art keywords
- seq
- nucleotide sequence
- lipolytic enzyme
- enzyme
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 126
- 108090000790 Enzymes Proteins 0.000 claims abstract description 353
- 102000004190 Enzymes Human genes 0.000 claims abstract description 349
- 230000002366 lipolytic effect Effects 0.000 claims abstract description 239
- 241000499912 Trichoderma reesei Species 0.000 claims abstract description 102
- 229940088598 enzyme Drugs 0.000 claims description 341
- 125000003729 nucleotide group Chemical group 0.000 claims description 277
- 239000002773 nucleotide Substances 0.000 claims description 276
- 108090000623 proteins and genes Proteins 0.000 claims description 136
- 230000014509 gene expression Effects 0.000 claims description 67
- 230000000694 effects Effects 0.000 claims description 65
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 58
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 40
- 239000013598 vector Substances 0.000 claims description 34
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 31
- 230000000295 complement effect Effects 0.000 claims description 31
- 238000012258 culturing Methods 0.000 claims description 27
- 230000009466 transformation Effects 0.000 claims description 27
- 235000013305 food Nutrition 0.000 claims description 23
- 238000006206 glycosylation reaction Methods 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 108010008885 Cellulose 1,4-beta-Cellobiosidase Proteins 0.000 claims description 17
- 230000013595 glycosylation Effects 0.000 claims description 16
- 229910001868 water Inorganic materials 0.000 claims description 14
- 230000001131 transforming effect Effects 0.000 claims description 12
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 11
- 239000013604 expression vector Substances 0.000 claims description 11
- 108010059892 Cellulase Proteins 0.000 claims description 9
- 239000012228 culture supernatant Substances 0.000 claims description 8
- 238000007865 diluting Methods 0.000 claims description 7
- 238000000746 purification Methods 0.000 claims description 7
- 239000004615 ingredient Substances 0.000 claims description 5
- 238000002955 isolation Methods 0.000 claims description 4
- 229940106157 cellulase Drugs 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 25
- 241000223259 Trichoderma Species 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 description 128
- 239000002609 medium Substances 0.000 description 84
- 108020004414 DNA Proteins 0.000 description 72
- 102000004169 proteins and genes Human genes 0.000 description 69
- 235000018102 proteins Nutrition 0.000 description 66
- 101710084376 Lipase 3 Proteins 0.000 description 57
- 239000012634 fragment Substances 0.000 description 50
- 238000000855 fermentation Methods 0.000 description 43
- 230000004151 fermentation Effects 0.000 description 43
- 108090001060 Lipase Proteins 0.000 description 42
- 102000004882 Lipase Human genes 0.000 description 38
- 239000004367 Lipase Substances 0.000 description 35
- 235000019421 lipase Nutrition 0.000 description 35
- 101150089778 pyr-4 gene Proteins 0.000 description 34
- 239000013612 plasmid Substances 0.000 description 31
- 239000000758 substrate Substances 0.000 description 31
- 238000003556 assay Methods 0.000 description 26
- 235000001014 amino acid Nutrition 0.000 description 25
- 229940024606 amino acid Drugs 0.000 description 24
- 241000228232 Aspergillus tubingensis Species 0.000 description 23
- 150000001413 amino acids Chemical group 0.000 description 23
- 238000004458 analytical method Methods 0.000 description 23
- 238000012217 deletion Methods 0.000 description 23
- 230000037430 deletion Effects 0.000 description 23
- 239000000523 sample Substances 0.000 description 23
- 239000000203 mixture Substances 0.000 description 22
- 108090000765 processed proteins & peptides Proteins 0.000 description 22
- 102000004196 processed proteins & peptides Human genes 0.000 description 20
- 101150114858 cbh2 gene Proteins 0.000 description 19
- 229920001184 polypeptide Polymers 0.000 description 19
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 18
- 235000010633 broth Nutrition 0.000 description 17
- 235000019626 lipase activity Nutrition 0.000 description 16
- 244000005700 microbiome Species 0.000 description 16
- 108091026890 Coding region Proteins 0.000 description 15
- 230000008569 process Effects 0.000 description 15
- 101000895926 Streptomyces plicatus Endo-beta-N-acetylglucosaminidase H Proteins 0.000 description 14
- 238000004520 electroporation Methods 0.000 description 14
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 13
- 239000001301 oxygen Substances 0.000 description 13
- 229910052760 oxygen Inorganic materials 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 239000000126 substance Substances 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 241000233866 Fungi Species 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 12
- 229910052799 carbon Inorganic materials 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 108091033319 polynucleotide Proteins 0.000 description 12
- 102000040430 polynucleotide Human genes 0.000 description 12
- 239000002157 polynucleotide Substances 0.000 description 12
- 239000000047 product Substances 0.000 description 12
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 12
- 238000006467 substitution reaction Methods 0.000 description 12
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 11
- UYXTWWCETRIEDR-UHFFFAOYSA-N Tributyrin Chemical compound CCCC(=O)OCC(OC(=O)CCC)COC(=O)CCC UYXTWWCETRIEDR-UHFFFAOYSA-N 0.000 description 11
- 125000000539 amino acid group Chemical group 0.000 description 11
- 230000008901 benefit Effects 0.000 description 11
- 235000013351 cheese Nutrition 0.000 description 11
- 238000010367 cloning Methods 0.000 description 11
- 238000001556 precipitation Methods 0.000 description 11
- 101710098247 Exoglucanase 1 Proteins 0.000 description 10
- 239000012141 concentrate Substances 0.000 description 10
- 230000012010 growth Effects 0.000 description 10
- 239000011159 matrix material Substances 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 239000000872 buffer Substances 0.000 description 9
- 230000002950 deficient Effects 0.000 description 9
- 235000013376 functional food Nutrition 0.000 description 9
- 239000002245 particle Substances 0.000 description 9
- SEHFUALWMUWDKS-UHFFFAOYSA-N 5-fluoroorotic acid Chemical compound OC(=O)C=1NC(=O)NC(=O)C=1F SEHFUALWMUWDKS-UHFFFAOYSA-N 0.000 description 8
- 101000899859 Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) Endoglucanase 1 Proteins 0.000 description 8
- 101710126559 Endoglucanase EG-II Proteins 0.000 description 8
- 239000002299 complementary DNA Substances 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 238000009396 hybridization Methods 0.000 description 8
- 230000035772 mutation Effects 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- -1 pyriylalanine Chemical compound 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- 230000028327 secretion Effects 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 7
- 101710098246 Exoglucanase 2 Proteins 0.000 description 7
- 108091034117 Oligonucleotide Proteins 0.000 description 7
- 108020004511 Recombinant DNA Proteins 0.000 description 7
- 101150052795 cbh-1 gene Proteins 0.000 description 7
- 230000022811 deglycosylation Effects 0.000 description 7
- 238000011049 filling Methods 0.000 description 7
- 108020001507 fusion proteins Proteins 0.000 description 7
- 102000037865 fusion proteins Human genes 0.000 description 7
- 238000003780 insertion Methods 0.000 description 7
- 230000037431 insertion Effects 0.000 description 7
- 230000000813 microbial effect Effects 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- DRCWOKJLSQUJPZ-DZGCQCFKSA-N (4ar,9as)-n-ethyl-1,4,9,9a-tetrahydrofluoren-4a-amine Chemical compound C1C2=CC=CC=C2[C@]2(NCC)[C@H]1CC=CC2 DRCWOKJLSQUJPZ-DZGCQCFKSA-N 0.000 description 6
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 6
- 241000228212 Aspergillus Species 0.000 description 6
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 6
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- 239000007995 HEPES buffer Substances 0.000 description 6
- 101001008429 Homo sapiens Nucleobindin-2 Proteins 0.000 description 6
- 230000004988 N-glycosylation Effects 0.000 description 6
- 102100027441 Nucleobindin-2 Human genes 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- 210000001938 protoplast Anatomy 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 235000015067 sauces Nutrition 0.000 description 6
- 238000000108 ultra-filtration Methods 0.000 description 6
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 6
- 229940045145 uridine Drugs 0.000 description 6
- 241000228245 Aspergillus niger Species 0.000 description 5
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 5
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 5
- 108010064785 Phospholipases Proteins 0.000 description 5
- 102000015439 Phospholipases Human genes 0.000 description 5
- 241000209140 Triticum Species 0.000 description 5
- 235000021307 Triticum Nutrition 0.000 description 5
- 239000012131 assay buffer Substances 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 229910001882 dioxygen Inorganic materials 0.000 description 5
- 235000021588 free fatty acids Nutrition 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 235000016709 nutrition Nutrition 0.000 description 5
- 239000008188 pellet Substances 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 238000002864 sequence alignment Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 238000010561 standard procedure Methods 0.000 description 5
- 230000009261 transgenic effect Effects 0.000 description 5
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 4
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 4
- 241000351920 Aspergillus nidulans Species 0.000 description 4
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 4
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
- 235000013361 beverage Nutrition 0.000 description 4
- 230000027455 binding Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 235000012970 cakes Nutrition 0.000 description 4
- 239000011575 calcium Substances 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000004590 computer program Methods 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 239000003599 detergent Substances 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 235000012041 food component Nutrition 0.000 description 4
- 239000005417 food ingredient Substances 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 150000004676 glycans Chemical group 0.000 description 4
- 230000003301 hydrolyzing effect Effects 0.000 description 4
- 235000015243 ice cream Nutrition 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 230000002797 proteolythic effect Effects 0.000 description 4
- 238000006722 reduction reaction Methods 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000000600 sorbitol Substances 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 230000002269 spontaneous effect Effects 0.000 description 4
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 4
- 229910052721 tungsten Inorganic materials 0.000 description 4
- 239000010937 tungsten Substances 0.000 description 4
- 244000215068 Acacia senegal Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 229920000084 Gum arabic Polymers 0.000 description 3
- 108091092195 Intron Proteins 0.000 description 3
- 239000007836 KH2PO4 Substances 0.000 description 3
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 3
- 102000011720 Lysophospholipase Human genes 0.000 description 3
- 108020002496 Lysophospholipase Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 3
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 3
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 3
- 102100033357 Pancreatic lipase-related protein 2 Human genes 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 108010043958 Peptoids Proteins 0.000 description 3
- 102100037883 Phospholipase B1, membrane-associated Human genes 0.000 description 3
- 108010058864 Phospholipases A2 Proteins 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 238000002105 Southern blotting Methods 0.000 description 3
- 244000299461 Theobroma cacao Species 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
- 239000013504 Triton X-100 Substances 0.000 description 3
- 239000000205 acacia gum Substances 0.000 description 3
- 235000010489 acacia gum Nutrition 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 101150069003 amdS gene Proteins 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 235000008429 bread Nutrition 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 235000011148 calcium chloride Nutrition 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 239000006071 cream Substances 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 235000014113 dietary fatty acids Nutrition 0.000 description 3
- 235000015872 dietary supplement Nutrition 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 229930195729 fatty acid Natural products 0.000 description 3
- 239000000194 fatty acid Substances 0.000 description 3
- 150000004665 fatty acids Chemical class 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 235000004213 low-fat Nutrition 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 235000010755 mineral Nutrition 0.000 description 3
- 239000011707 mineral Substances 0.000 description 3
- 239000007003 mineral medium Substances 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- 239000002417 nutraceutical Substances 0.000 description 3
- 235000021436 nutraceutical agent Nutrition 0.000 description 3
- 239000002751 oligonucleotide probe Substances 0.000 description 3
- 229960003104 ornithine Drugs 0.000 description 3
- 229960005190 phenylalanine Drugs 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- JEGLJLCZRAWOBI-UHFFFAOYSA-N rac-1,2-di-O-dodecanylglycero-3-glutaric acid 6'-methylresorufin ester Chemical compound C1=CC(=O)C=C2OC3=C(C)C(OC(=O)CCCC(=O)OCC(COCCCCCCCCCCCC)OCCCCCCCCCCCC)=CC=C3N=C21 JEGLJLCZRAWOBI-UHFFFAOYSA-N 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000011573 trace mineral Substances 0.000 description 3
- 235000013619 trace mineral Nutrition 0.000 description 3
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 108010084185 Cellulases Proteins 0.000 description 2
- 102000005575 Cellulases Human genes 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102100022624 Glucoamylase Human genes 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- 102000005720 Glutathione transferase Human genes 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- QWCKQJZIFLGMSD-VKHMYHEASA-N L-alpha-aminobutyric acid Chemical compound CC[C@H](N)C(O)=O QWCKQJZIFLGMSD-VKHMYHEASA-N 0.000 description 2
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 241000235648 Pichia Species 0.000 description 2
- 108010059820 Polygalacturonase Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- HIWPGCMGAMJNRG-ACCAVRKYSA-N Sophorose Natural products O([C@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HIWPGCMGAMJNRG-ACCAVRKYSA-N 0.000 description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical group OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical group C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000005804 alkylation reaction Methods 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 230000006229 amino acid addition Effects 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 229940025131 amylases Drugs 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 235000015173 baked goods and baking mixes Nutrition 0.000 description 2
- HIWPGCMGAMJNRG-UHFFFAOYSA-N beta-sophorose Natural products OC1C(O)C(CO)OC(O)C1OC1C(O)C(O)C(O)C(CO)O1 HIWPGCMGAMJNRG-UHFFFAOYSA-N 0.000 description 2
- 235000015895 biscuits Nutrition 0.000 description 2
- 229910052796 boron Inorganic materials 0.000 description 2
- 238000004422 calculation algorithm Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 2
- 235000019219 chocolate Nutrition 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 235000014510 cooky Nutrition 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 235000011850 desserts Nutrition 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 101150066032 egl-1 gene Proteins 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 108010018734 hexose oxidase Proteins 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000012669 liquid formulation Substances 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical compound CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 230000005257 nucleotidylation Effects 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 238000012510 peptide mapping method Methods 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- SXADIBFZNXBEGI-UHFFFAOYSA-N phosphoramidous acid Chemical compound NP(O)O SXADIBFZNXBEGI-UHFFFAOYSA-N 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- PZDOWFGHCNHPQD-VNNZMYODSA-N sophorose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](C=O)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O PZDOWFGHCNHPQD-VNNZMYODSA-N 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 238000003828 vacuum filtration Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 235000013618 yogurt Nutrition 0.000 description 2
- ZDRLKQLULCHOAJ-SECBINFHSA-N (2S)-2-amino-2,3,3-trifluoro-3-(4-hydroxyphenyl)propanoic acid Chemical compound FC([C@](N)(C(=O)O)F)(C1=CC=C(C=C1)O)F ZDRLKQLULCHOAJ-SECBINFHSA-N 0.000 description 1
- IYKLZBIWFXPUCS-VIFPVBQESA-N (2s)-2-(naphthalen-1-ylamino)propanoic acid Chemical compound C1=CC=C2C(N[C@@H](C)C(O)=O)=CC=CC2=C1 IYKLZBIWFXPUCS-VIFPVBQESA-N 0.000 description 1
- WNNNWFKQCKFSDK-BYPYZUCNSA-N (2s)-2-aminopent-4-enoic acid Chemical compound OC(=O)[C@@H](N)CC=C WNNNWFKQCKFSDK-BYPYZUCNSA-N 0.000 description 1
- GTVVZTAFGPQSPC-QMMMGPOBSA-N (2s)-2-azaniumyl-3-(4-nitrophenyl)propanoate Chemical compound OC(=O)[C@@H](N)CC1=CC=C([N+]([O-])=O)C=C1 GTVVZTAFGPQSPC-QMMMGPOBSA-N 0.000 description 1
- BWKMGYQJPOAASG-VIFPVBQESA-N (3s)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid Chemical compound C1=CC=C2CN[C@H](C(=O)O)CC2=C1 BWKMGYQJPOAASG-VIFPVBQESA-N 0.000 description 1
- 101150084750 1 gene Proteins 0.000 description 1
- KDYAPQVYJXUQNY-OPHDRXFHSA-N 1,2-di-(alpha-linolenoyl)-3-[alpha-D-galactosyl-(1->6)-beta-D-galactosyl]-sn-glycerol Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](OC[C@@H](COC(=O)CCCCCCC\C=C/C\C=C/C\C=C/CC)OC(=O)CCCCCCC\C=C/C\C=C/C\C=C/CC)O[C@@H]1CO[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 KDYAPQVYJXUQNY-OPHDRXFHSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- BLCJBICVQSYOIF-UHFFFAOYSA-N 2,2-diaminobutanoic acid Chemical compound CCC(N)(N)C(O)=O BLCJBICVQSYOIF-UHFFFAOYSA-N 0.000 description 1
- WTOFYLAWDLQMBZ-UHFFFAOYSA-N 2-azaniumyl-3-thiophen-2-ylpropanoate Chemical compound OC(=O)C(N)CC1=CC=CS1 WTOFYLAWDLQMBZ-UHFFFAOYSA-N 0.000 description 1
- 108020005065 3' Flanking Region Proteins 0.000 description 1
- PDLPTSJWDUCMKS-UHFFFAOYSA-N 3-[4-(3-sulfopropyl)piperazin-1-yl]propane-1-sulfonic acid Chemical compound OS(=O)(=O)CCCN1CCN(CCCS(O)(=O)=O)CC1 PDLPTSJWDUCMKS-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- NIGWMJHCCYYCSF-QMMMGPOBSA-N 4-chloro-L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(Cl)C=C1 NIGWMJHCCYYCSF-QMMMGPOBSA-N 0.000 description 1
- 108010011619 6-Phytase Proteins 0.000 description 1
- XDOLZJYETYVRKV-UHFFFAOYSA-N 7-Aminoheptanoic acid Chemical compound NCCCCCCC(O)=O XDOLZJYETYVRKV-UHFFFAOYSA-N 0.000 description 1
- 108010013043 Acetylesterase Proteins 0.000 description 1
- 108700016155 Acyl transferases Proteins 0.000 description 1
- 102000057234 Acyl transferases Human genes 0.000 description 1
- 102000004400 Aminopeptidases Human genes 0.000 description 1
- 108090000915 Aminopeptidases Proteins 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000228193 Aspergillus clavatus Species 0.000 description 1
- 241001507865 Aspergillus fischeri Species 0.000 description 1
- 241001225321 Aspergillus fumigatus Species 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 240000001009 Aspergillus oryzae RIB40 Species 0.000 description 1
- 235000013023 Aspergillus oryzae RIB40 Nutrition 0.000 description 1
- 241001465318 Aspergillus terreus Species 0.000 description 1
- 101150076489 B gene Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- 101000583086 Bunodosoma granuliferum Delta-actitoxin-Bgr2b Proteins 0.000 description 1
- 101100011368 Caenorhabditis elegans egl-1 gene Proteins 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 102000004308 Carboxylic Ester Hydrolases Human genes 0.000 description 1
- 108090000863 Carboxylic Ester Hydrolases Proteins 0.000 description 1
- 108010006303 Carboxypeptidases Proteins 0.000 description 1
- 102000005367 Carboxypeptidases Human genes 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010022172 Chitinases Proteins 0.000 description 1
- 102000012286 Chitinases Human genes 0.000 description 1
- 241000078532 Chlorococcum polymorphum Species 0.000 description 1
- 108090000746 Chymosin Proteins 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241001362614 Crassa Species 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N D-alanine Chemical compound C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 101100109110 Danio rerio aph1b gene Proteins 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 101001096557 Dickeya dadantii (strain 3937) Rhamnogalacturonate lyase Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 108020000949 Fungal DNA Proteins 0.000 description 1
- 101150108358 GLAA gene Proteins 0.000 description 1
- 102100039556 Galectin-4 Human genes 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 108050008938 Glucoamylases Proteins 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 101000608765 Homo sapiens Galectin-4 Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 101150017040 I gene Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102100024319 Intestinal-type alkaline phosphatase Human genes 0.000 description 1
- 101710184243 Intestinal-type alkaline phosphatase Proteins 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- SNDPXSYFESPGGJ-BYPYZUCNSA-N L-2-aminopentanoic acid Chemical compound CCC[C@H](N)C(O)=O SNDPXSYFESPGGJ-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ZGUNAGUHMKGQNY-ZETCQYMHSA-N L-alpha-phenylglycine zwitterion Chemical compound OC(=O)[C@@H](N)C1=CC=CC=C1 ZGUNAGUHMKGQNY-ZETCQYMHSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- UCUNFLYVYCGDHP-BYPYZUCNSA-N L-methionine sulfone Chemical compound CS(=O)(=O)CC[C@H](N)C(O)=O UCUNFLYVYCGDHP-BYPYZUCNSA-N 0.000 description 1
- UCUNFLYVYCGDHP-UHFFFAOYSA-N L-methionine sulfone Natural products CS(=O)(=O)CCC(N)C(O)=O UCUNFLYVYCGDHP-UHFFFAOYSA-N 0.000 description 1
- SNDPXSYFESPGGJ-UHFFFAOYSA-N L-norVal-OH Natural products CCCC(N)C(O)=O SNDPXSYFESPGGJ-UHFFFAOYSA-N 0.000 description 1
- DZLNHFMRPBPULJ-VKHMYHEASA-N L-thioproline Chemical compound OC(=O)[C@@H]1CSCN1 DZLNHFMRPBPULJ-VKHMYHEASA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 108010029541 Laccase Proteins 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 108010054377 Mannosidases Proteins 0.000 description 1
- 102000001696 Mannosidases Human genes 0.000 description 1
- 108010006519 Molecular Chaperones Proteins 0.000 description 1
- 102000005431 Molecular Chaperones Human genes 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- 102100036617 Monoacylglycerol lipase ABHD2 Human genes 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical group CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 229910014130 Na—P Inorganic materials 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 241000320412 Ogataea angusta Species 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- PEMUHKUIQHFMTH-UHFFFAOYSA-N P-Bromo-DL-phenylalanine Chemical compound OC(=O)C(N)CC1=CC=C(Br)C=C1 PEMUHKUIQHFMTH-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000736122 Parastagonospora nodorum Species 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 241000574469 Penicillium allii Species 0.000 description 1
- 241001507683 Penicillium aurantiogriseum Species 0.000 description 1
- 241000228150 Penicillium chrysogenum Species 0.000 description 1
- 108700020962 Peroxidase Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 102000004861 Phosphoric Diester Hydrolases Human genes 0.000 description 1
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108010039918 Polylysine Chemical group 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 108090001066 Racemases and epimerases Proteins 0.000 description 1
- 102000004879 Racemases and epimerases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 101001095983 Staphylococcus aureus Protein rep Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 235000019486 Sunflower oil Nutrition 0.000 description 1
- 241001523006 Talaromyces marneffei Species 0.000 description 1
- 235000009470 Theobroma cacao Nutrition 0.000 description 1
- 241000223257 Thermomyces Species 0.000 description 1
- 101000984201 Thermomyces lanuginosus Lipase Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 108060008539 Transglutaminase Proteins 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 102000003425 Tyrosinase Human genes 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- KYOBSHFOBAOFBF-UHFFFAOYSA-N UMP Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1C(O)=O KYOBSHFOBAOFBF-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 108010048241 acetamidase Proteins 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000002730 additional effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 229960002684 aminocaproic acid Drugs 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 150000003868 ammonium compounds Chemical class 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 229940091771 aspergillus fumigatus Drugs 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000021168 barbecue Nutrition 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 235000015496 breakfast cereal Nutrition 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 235000012839 cake mixes Nutrition 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 101150102092 ccdB gene Proteins 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 235000020140 chocolate milk drink Nutrition 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 229940080701 chymosin Drugs 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 238000012411 cloning technique Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 235000013353 coffee beverage Nutrition 0.000 description 1
- 238000002485 combustion reaction Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005112 continuous flow technique Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 235000015142 cultured sour cream Nutrition 0.000 description 1
- 108010005400 cutinase Proteins 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 229940119679 deoxyribonucleases Drugs 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 235000012489 doughnuts Nutrition 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 101150003727 egl2 gene Proteins 0.000 description 1
- 108010091371 endoglucanase 1 Proteins 0.000 description 1
- 108010091384 endoglucanase 2 Proteins 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 210000005254 filamentous fungi cell Anatomy 0.000 description 1
- 235000013332 fish product Nutrition 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 150000004820 halides Chemical class 0.000 description 1
- 230000008821 health effect Effects 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 108010002430 hemicellulase Proteins 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 235000008446 instant noodles Nutrition 0.000 description 1
- 235000014109 instant soup Nutrition 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000020124 milk-based beverage Nutrition 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000007498 myristoylation Effects 0.000 description 1
- GNOLWGAJQVLBSM-UHFFFAOYSA-N n,n,5,7-tetramethyl-1,2,3,4-tetrahydronaphthalen-1-amine Chemical compound C1=C(C)C=C2C(N(C)C)CCCC2=C1C GNOLWGAJQVLBSM-UHFFFAOYSA-N 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 235000012149 noodles Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- KYOBSHFOBAOFBF-ZAKLUEHWSA-N orotidine-5'-monophosphate Chemical compound O[C@@H]1[C@@H](O)[C@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1C(O)=O KYOBSHFOBAOFBF-ZAKLUEHWSA-N 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 235000014594 pastries Nutrition 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 108010087558 pectate lyase Proteins 0.000 description 1
- 108010072638 pectinacetylesterase Proteins 0.000 description 1
- 102000004251 pectinacetylesterase Human genes 0.000 description 1
- 108020004410 pectinesterase Proteins 0.000 description 1
- 230000002351 pectolytic effect Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920000656 polylysine Chemical group 0.000 description 1
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 235000013613 poultry product Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229940043267 rhodamine b Drugs 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- PCMORTLOPMLEFB-ONEGZZNKSA-N sinapic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-ONEGZZNKSA-N 0.000 description 1
- PCMORTLOPMLEFB-UHFFFAOYSA-N sinapinic acid Natural products COC1=CC(C=CC(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-UHFFFAOYSA-N 0.000 description 1
- 235000009561 snack bars Nutrition 0.000 description 1
- 235000011888 snacks Nutrition 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 235000014214 soft drink Nutrition 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000002600 sunflower oil Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 235000013616 tea Nutrition 0.000 description 1
- VKFFEYLSKIYTSJ-UHFFFAOYSA-N tetraazanium;phosphonato phosphate Chemical compound [NH4+].[NH4+].[NH4+].[NH4+].[O-]P([O-])(=O)OP([O-])([O-])=O VKFFEYLSKIYTSJ-UHFFFAOYSA-N 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- OFVLGDICTFRJMM-WESIUVDSSA-N tetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O OFVLGDICTFRJMM-WESIUVDSSA-N 0.000 description 1
- 239000000892 thaumatin Substances 0.000 description 1
- 235000010436 thaumatin Nutrition 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000000954 titration curve Methods 0.000 description 1
- 235000012184 tortilla Nutrition 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000005809 transesterification reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 102000003601 transglutaminase Human genes 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 235000021119 whey protein Nutrition 0.000 description 1
- 235000008924 yoghurt drink Nutrition 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
- 235000009529 zinc sulphate Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/14—Yeasts or derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
Abstract
The present invention relates to a method for the production of a lipolytic enzyme in Trichoderma reesei and the lipolytic enzyme obtainable therefrom. In addition, the present invention relates to the use of Trichoderma to express a lipolytic enzyme.
Description
METHOD
FIELD OF THE PRESENT INVENTION
The present invention relates to a method for the production of a lipolytic enzyme in Trichoderma reesei and the lipolytic enzyme obtainable there from. In addition, the present invention relates to the use of Trichoderma to express a lipolytic enzyme.
BACKGROUND OF THE PRESENT INVENTION
Lipases (EC 3.1..13), which can be defined as carboxylesterases which catalyze the hydrolysis of acylglycerols, are physiologically very important enzymes as one of the three major digestive enzymes together with amylases and proteases. They hydrolyse lipids to glycerol and fatty acids, but can also function in esterification or transesterification reactions.
Lipases have applications in several industrial processes such as processing of oils and fats, detergent manufacturing, paper processing and in the cheese making and baking industries.
WO 98/45453 discloses a lipolytic enzyme (and variants thereof) derived from the filamentous fungus Aspergillus tubingensis- This enzyme is sometimes referred to as "lipase 3 WO 98/45453 characterizes several of the physicochemical characteristics of lipase 3. Uses of this lipolytic enzyme for improving the properties of bread were also described, in particular for improving the properties of bread, WO 98/45453 also described the cloning and expression of lipase 3 and its variants in Aspergillus tubirrgensis. It was found that this lipolytic enzyme could be overexpressed in Aspergillus tubingensis, however the enzyme was overglycosylated in A..
tubingensis which, in some situations, can decrease its activity, There is a need for a method for the production of lipase 3 and its variants and other lipolytic enzymes on a commercial scale and using expression hosts which provide high levels of protein expression and yield.. In addition, there is also a need to overcome the
FIELD OF THE PRESENT INVENTION
The present invention relates to a method for the production of a lipolytic enzyme in Trichoderma reesei and the lipolytic enzyme obtainable there from. In addition, the present invention relates to the use of Trichoderma to express a lipolytic enzyme.
BACKGROUND OF THE PRESENT INVENTION
Lipases (EC 3.1..13), which can be defined as carboxylesterases which catalyze the hydrolysis of acylglycerols, are physiologically very important enzymes as one of the three major digestive enzymes together with amylases and proteases. They hydrolyse lipids to glycerol and fatty acids, but can also function in esterification or transesterification reactions.
Lipases have applications in several industrial processes such as processing of oils and fats, detergent manufacturing, paper processing and in the cheese making and baking industries.
WO 98/45453 discloses a lipolytic enzyme (and variants thereof) derived from the filamentous fungus Aspergillus tubingensis- This enzyme is sometimes referred to as "lipase 3 WO 98/45453 characterizes several of the physicochemical characteristics of lipase 3. Uses of this lipolytic enzyme for improving the properties of bread were also described, in particular for improving the properties of bread, WO 98/45453 also described the cloning and expression of lipase 3 and its variants in Aspergillus tubirrgensis. It was found that this lipolytic enzyme could be overexpressed in Aspergillus tubingensis, however the enzyme was overglycosylated in A..
tubingensis which, in some situations, can decrease its activity, There is a need for a method for the production of lipase 3 and its variants and other lipolytic enzymes on a commercial scale and using expression hosts which provide high levels of protein expression and yield.. In addition, there is also a need to overcome the
2 problem of decreased enzyme activity due to overglycosylation of the lipolytic enzyme as seen for example when it is overexpressed in A. tubingensis..
SUMMARY ASPECTS OF THE PRESENT INVENTION
It has surprisingly been found that Trichoderma reesei is a highly efficient expression host for lipolytic enzymes - in particular lipase 3 and its variants and other lipolytic enzymes Accordingly, in a first aspect of the present invention there is provided a method of producing a lipolytic enzyme comprising the steps of.
(i) providing a Trichoderma reesei cell comprising a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID
NO: 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40%
sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a Iipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO. 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO. 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions; and (ii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said Iipolytic enzyme.
SUMMARY ASPECTS OF THE PRESENT INVENTION
It has surprisingly been found that Trichoderma reesei is a highly efficient expression host for lipolytic enzymes - in particular lipase 3 and its variants and other lipolytic enzymes Accordingly, in a first aspect of the present invention there is provided a method of producing a lipolytic enzyme comprising the steps of.
(i) providing a Trichoderma reesei cell comprising a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID
NO: 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40%
sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a Iipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO. 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO. 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions; and (ii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said Iipolytic enzyme.
3 In a further aspect of the present invention there is provided a method of producing a lipolytic enzyme comprising the steps of.
(i) transfecting or transforming a Trichoderma reesei cell with a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID
NO. 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO. 4 or a nucleotide sequence which has at least '15 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4, and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO., 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4 or the complement of any thereof under stringent conditions;
(ii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme..
In a further aspect of the present invention there is provided a method of producing a lipolytic enzyme comprising the steps of:
(i) transfecting or transforming a Trichoderma reesei cell with a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID
NO: 1 or 2; and/or
(i) transfecting or transforming a Trichoderma reesei cell with a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID
NO. 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO. 4 or a nucleotide sequence which has at least '15 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4, and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO., 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4 or the complement of any thereof under stringent conditions;
(ii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme..
In a further aspect of the present invention there is provided a method of producing a lipolytic enzyme comprising the steps of:
(i) transfecting or transforming a Trichoderma reesei cell with a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID
NO: 1 or 2; and/or
4 b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO. 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO., 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions;
(ii) repeating step (i) on the cell to sequentially transfect or transform the cell with at least one additional heterologous nucleotide sequence as defined in (i)(a), (i)(b) or (i)(c) (e.g., .such as a heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO, 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID NO-I or 2); and (iii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme.
The present invention yet further provides a lipolytic enzyme obtainable by a method of the present invention.
There is also provided by the present invention, a foodstuff for human consumption comprising said lipolytic enzyme obtainable by a method of the present invention.
The present invention yet further provides a transformed or transfected Trichoderma reesei cell comprising:
a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme protein having at least 40% sequence identity to SEQ ID NO. 1 or 2, and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40%
sequence identity to SEQ ID NO, 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme
(ii) repeating step (i) on the cell to sequentially transfect or transform the cell with at least one additional heterologous nucleotide sequence as defined in (i)(a), (i)(b) or (i)(c) (e.g., .such as a heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO, 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID NO-I or 2); and (iii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme.
The present invention yet further provides a lipolytic enzyme obtainable by a method of the present invention.
There is also provided by the present invention, a foodstuff for human consumption comprising said lipolytic enzyme obtainable by a method of the present invention.
The present invention yet further provides a transformed or transfected Trichoderma reesei cell comprising:
a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme protein having at least 40% sequence identity to SEQ ID NO. 1 or 2, and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40%
sequence identity to SEQ ID NO, 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme
5 wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO. 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO. 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions.
In another aspect the present invention provides an expression vector comprising i) at least one nucleotide sequence which nucleotide sequence:
a) encodes a lipolytic enzyme protein having at least 40% sequence identity to SEQ ID NO. 1 or 2; and/or b) encodes a lipolytic enzyme and comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40%
sequence identity to SEQ ID NO. 3 or SEQ ID NO. 4; and/or c) encodes a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ 1D NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions; and ii) at least one cellobiohydrolase promoter, wherein said at least one nucleotide sequence is under the control of said at least one cellobiohydrolase promoter-In another aspect of the present invention, there is provided a use of a Trichoderma reesei cell in the expression of a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID NO., 1 or 2; and/or
In another aspect the present invention provides an expression vector comprising i) at least one nucleotide sequence which nucleotide sequence:
a) encodes a lipolytic enzyme protein having at least 40% sequence identity to SEQ ID NO. 1 or 2; and/or b) encodes a lipolytic enzyme and comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40%
sequence identity to SEQ ID NO. 3 or SEQ ID NO. 4; and/or c) encodes a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ 1D NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions; and ii) at least one cellobiohydrolase promoter, wherein said at least one nucleotide sequence is under the control of said at least one cellobiohydrolase promoter-In another aspect of the present invention, there is provided a use of a Trichoderma reesei cell in the expression of a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID NO., 1 or 2; and/or
6 b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40%
sequence identity to SEQ I D NO. 3 or SEQ I D NO: 4, and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions;
for improving one or more of the following. expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity or yield.
Surprisingly, we have found that Trichoderma reesei is capable of producing the lipolytic enzymes in significantly high yields.
Furthermore, we have found that the lipolytic enzymes produced by the methodology of the present invention are surprisingly not overglycosylated and therefore have good enzyme activity.
Bradner et at (in Current Genetics, 44: 224-230, (2003)) used Trichoderma reesei as an expression host organism in their search for previously unknown lipolytic enzymes. A
lipase from an Antarctic isolate of Penicillium allii was cloned and expressed in Trichoderma reesei. Bradner et al. concluded that the methods described would be useful for prospecting for potentially novel lipase genes but no suggestion was made that T. reesei could be used for overexpression of proteins.
In another aspect the present invention provides a method of producing a lipolytic enzyme comprising the steps of:
(i) providing a transformed or transfected Trichoderma reesei cell comprising at least one heterologous nucleotide sequence encoding a lipolytic enzyme;
sequence identity to SEQ I D NO. 3 or SEQ I D NO: 4, and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions;
for improving one or more of the following. expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity or yield.
Surprisingly, we have found that Trichoderma reesei is capable of producing the lipolytic enzymes in significantly high yields.
Furthermore, we have found that the lipolytic enzymes produced by the methodology of the present invention are surprisingly not overglycosylated and therefore have good enzyme activity.
Bradner et at (in Current Genetics, 44: 224-230, (2003)) used Trichoderma reesei as an expression host organism in their search for previously unknown lipolytic enzymes. A
lipase from an Antarctic isolate of Penicillium allii was cloned and expressed in Trichoderma reesei. Bradner et al. concluded that the methods described would be useful for prospecting for potentially novel lipase genes but no suggestion was made that T. reesei could be used for overexpression of proteins.
In another aspect the present invention provides a method of producing a lipolytic enzyme comprising the steps of:
(i) providing a transformed or transfected Trichoderma reesei cell comprising at least one heterologous nucleotide sequence encoding a lipolytic enzyme;
7 (ii) culturing the cell at a pH 4 to pH 5.5 under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme;
(iii) isolating, purifying or concentrating the enzyme in a medium at pH 5.5 to pH
6.5.
In this aspect, preferably the lipolytic enzyme:
a) comprises an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or comprises an amino acid sequence which has at least 40% sequence identity to SEQ ID NO: 1 or 2; and/or b) is encoded by a nucleotide comprising the sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or comprising a nucleotide sequence which has at least 40%
sequence identity to SEQ ID NO. 3 or SEQ ID NO: 4; and/or c) is encoded by a nucleotide sequence comprising a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or comprises a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID
NO: 4 or the complement of any thereof under stringent conditions-DETAILED ASPECTS OF THE PRESENT INVENTION
Preferably, the heterologous nucleotide sequence is operably connected (directly or indirectly) to a promoter sequence such that the heterologous nucleotide sequence is under the control of the promoter sequence, The promoter sequence may be any suitable promoter - such as a promoter that is naturally associated with the nucleotide sequence coding for the lipolytic enzyme and/or a heterologous promoter, such as a cellobiohydrolase 1 promoter sequence or Tef promoter or a glucoamylase promoter. Other Trichoderma promoter sequences derived from stp (United States Serial No. 12/193,614), cbh2, egl1, eg12, gpdl genes, can be linked to the nucleotide sequence of the lipolytic enzyme.
(iii) isolating, purifying or concentrating the enzyme in a medium at pH 5.5 to pH
6.5.
In this aspect, preferably the lipolytic enzyme:
a) comprises an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or comprises an amino acid sequence which has at least 40% sequence identity to SEQ ID NO: 1 or 2; and/or b) is encoded by a nucleotide comprising the sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or comprising a nucleotide sequence which has at least 40%
sequence identity to SEQ ID NO. 3 or SEQ ID NO: 4; and/or c) is encoded by a nucleotide sequence comprising a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or comprises a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID
NO: 4 or the complement of any thereof under stringent conditions-DETAILED ASPECTS OF THE PRESENT INVENTION
Preferably, the heterologous nucleotide sequence is operably connected (directly or indirectly) to a promoter sequence such that the heterologous nucleotide sequence is under the control of the promoter sequence, The promoter sequence may be any suitable promoter - such as a promoter that is naturally associated with the nucleotide sequence coding for the lipolytic enzyme and/or a heterologous promoter, such as a cellobiohydrolase 1 promoter sequence or Tef promoter or a glucoamylase promoter. Other Trichoderma promoter sequences derived from stp (United States Serial No. 12/193,614), cbh2, egl1, eg12, gpdl genes, can be linked to the nucleotide sequence of the lipolytic enzyme.
8 For some embodiments the nucleotide sequence coding for the lipolytic enzyme may include one or more introns..
For some embodiments the nucleotide sequence coding for the lipolytic enzyme is a genomic sequence.
For some embodiments the nucleotide sequence coding for the lipolytic enzyme is a cDNA sequence.
The method of the present invention may comprise the additional step of isolating and/or purifying and/or recovering the lipolytic enzyme For some embodiments the level of expressed lipolytic enzyme is high enough so that one can use the broth medium into which the enzyme has been secreted (preferably after removal of the cell(s)) or in a concentrate form (preferably after removal of the cell(s)).
Therefore, in a preferred aspect, the method of the present invention includes the following additional step(s) of isolating and/or purifying and/or recovering the lipolytic enzyme.
In a more preferred aspect, the method of the present invention includes the following additional step(s) of removing the cell(s) from the medium (e.g. broth) into which the enzyme has been secreted.
In a more preferred aspect, the method of the present invention includes the following additional step(s) of removing the cell(s) from the medium (e.g. broth) into which the enzyme has been secreted; and then concentrating the medium,.
The cell(s) may be removed from the medium via suitable separation techniques -e g.
suitable filtration techniques and/or centrifugation techniques. A particular example is use of ultra-filtration to prepare UFCs (ultra-filtration concentrates).
In one embodiment, the pH of the medium for culturing is about pH 4 to about 5,5, preferably about pH 4.
For some embodiments the nucleotide sequence coding for the lipolytic enzyme is a genomic sequence.
For some embodiments the nucleotide sequence coding for the lipolytic enzyme is a cDNA sequence.
The method of the present invention may comprise the additional step of isolating and/or purifying and/or recovering the lipolytic enzyme For some embodiments the level of expressed lipolytic enzyme is high enough so that one can use the broth medium into which the enzyme has been secreted (preferably after removal of the cell(s)) or in a concentrate form (preferably after removal of the cell(s)).
Therefore, in a preferred aspect, the method of the present invention includes the following additional step(s) of isolating and/or purifying and/or recovering the lipolytic enzyme.
In a more preferred aspect, the method of the present invention includes the following additional step(s) of removing the cell(s) from the medium (e.g. broth) into which the enzyme has been secreted.
In a more preferred aspect, the method of the present invention includes the following additional step(s) of removing the cell(s) from the medium (e.g. broth) into which the enzyme has been secreted; and then concentrating the medium,.
The cell(s) may be removed from the medium via suitable separation techniques -e g.
suitable filtration techniques and/or centrifugation techniques. A particular example is use of ultra-filtration to prepare UFCs (ultra-filtration concentrates).
In one embodiment, the pH of the medium for culturing is about pH 4 to about 5,5, preferably about pH 4.
9 In one embodiment, the pH of the medium for culturing is about pH 4.
In a preferred embodiment, prior to the isolation and/or purification and/or concentration of the enzyme, the pH of the medium is raised at the end of the fermentation run when sufficient levels of the secreted, soluble enzyme are reached.
In one embodiment, the pH of the medium for isolating and/or purifying and/or concentrating the enzyme is above pH 5.5 to about pH 6..5.
Thus, in one embodiment, the pH of the medium for culturing is about pH 4 and then the pH of the medium is raised such that the pH of the medium for isolating and/or purifying and/or concentrating the enzyme is above pH 5..5 to about pH 6.5.
Thus, in one embodiment, the pH of the medium for culturing is about pH 4 and then the pH of the medium is raised such that the pH of the medium for isolating and/or purifying and/or concentrating the enzyme is above about pH 6 to about pH 6.5.
In a preferred embodiment, the pH of the medium for culturing is about pH 4,5 and then the pH of the medium is raised such that the pH of the medium for isolating and/or purifying and/or concentrating the enzyme is about pH 6, Preferably the Trichoderma reesei cell is provided by transforming it with, or is transformed with, the nucleotide sequence using electroporation, such as by the electroporation methodology disclosed in WO 2008/153712 A2.
In another embodiment, the Trichoderma reesei cell may be provided by transforming it with, or may be transformed with, the nucleotide sequence using biolistic transformation, Suitably there may be at least one heterologous nucleotide sequence encoding the lipolytic enzyme in the Trichoderma reesei cell. In some embodiments, there may be two or more copies (I e., multiple copies) of the or each heterologous nucleotide sequence encoding the lipolytic enzyme according to the present invention in the Trichoderma reesei cell. For example, in one embodiment there may be at least two nucleotide sequences encoding the lipolytic enzyme in the Trichoderma reesei. In other embodiments there may be at least three, such as at least four, such as at least five, or such as at least six heterologous nucleotide sequences encoding said lipolytic enzyme.
Suitably there may be up to about six, preferably up to about seven, preferably up to about eight, preferably up to about ten heterologous nucleotide sequences encoding the 5 lipolytic enzyme in the Trichoderma reesei cell. In some embodiments each heterologous nucleotide sequence is associated with and is under the control of a promoter. Suitably each heterologous nucleotide sequence may have a separate promoter associated with it and be under the control of that promoter, The promoters may be the same or different..
Thus, in one embodiment the Trichoderma reesei cell may comprise or be transfected or transformed with at least 2 heterologous nucleotide sequences encoding the lipolytic enzyme. In another embodiment the Trichoderma reesei cell may comprise or be transfected or transformed with at least 3, or at least 4, or at least 5 or at least 6 heterologous nucleotide sequences encoding the lipolytic enzyme. In one embodiment there is up to about 6 heterologous nucleotide sequences encoding the lipolytic enzyme in accordance with the present invention.. In some embodiments there may be up to about 10 heterologous nucleotide sequences encoding the lipolytic enzyme in accordance with the present invention. In some embodiments each heterologous nucleotide sequence is associated with and under the control of a promoter.
Suitably each heterologous nucleotide sequence may have separate promoter associated with it and be under the control of that promoter.
Suitably, the (or each) heterologous nucleotide sequence may comprise a nucleotide sequence which encodes a signal peptide, which nucleotide sequence encoding said signal peptide is operably linked to said nucleotide sequence encoding said lipolytic enzyme. If there are multiple heterologous nucleotide sequences and wherein more than one has a signal sequence associated therewith, then the signal sequences may be the same or different..
Suitably the lipolytic enzyme may comprise an endogenous or exogenous signal peptide. When the signal peptide is endogenous - it means that the signal peptide is that which is naturally linked with the lipolytic enzyme when produced naturally For example, the signal peptide may be the signal peptide in Aspergillus tubingensis which is naturally linked with the lipolytic enzyme when found in Aspergillus tubingensis.
The term "heterologous" as used herein means that it is does not occur naturally in the Trichoderma reesei cell. In other words, it is exogenous to the Trichoderma reesei cell.
For example the term "heterologous nucleotide sequence" as used herein means that the nucleotide sequence does not occur naturally in the Trichoderma reesei cell.. In other words, the nucleotide sequence is exogenous to the Trichoderma reesei cell. The term also includes multiple copies of the naturally occurring sequence as such additional multiple copies would be heterologous.
In one embodiment preferably the heterologous nucleotide sequence is obtained or obtainable from a microorganism, particularly a fungi In one embodiment preferably the heterologous nucleotide sequence is obtained or obtainable from Aspergillus, particularly Aspergillus tubingensis., In a further aspect of the present invention there is provided a method of producing a lipolytic enzyme comprising the steps of.
(i) providing a Trichoderma reesei cell comprising a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID
NO: 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO. 4 or a nucleotide sequence which is at least 40%
sequence identity to SEQ ID NO: 3 or SEQ ID NO, 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO. 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions; and (ii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme; and wherein said Trichoderma reesei cell has at least two genes encoding non-lipolytic enzymes suppressed.
In a further aspect of the present invention there is provided a method of producing a lipolytic enzyme comprising the steps of:
(i) transfecting or transforming a Trichoderma reesei cell with a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID
NO: 1 or 2, and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO. 3 or SEQ 1D NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO. 3 or SEQ ID NO, 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ 1D NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ 1D NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions;
(ii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme; and wherein said Trichoderma reesei cell has at least two genes encoding non-lipolytic enzymes suppressed.
In a further aspect of the present invention there is provided a method of producing a lipolytic enzyme comprising the steps of, (i) transfecting or transforming a Trichoderma reesei cell with a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO. 2 or an amino acid sequence which has at least 30% sequence identity to SEQ ID
NO. 1 or 2; andlor b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4 or the complement of any thereof under stringent conditions;
(ii) repeating step (i) on the cell to sequentially transfect or transform the cell with at least one additional heterologous nucleotide sequence as defined in (i)(a), (i)(b) or (i)(c) (e.g. such as a heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID NO:
1 or 2); and (iii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme; and wherein said Trichoderma reesei cell has at least two genes encoding non-lipolytic enzymes suppressed.
The present invention yet further provides a lipolytic enzyme obtainable by the methods of the present invention There is also provided by the present invention, a foodstuff for human consumption comprising said lipolytic enzyme obtainable by a method of the present invention The present invention yet further provides a transformed or transfected Trichoderma reesei cell comprising:
a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme protein having at least 40% sequence identity to SEQ ID NO. 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40%
sequence identity to SEQ ID NO: 3 or SEQ ID NO': 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ 1D NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ 1D NO., 4 or the complement of any thereof under stringent conditions; and wherein said Trichoderma reesei cell has at least two genes encoding non-lipolytic enzymes suppressed.
Preferably, at least three genes encoding non-lipolytic enzymes suppressed.
Preferably, at least four genes encoding non-lipolytic enzymes suppressed.
"Suppressed' means that the cell does not express the relevant non-lipolytic enzyme at the same level as the non-transformeditransfected cell. In some embodiments, "suppressed" means that the cell does not express the relevant non-lipolytic enzyme..
The suppression may be brought about by techniques known in the art, such as by deletions.
Preferably, at least one of the genes encoding a non-lipolytic enzyme that is suppressed is a cellulase gene.
5 Preferably, at least two of the genes encoding a non-lipolytic enzyme that is suppressed are cellulase genes, An example of at least one of the genes encoding a non-lipolytic enzyme that is suppressed is a gene encoding a cellobiohydrolases (e.g. CBHI or CBHII).
Another example of at least one of the genes encoding a non-lipolytic enzyme that is suppressed is a gene encoding an endoglucanases (e,g. EGI and EGII)..
In some embodiments the Trichoderma reesei cell of the present invention is a cell in which the endogenous genes encoding one or both cellobiohydrolases (CBHI and CBHII) and/or one or both of the endoglucanases (EGI and EGII) are deleted or disrupted Suitably the Trichoderma reesei cell may be a non-GMM cell or derivative thereof, for example a derivative of the strain RL-P37. Suitably the Trichoderma reesei cell may be a derivative of the strain RIL-P37 that is produced using the method set out in Example 10..
In some embodiments, the heterologous nucleotide sequence may encode a lipolytic enzyme comprising an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98%
sequence identity to SEQ ID NO: 1 or SEQ ID No. 2 or a sequence comprising one or several amino acid additions, deletions or substitutions compared to SEQ 1D
NO: 1 or SEQ I D No, 2.
In some embodiments the heterologous nucleotide sequence may encode a lipolytic enzyme and comprises a nucleotide sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98%
sequence identity to SEQ ID NO: 3 or SEQ ID No, 4 or a sequence comprising one or several nucleotide additions, deletions or substitutions compared to SEQ ID
NO. 3 or SEQ ID No 4.
Preferably, the heterologous nucleotide sequence encodes a lipolytic enzyme comprising an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to SEQ ID
NO: 1 or SEQ ID No.. 2 or a sequence comprising one or several amino acid additions, deletions or substitutions compared to SEQ ID NO. 1 or SEQ ID No_ 2.
Preferably, the heterologous nucleotide sequence encodes a lipolytic enzyme and comprises a nucleotide sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to SEQ ID
NO: 3 or SEQ ID No. 4 or a sequence comprising one or several nucleotide additions, deletions or substitutions compared to SEQ ID NO: 3 or SEQ 1D No. 4..
The amino acid sequence identity of a number of lipolytic enzymes to the lipase from Aspergillus tubingensis having the sequence shown in SEQ ID NO: 3 (also called herein lipase 3) are shown in Table 1 below.
Table 1 - Lipolytic enzymes with sequence identity to the lipolytic enzyme from Aspergillus tubingensis (lipase 3).
Lipases from different fungi Accession No Amino acid sequence identit A. ni er CBS 513.88 XP 001397501.1 93 Aspergillus niger ABG73613.1 93 Aspergillus niger ABG37906.1 93 Aspergillus nidulans XP681315.1 61 Aspergillus clavatus NRRL XP_001276337A1 57 Neosartorya fischeri NRRL XP0012663291 60 Aspergillus fumigatus XP748138.1 59 Af293 Aspergillus oryzae RIB40 XP 001818694.1 56 Aspergillus terreus XP001218444,1 54 Penicillium chrysogenum CAP96359.1 55 Wisconsin 54-1255 Aspergillus niger ABG73614.1 54 Aspergillus niger XP 001393532.1 53 Thermomyces 059952 1 50 lanuginosus Penicillium marneffei ATCC XP 0021471441 49 Aspergillus oryzae R XP 001824529.1 44 Phaeosphaeria nodorum 5N15 XP 001796872.1 45 Penicillium cyclopium P61869.1 42 Penicillium camembe.rti. 1T1A A 42 The Trichoderma reesei host cell used in the present invention may be any Trichoderma reesei cell. The cell may be considered to be a wild type Trichoderma reesei cell. The Trichoderma reesei cell may be one from which genes encoding one or more secreted cellobiohydrolases (CBHI or CBH1I) has/have been deleted or disrupted so that they are not expressed Suitably the Trichoderma reesei cell may be a non-genetically modified cell or derivative thereof, for example a derivative of the strain RL-P37.
Suitably the Trichoderma reesei cell may be a derivative of the strain RL-P37 that is produced using the method set out in Example 10, The present invention may be carried out in a fermentor which may comprise about 3 liters to about 20 liters culture mediae In another embodiment the present invention is carried out as a 10-16 liter, preferably a 14 liter scale fermentation.. In one embodiment, preferably the fermentation is carried out with more than about 12 liters, preferably more than about 14 liters.
The Trichoderma reesei host cell is preferably suitable for use in large scale fermentation..
In preferred embodiments, the present invention is carried out on a commercial scale.
In this respect, the fermentation of the present invention is carried out at more than about 50,000 litres scale, preferably more than about 80,000 litres scale, preferably at more than about 200,000 litre scale fermentation..
In one embodiment the total protein produced by the method of the present invention is well in excess of about 20 g/liter.
Of the total protein produced in the present invention the majority is the desired lipolytic enzyme. In one embodiment the total secreted protein produced by the method of the present invention comprises at least 50% of the desired lipolytic enzyme. In one embodiment the total secreted protein produced by the method of the present invention comprises at least 60% of the desired lipolytic enzyme. In one embodiment the total secreted protein produced by the method of the present invention comprises at least 70% of the desired lipolytic enzyme. In one embodiment the total secreted protein produced by the method of the present invention comprises at least 80% of the desired lipolytic enzyme..
Suitably the present invention may comprise selection of transformants which have been screened for production of the lipolytic enzyme (high producers of the desired enzyme are preferentially selected).
In one embodiment suitably the present invention may comprise a first transformation step wherein a Trichoderma reesei cell is transformed with at least one heterologous nucleotide sequence encoding the lipolytic enzyme defined herein, selection of transformants which have been screened for production of the lipolytic enzyme (high producers of the desired enzyme are preferentially selected), and a second transformation step (i.e., retransformation step) of a selected transformant with at least one heterologous nucleotide sequence encoding the lipolytic enzyme defined herein, followed by further selection of new transformants which have been screened for production of the lipolytic enzyme (high producers of the desired enzyme are preferentially selected)-In some embodiments the lipolytic enzyme produced by the present invention maybe glycosylated. In some embodiments, the lipolytic enzyme may be N-glycosylated at N32 (when numbered using SEQ ID NO., 2) or at an equivalent position for other lipolytic enzymes according to the invention. This aspect may impart significant advantages in that the activity of the enzyme is not disrupted or reduced by glycosylation of the enzyme. Without wishing to be bound by theory, reduction in activity of the lipolytic enzyme can be seen when it is produced in other hosts such as A, tubingensis and is thought to be due to over-glycosylation of the enzyme at at least the N242 site-The lipolytic enzyme produced by the present invention is therefore distinguishable from the lipolytic enzyme produced in other hosts, e.g. A, tubingensis, because of the degree of glycosylation of the enzyme, particularly at the N32 site. In some embodiments of the present invention the enzyme has glycosylation at, at least, the N32 site.
Suitably the lipolytic enzyme may be produced with a signal peptide. In other words the heterologous nucleotide sequence used in the present invention comprises a portion thereof which encodes a signal peptide.
The signal peptide may be used to direct secretion of the lipolytic enzyme through a particular cell membrane. The signal peptide sequences may be endogenous or exogenous to the lipolytic enzyme coding sequence. For instance, the signal peptide may be the signal peptide which is endogenous to the lipolytic enzyme is Aspergillus tubingensis.. Alternatively, the coding sequence for the signal peptide may be obtained (or obtainable) from a cellobiohydrolase gene of Trichoderma reesei -15 However, any signal peptide coding sequence capable of directing the expressed lipolytic enzyme into the secretory pathway of a Trlchoderma reesei cell of choice may be used When we refer to improving one or more of the following: expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity and/or yield this is compared with conventional methods of expressing this lipolytic enzyme. For example, in the present invention there is provided an improved expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity and/or yield compared with production of the lipolytic enzyme in another host organism (i.e. a host organism other than T reesei). In particular, there is an improved expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity and/or yield of the lipolytic enzyme by the present invention (i.e. produced in the Trichoderma reesei cell) as compared with expression of the same lipolytic enzyme in an Aspergillus tubingensis cell (for example as taught in W098/45453, as incorporated herein by reference).
The term "improved glycosylation" as used herein means that, preferably, glycosylation occurs at N32 (when numbered using SEQ ID NO: 2). Without wishing to be bound by theory, in some situations, the lipolytic enzyme produced in host cells other than T.
reesei (and particularly in Aspergillus tubingensis (e.g, as taught in W098145453)) may be glycosylated (or overglycosylated), particularly at N242.. Therefore, the lipolytic enzyme produced in host cells other than T. reesei and particularly in Aspergillus tubingensis (e g, as taught in W098145453) may be glycosylated at both N32 and sites, However, and without wishing to be bound by theory, the N242 site is in the vicinity of one of the active site residues, namely His258 of SEQ ID No 2.
Thus, it is 5 believed that glycosylation (or overglycosylation) at the N242 site can lead to a reduced activity (i.e.. lipase activity) of the enzyme.. The lipolytic enzyme of the present invention does not have reduced activity. Glycosylation of the lipolytic enzyme of the present invention may occur at the N32 site which is away from the active site residues, such as His258..
The term "improved enzyme activity" as used herein means that the activity is the same as or greater than the lipase activity of the lipolytic enzyme produced naturally by Aspergillus tubingensis.
By enzyme activity we mean at least lipase activity Enzyme activity (e.g.
lipase activity) can be measured using the relevant protocols set out below in the Examples section,.
It has been surprisingly found that the lipolytic enzyme produced in accordance with the present invention is easy to isolate from the medium into which it has been excreted -i.e, the culture (fermentation) broth - as high expression levels are obtained,. Figure 2 shows a schematic for the method of the present invention.
Thus, according to a preferred aspect of the present invention, the method of the present invention may involve one or more of the following steps to the medium into which the enzyme of the present invention has been secreted following culturing of the cell: diluting the medium (preferably with water); separating the cell(s) from the medium; concentrating the medium (preferably wherein said medium is cell-free);
granulating said medium (preferably wherein said medium is cell-free)..
In a preferred aspect of the present invention, the method of the present invention involves the following steps to the medium into which the enzyme of the present invention has been secreted following culturing of the cell: diluting the medium (preferably with water); separating the cell(s) from the medium; concentrating the medium (preferably wherein said medium is cell-free); and optionally granulating said medium (preferably wherein said medium is cell-free).
In a preferred aspect of the present invention, the method of the present invention involves the following steps to the medium into which the enzyme of the present invention has been secreted following culturing of the cell: diluting the medium (preferably with water); separating the cell(s) from the medium, concentrating the medium (preferably wherein said medium is cell-free); and granulating said medium (preferably wherein said medium is cell-free)..
In a preferred aspect of the present invention, the method of the present invention involves the following steps to the medium into which the enzyme of the present invention has been secreted following culturing of the cell: diluting the medium with water; separating the cell(s) from the medium; concentrating the medium wherein said medium is cell-free, and optionally granulating said medium wherein said medium is cell-free, In a preferred aspect of the present invention, the method of the present invention involves the following steps to the medium into which the enzyme of the present invention has been secreted following culturing of the cell. diluting the medium with water; separating the cell(s) from the medium; concentrating the medium wherein said medium is cell-free; and granulating said medium wherein said medium is cell-free, According to further aspects of the present invention the enzyme of the present invention is then used in a method to prepare a food or foodstuff intended for human consumption said method comprising admixing said enzyme with a suitable food or foodstuff ingredient. Preferably, said enzyme is in the medium into which the enzyme of the present invention has been secreted following culturing of the cell.
Preferably said medium is cell-free (i.e, the cell(s) have been separated from the medium).
Preferably said medium is concentrated.. In some embodiments, preferably the medium is granulated.
Preferably the lipase precipitates out of solution in the fermentation broth, Preferably, the lipase precipitate is re-solubilised by pH adjustment Preferably the pH is adjusted to a pH above the pH of the fermentation broth.
ADVANTAGES
In addition to the advantages mentioned above, another advantage of the present invention is that it enables commercial scale production of the lipolytic enzyme. The method of the present invention allows for the lipolytic enzyme to be produced in a high yield.
One advantage of the present invention is that it has surprisingly been found that it is possible to go directly from the transformation and screening step (i.e.. say from the microtitre plate) directly to large scale fermentation (e.g.. at least 14 litre fermentation) This is surprisingly possible because the screening step (particularly the microtitre plate results) are highly predictive of good performance in the large scale fermentation, This contrasts with conventional methods where it is often necessary to cultivate the strain in flasks before moving to larger scale fermentation) This has significant advantages in shortening the production time and/or simplifying the overall procedure and/or reducing costs..
A further advantage of the present invention is that it provides an enhanced/increased expression and/or an improved yield of the lipolytic enzyme compared with conventional methods of expressing this lipolytic enzyme, For example, in the present invention there is provided an enhanced/increased expression and/or an improved yield of the lipolytic enzyme compared with production of the lipolytic in another host organism (i.
e, a host organism other than T. reeser). In particular, there is an increased expression and/or an improved yield of the lipolytic enzyme by the present invention (i.e.
produced in the Trichoderma reesei cell) as compared with expression of the same lipolytic enzyme in an Aspergillus tubingensis cell (for example as taught in W098145453, as incorporated herein by reference)..
A further advantage of the present invention is that the lipolytic enzyme produced in accordance with the present invention is easy to produce and isolate and/or purify and/or concentrate.
A further advantage of the present invention is that the lipolytic enzyme produced in accordance with the present invention is easy to re-solubilise.
A further advantage of the present invention is that the lipolytic enzyme produced in accordance with the present invention may be used as a granulate or as a solution..
LIPOLYTIC ENZYME
The term "lipolytic enzyme" as used herein means an enzyme with triacylglycerol hydrolysing activity (classified as E,C 3 1..1.3), Suitably, the lipolytic enzyme of the present invention may exhibit one or more of the following additional activities: glycolipase activity (E,C. 3.1..1.26), phospholipase A2 activity (E.,C. 3.1.1.4), phospholipase Al activity (E.C 3..1.1.32) or phospholipase B
activity (E.C. 3.1..1.5).. The term "glycolipase activity" as used herein encompasses "galactolipase activity",.
Suitably, the lipolytic enzyme according to the present invention may have at least one or more of the following activities: glycolipase activity (E G. 3.11.26) and/or phospholipase Al activity (E,C.. 3.1.1 32) and/or phospholipase A2 activity (E.C.
3 1.1.4) and/or phospholipase B activity (E,C_ 3.1..1.5) ISOLATED
In one aspect, preferably the lipolytic enzyme according to the present invention is in an isolated form. The term "isolated" means that the lipolytic enzyme is at least substantially free from at least one other component with which the lipolytic enzyme is naturally associated in nature and as found in nature. The term "isolated" may mean that the lipolytic enzyme is at least substantially free from at least one other component in the culture media in which it is produced. The lipolytic enzyme of the present invention may be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated or with which the enzyme may be produced in the T reesei host. Thus, for example it may be substantially free of the cell(s) or one or more potentially contaminating polypeptides and/or nucleic acid molecules The lipolytic enzyme may be isolated by separating the cell(s) from the broth during or after fermentation so that the lipolytic enzyme remains in the broth.. The lipolytic enzyme may be isolated by subjecting the fermentation broth to cell separation by vacuum filtration.
PURIFIED
In one aspect, preferably the lipolytic enzyme according to the present invention is in a purified form. The term "purified" means that the given component is present at a high level. The component is desirably the predominant component present in a composition. Preferably, it is present at a level of at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80% said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration.. For some embodiments the amount is at least about 85% said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration..
CONCENTRATE
In one aspect, preferably the lipolytic enzyme according to the present invention is used as a concentrate. The concentrate may be a concentrated form of the medium into which the enzyme has been excreted. Preferably, the concentrate may be a concentrated form of the medium into which the enzyme has been secreted and wherein the cell(s) have been removed NUCLEOTIDE SEQUENCE
The scope of the present invention encompasses nucleotide sequences encoding proteins having the specific properties and/or parameters as defined herein, The term "nucleotide sequence" as used herein refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homologues, fragments and derivatives thereof (such as portions thereof).. The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or anti-sense strand.
The term "nucleotide sequence" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA sequence coding for the present invention.
in a preferred embodiment, the nucleotide sequence when relating to and when encompassed by the per se scope of the present invention does not include the native nucleotide sequence according to the present invention when in its natural 5 environment and when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment. For ease of reference, we shall call this preferred embodiment the "non-native nucleotide sequence", In this regard, the term "native nucleotide sequence" means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is
In a preferred embodiment, prior to the isolation and/or purification and/or concentration of the enzyme, the pH of the medium is raised at the end of the fermentation run when sufficient levels of the secreted, soluble enzyme are reached.
In one embodiment, the pH of the medium for isolating and/or purifying and/or concentrating the enzyme is above pH 5.5 to about pH 6..5.
Thus, in one embodiment, the pH of the medium for culturing is about pH 4 and then the pH of the medium is raised such that the pH of the medium for isolating and/or purifying and/or concentrating the enzyme is above pH 5..5 to about pH 6.5.
Thus, in one embodiment, the pH of the medium for culturing is about pH 4 and then the pH of the medium is raised such that the pH of the medium for isolating and/or purifying and/or concentrating the enzyme is above about pH 6 to about pH 6.5.
In a preferred embodiment, the pH of the medium for culturing is about pH 4,5 and then the pH of the medium is raised such that the pH of the medium for isolating and/or purifying and/or concentrating the enzyme is about pH 6, Preferably the Trichoderma reesei cell is provided by transforming it with, or is transformed with, the nucleotide sequence using electroporation, such as by the electroporation methodology disclosed in WO 2008/153712 A2.
In another embodiment, the Trichoderma reesei cell may be provided by transforming it with, or may be transformed with, the nucleotide sequence using biolistic transformation, Suitably there may be at least one heterologous nucleotide sequence encoding the lipolytic enzyme in the Trichoderma reesei cell. In some embodiments, there may be two or more copies (I e., multiple copies) of the or each heterologous nucleotide sequence encoding the lipolytic enzyme according to the present invention in the Trichoderma reesei cell. For example, in one embodiment there may be at least two nucleotide sequences encoding the lipolytic enzyme in the Trichoderma reesei. In other embodiments there may be at least three, such as at least four, such as at least five, or such as at least six heterologous nucleotide sequences encoding said lipolytic enzyme.
Suitably there may be up to about six, preferably up to about seven, preferably up to about eight, preferably up to about ten heterologous nucleotide sequences encoding the 5 lipolytic enzyme in the Trichoderma reesei cell. In some embodiments each heterologous nucleotide sequence is associated with and is under the control of a promoter. Suitably each heterologous nucleotide sequence may have a separate promoter associated with it and be under the control of that promoter, The promoters may be the same or different..
Thus, in one embodiment the Trichoderma reesei cell may comprise or be transfected or transformed with at least 2 heterologous nucleotide sequences encoding the lipolytic enzyme. In another embodiment the Trichoderma reesei cell may comprise or be transfected or transformed with at least 3, or at least 4, or at least 5 or at least 6 heterologous nucleotide sequences encoding the lipolytic enzyme. In one embodiment there is up to about 6 heterologous nucleotide sequences encoding the lipolytic enzyme in accordance with the present invention.. In some embodiments there may be up to about 10 heterologous nucleotide sequences encoding the lipolytic enzyme in accordance with the present invention. In some embodiments each heterologous nucleotide sequence is associated with and under the control of a promoter.
Suitably each heterologous nucleotide sequence may have separate promoter associated with it and be under the control of that promoter.
Suitably, the (or each) heterologous nucleotide sequence may comprise a nucleotide sequence which encodes a signal peptide, which nucleotide sequence encoding said signal peptide is operably linked to said nucleotide sequence encoding said lipolytic enzyme. If there are multiple heterologous nucleotide sequences and wherein more than one has a signal sequence associated therewith, then the signal sequences may be the same or different..
Suitably the lipolytic enzyme may comprise an endogenous or exogenous signal peptide. When the signal peptide is endogenous - it means that the signal peptide is that which is naturally linked with the lipolytic enzyme when produced naturally For example, the signal peptide may be the signal peptide in Aspergillus tubingensis which is naturally linked with the lipolytic enzyme when found in Aspergillus tubingensis.
The term "heterologous" as used herein means that it is does not occur naturally in the Trichoderma reesei cell. In other words, it is exogenous to the Trichoderma reesei cell.
For example the term "heterologous nucleotide sequence" as used herein means that the nucleotide sequence does not occur naturally in the Trichoderma reesei cell.. In other words, the nucleotide sequence is exogenous to the Trichoderma reesei cell. The term also includes multiple copies of the naturally occurring sequence as such additional multiple copies would be heterologous.
In one embodiment preferably the heterologous nucleotide sequence is obtained or obtainable from a microorganism, particularly a fungi In one embodiment preferably the heterologous nucleotide sequence is obtained or obtainable from Aspergillus, particularly Aspergillus tubingensis., In a further aspect of the present invention there is provided a method of producing a lipolytic enzyme comprising the steps of.
(i) providing a Trichoderma reesei cell comprising a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID
NO: 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO. 4 or a nucleotide sequence which is at least 40%
sequence identity to SEQ ID NO: 3 or SEQ ID NO, 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO. 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions; and (ii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme; and wherein said Trichoderma reesei cell has at least two genes encoding non-lipolytic enzymes suppressed.
In a further aspect of the present invention there is provided a method of producing a lipolytic enzyme comprising the steps of:
(i) transfecting or transforming a Trichoderma reesei cell with a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID
NO: 1 or 2, and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO. 3 or SEQ 1D NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO. 3 or SEQ ID NO, 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ 1D NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ 1D NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions;
(ii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme; and wherein said Trichoderma reesei cell has at least two genes encoding non-lipolytic enzymes suppressed.
In a further aspect of the present invention there is provided a method of producing a lipolytic enzyme comprising the steps of, (i) transfecting or transforming a Trichoderma reesei cell with a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO. 2 or an amino acid sequence which has at least 30% sequence identity to SEQ ID
NO. 1 or 2; andlor b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4 or the complement of any thereof under stringent conditions;
(ii) repeating step (i) on the cell to sequentially transfect or transform the cell with at least one additional heterologous nucleotide sequence as defined in (i)(a), (i)(b) or (i)(c) (e.g. such as a heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID NO:
1 or 2); and (iii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme; and wherein said Trichoderma reesei cell has at least two genes encoding non-lipolytic enzymes suppressed.
The present invention yet further provides a lipolytic enzyme obtainable by the methods of the present invention There is also provided by the present invention, a foodstuff for human consumption comprising said lipolytic enzyme obtainable by a method of the present invention The present invention yet further provides a transformed or transfected Trichoderma reesei cell comprising:
a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme protein having at least 40% sequence identity to SEQ ID NO. 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40%
sequence identity to SEQ ID NO: 3 or SEQ ID NO': 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ 1D NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ 1D NO., 4 or the complement of any thereof under stringent conditions; and wherein said Trichoderma reesei cell has at least two genes encoding non-lipolytic enzymes suppressed.
Preferably, at least three genes encoding non-lipolytic enzymes suppressed.
Preferably, at least four genes encoding non-lipolytic enzymes suppressed.
"Suppressed' means that the cell does not express the relevant non-lipolytic enzyme at the same level as the non-transformeditransfected cell. In some embodiments, "suppressed" means that the cell does not express the relevant non-lipolytic enzyme..
The suppression may be brought about by techniques known in the art, such as by deletions.
Preferably, at least one of the genes encoding a non-lipolytic enzyme that is suppressed is a cellulase gene.
5 Preferably, at least two of the genes encoding a non-lipolytic enzyme that is suppressed are cellulase genes, An example of at least one of the genes encoding a non-lipolytic enzyme that is suppressed is a gene encoding a cellobiohydrolases (e.g. CBHI or CBHII).
Another example of at least one of the genes encoding a non-lipolytic enzyme that is suppressed is a gene encoding an endoglucanases (e,g. EGI and EGII)..
In some embodiments the Trichoderma reesei cell of the present invention is a cell in which the endogenous genes encoding one or both cellobiohydrolases (CBHI and CBHII) and/or one or both of the endoglucanases (EGI and EGII) are deleted or disrupted Suitably the Trichoderma reesei cell may be a non-GMM cell or derivative thereof, for example a derivative of the strain RL-P37. Suitably the Trichoderma reesei cell may be a derivative of the strain RIL-P37 that is produced using the method set out in Example 10..
In some embodiments, the heterologous nucleotide sequence may encode a lipolytic enzyme comprising an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98%
sequence identity to SEQ ID NO: 1 or SEQ ID No. 2 or a sequence comprising one or several amino acid additions, deletions or substitutions compared to SEQ 1D
NO: 1 or SEQ I D No, 2.
In some embodiments the heterologous nucleotide sequence may encode a lipolytic enzyme and comprises a nucleotide sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98%
sequence identity to SEQ ID NO: 3 or SEQ ID No, 4 or a sequence comprising one or several nucleotide additions, deletions or substitutions compared to SEQ ID
NO. 3 or SEQ ID No 4.
Preferably, the heterologous nucleotide sequence encodes a lipolytic enzyme comprising an amino acid sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to SEQ ID
NO: 1 or SEQ ID No.. 2 or a sequence comprising one or several amino acid additions, deletions or substitutions compared to SEQ ID NO. 1 or SEQ ID No_ 2.
Preferably, the heterologous nucleotide sequence encodes a lipolytic enzyme and comprises a nucleotide sequence having at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to SEQ ID
NO: 3 or SEQ ID No. 4 or a sequence comprising one or several nucleotide additions, deletions or substitutions compared to SEQ ID NO: 3 or SEQ 1D No. 4..
The amino acid sequence identity of a number of lipolytic enzymes to the lipase from Aspergillus tubingensis having the sequence shown in SEQ ID NO: 3 (also called herein lipase 3) are shown in Table 1 below.
Table 1 - Lipolytic enzymes with sequence identity to the lipolytic enzyme from Aspergillus tubingensis (lipase 3).
Lipases from different fungi Accession No Amino acid sequence identit A. ni er CBS 513.88 XP 001397501.1 93 Aspergillus niger ABG73613.1 93 Aspergillus niger ABG37906.1 93 Aspergillus nidulans XP681315.1 61 Aspergillus clavatus NRRL XP_001276337A1 57 Neosartorya fischeri NRRL XP0012663291 60 Aspergillus fumigatus XP748138.1 59 Af293 Aspergillus oryzae RIB40 XP 001818694.1 56 Aspergillus terreus XP001218444,1 54 Penicillium chrysogenum CAP96359.1 55 Wisconsin 54-1255 Aspergillus niger ABG73614.1 54 Aspergillus niger XP 001393532.1 53 Thermomyces 059952 1 50 lanuginosus Penicillium marneffei ATCC XP 0021471441 49 Aspergillus oryzae R XP 001824529.1 44 Phaeosphaeria nodorum 5N15 XP 001796872.1 45 Penicillium cyclopium P61869.1 42 Penicillium camembe.rti. 1T1A A 42 The Trichoderma reesei host cell used in the present invention may be any Trichoderma reesei cell. The cell may be considered to be a wild type Trichoderma reesei cell. The Trichoderma reesei cell may be one from which genes encoding one or more secreted cellobiohydrolases (CBHI or CBH1I) has/have been deleted or disrupted so that they are not expressed Suitably the Trichoderma reesei cell may be a non-genetically modified cell or derivative thereof, for example a derivative of the strain RL-P37.
Suitably the Trichoderma reesei cell may be a derivative of the strain RL-P37 that is produced using the method set out in Example 10, The present invention may be carried out in a fermentor which may comprise about 3 liters to about 20 liters culture mediae In another embodiment the present invention is carried out as a 10-16 liter, preferably a 14 liter scale fermentation.. In one embodiment, preferably the fermentation is carried out with more than about 12 liters, preferably more than about 14 liters.
The Trichoderma reesei host cell is preferably suitable for use in large scale fermentation..
In preferred embodiments, the present invention is carried out on a commercial scale.
In this respect, the fermentation of the present invention is carried out at more than about 50,000 litres scale, preferably more than about 80,000 litres scale, preferably at more than about 200,000 litre scale fermentation..
In one embodiment the total protein produced by the method of the present invention is well in excess of about 20 g/liter.
Of the total protein produced in the present invention the majority is the desired lipolytic enzyme. In one embodiment the total secreted protein produced by the method of the present invention comprises at least 50% of the desired lipolytic enzyme. In one embodiment the total secreted protein produced by the method of the present invention comprises at least 60% of the desired lipolytic enzyme. In one embodiment the total secreted protein produced by the method of the present invention comprises at least 70% of the desired lipolytic enzyme. In one embodiment the total secreted protein produced by the method of the present invention comprises at least 80% of the desired lipolytic enzyme..
Suitably the present invention may comprise selection of transformants which have been screened for production of the lipolytic enzyme (high producers of the desired enzyme are preferentially selected).
In one embodiment suitably the present invention may comprise a first transformation step wherein a Trichoderma reesei cell is transformed with at least one heterologous nucleotide sequence encoding the lipolytic enzyme defined herein, selection of transformants which have been screened for production of the lipolytic enzyme (high producers of the desired enzyme are preferentially selected), and a second transformation step (i.e., retransformation step) of a selected transformant with at least one heterologous nucleotide sequence encoding the lipolytic enzyme defined herein, followed by further selection of new transformants which have been screened for production of the lipolytic enzyme (high producers of the desired enzyme are preferentially selected)-In some embodiments the lipolytic enzyme produced by the present invention maybe glycosylated. In some embodiments, the lipolytic enzyme may be N-glycosylated at N32 (when numbered using SEQ ID NO., 2) or at an equivalent position for other lipolytic enzymes according to the invention. This aspect may impart significant advantages in that the activity of the enzyme is not disrupted or reduced by glycosylation of the enzyme. Without wishing to be bound by theory, reduction in activity of the lipolytic enzyme can be seen when it is produced in other hosts such as A, tubingensis and is thought to be due to over-glycosylation of the enzyme at at least the N242 site-The lipolytic enzyme produced by the present invention is therefore distinguishable from the lipolytic enzyme produced in other hosts, e.g. A, tubingensis, because of the degree of glycosylation of the enzyme, particularly at the N32 site. In some embodiments of the present invention the enzyme has glycosylation at, at least, the N32 site.
Suitably the lipolytic enzyme may be produced with a signal peptide. In other words the heterologous nucleotide sequence used in the present invention comprises a portion thereof which encodes a signal peptide.
The signal peptide may be used to direct secretion of the lipolytic enzyme through a particular cell membrane. The signal peptide sequences may be endogenous or exogenous to the lipolytic enzyme coding sequence. For instance, the signal peptide may be the signal peptide which is endogenous to the lipolytic enzyme is Aspergillus tubingensis.. Alternatively, the coding sequence for the signal peptide may be obtained (or obtainable) from a cellobiohydrolase gene of Trichoderma reesei -15 However, any signal peptide coding sequence capable of directing the expressed lipolytic enzyme into the secretory pathway of a Trlchoderma reesei cell of choice may be used When we refer to improving one or more of the following: expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity and/or yield this is compared with conventional methods of expressing this lipolytic enzyme. For example, in the present invention there is provided an improved expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity and/or yield compared with production of the lipolytic enzyme in another host organism (i.e. a host organism other than T reesei). In particular, there is an improved expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity and/or yield of the lipolytic enzyme by the present invention (i.e. produced in the Trichoderma reesei cell) as compared with expression of the same lipolytic enzyme in an Aspergillus tubingensis cell (for example as taught in W098/45453, as incorporated herein by reference).
The term "improved glycosylation" as used herein means that, preferably, glycosylation occurs at N32 (when numbered using SEQ ID NO: 2). Without wishing to be bound by theory, in some situations, the lipolytic enzyme produced in host cells other than T.
reesei (and particularly in Aspergillus tubingensis (e.g, as taught in W098145453)) may be glycosylated (or overglycosylated), particularly at N242.. Therefore, the lipolytic enzyme produced in host cells other than T. reesei and particularly in Aspergillus tubingensis (e g, as taught in W098145453) may be glycosylated at both N32 and sites, However, and without wishing to be bound by theory, the N242 site is in the vicinity of one of the active site residues, namely His258 of SEQ ID No 2.
Thus, it is 5 believed that glycosylation (or overglycosylation) at the N242 site can lead to a reduced activity (i.e.. lipase activity) of the enzyme.. The lipolytic enzyme of the present invention does not have reduced activity. Glycosylation of the lipolytic enzyme of the present invention may occur at the N32 site which is away from the active site residues, such as His258..
The term "improved enzyme activity" as used herein means that the activity is the same as or greater than the lipase activity of the lipolytic enzyme produced naturally by Aspergillus tubingensis.
By enzyme activity we mean at least lipase activity Enzyme activity (e.g.
lipase activity) can be measured using the relevant protocols set out below in the Examples section,.
It has been surprisingly found that the lipolytic enzyme produced in accordance with the present invention is easy to isolate from the medium into which it has been excreted -i.e, the culture (fermentation) broth - as high expression levels are obtained,. Figure 2 shows a schematic for the method of the present invention.
Thus, according to a preferred aspect of the present invention, the method of the present invention may involve one or more of the following steps to the medium into which the enzyme of the present invention has been secreted following culturing of the cell: diluting the medium (preferably with water); separating the cell(s) from the medium; concentrating the medium (preferably wherein said medium is cell-free);
granulating said medium (preferably wherein said medium is cell-free)..
In a preferred aspect of the present invention, the method of the present invention involves the following steps to the medium into which the enzyme of the present invention has been secreted following culturing of the cell: diluting the medium (preferably with water); separating the cell(s) from the medium; concentrating the medium (preferably wherein said medium is cell-free); and optionally granulating said medium (preferably wherein said medium is cell-free).
In a preferred aspect of the present invention, the method of the present invention involves the following steps to the medium into which the enzyme of the present invention has been secreted following culturing of the cell: diluting the medium (preferably with water); separating the cell(s) from the medium, concentrating the medium (preferably wherein said medium is cell-free); and granulating said medium (preferably wherein said medium is cell-free)..
In a preferred aspect of the present invention, the method of the present invention involves the following steps to the medium into which the enzyme of the present invention has been secreted following culturing of the cell: diluting the medium with water; separating the cell(s) from the medium; concentrating the medium wherein said medium is cell-free, and optionally granulating said medium wherein said medium is cell-free, In a preferred aspect of the present invention, the method of the present invention involves the following steps to the medium into which the enzyme of the present invention has been secreted following culturing of the cell. diluting the medium with water; separating the cell(s) from the medium; concentrating the medium wherein said medium is cell-free; and granulating said medium wherein said medium is cell-free, According to further aspects of the present invention the enzyme of the present invention is then used in a method to prepare a food or foodstuff intended for human consumption said method comprising admixing said enzyme with a suitable food or foodstuff ingredient. Preferably, said enzyme is in the medium into which the enzyme of the present invention has been secreted following culturing of the cell.
Preferably said medium is cell-free (i.e, the cell(s) have been separated from the medium).
Preferably said medium is concentrated.. In some embodiments, preferably the medium is granulated.
Preferably the lipase precipitates out of solution in the fermentation broth, Preferably, the lipase precipitate is re-solubilised by pH adjustment Preferably the pH is adjusted to a pH above the pH of the fermentation broth.
ADVANTAGES
In addition to the advantages mentioned above, another advantage of the present invention is that it enables commercial scale production of the lipolytic enzyme. The method of the present invention allows for the lipolytic enzyme to be produced in a high yield.
One advantage of the present invention is that it has surprisingly been found that it is possible to go directly from the transformation and screening step (i.e.. say from the microtitre plate) directly to large scale fermentation (e.g.. at least 14 litre fermentation) This is surprisingly possible because the screening step (particularly the microtitre plate results) are highly predictive of good performance in the large scale fermentation, This contrasts with conventional methods where it is often necessary to cultivate the strain in flasks before moving to larger scale fermentation) This has significant advantages in shortening the production time and/or simplifying the overall procedure and/or reducing costs..
A further advantage of the present invention is that it provides an enhanced/increased expression and/or an improved yield of the lipolytic enzyme compared with conventional methods of expressing this lipolytic enzyme, For example, in the present invention there is provided an enhanced/increased expression and/or an improved yield of the lipolytic enzyme compared with production of the lipolytic in another host organism (i.
e, a host organism other than T. reeser). In particular, there is an increased expression and/or an improved yield of the lipolytic enzyme by the present invention (i.e.
produced in the Trichoderma reesei cell) as compared with expression of the same lipolytic enzyme in an Aspergillus tubingensis cell (for example as taught in W098145453, as incorporated herein by reference)..
A further advantage of the present invention is that the lipolytic enzyme produced in accordance with the present invention is easy to produce and isolate and/or purify and/or concentrate.
A further advantage of the present invention is that the lipolytic enzyme produced in accordance with the present invention is easy to re-solubilise.
A further advantage of the present invention is that the lipolytic enzyme produced in accordance with the present invention may be used as a granulate or as a solution..
LIPOLYTIC ENZYME
The term "lipolytic enzyme" as used herein means an enzyme with triacylglycerol hydrolysing activity (classified as E,C 3 1..1.3), Suitably, the lipolytic enzyme of the present invention may exhibit one or more of the following additional activities: glycolipase activity (E,C. 3.1..1.26), phospholipase A2 activity (E.,C. 3.1.1.4), phospholipase Al activity (E.C 3..1.1.32) or phospholipase B
activity (E.C. 3.1..1.5).. The term "glycolipase activity" as used herein encompasses "galactolipase activity",.
Suitably, the lipolytic enzyme according to the present invention may have at least one or more of the following activities: glycolipase activity (E G. 3.11.26) and/or phospholipase Al activity (E,C.. 3.1.1 32) and/or phospholipase A2 activity (E.C.
3 1.1.4) and/or phospholipase B activity (E,C_ 3.1..1.5) ISOLATED
In one aspect, preferably the lipolytic enzyme according to the present invention is in an isolated form. The term "isolated" means that the lipolytic enzyme is at least substantially free from at least one other component with which the lipolytic enzyme is naturally associated in nature and as found in nature. The term "isolated" may mean that the lipolytic enzyme is at least substantially free from at least one other component in the culture media in which it is produced. The lipolytic enzyme of the present invention may be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated or with which the enzyme may be produced in the T reesei host. Thus, for example it may be substantially free of the cell(s) or one or more potentially contaminating polypeptides and/or nucleic acid molecules The lipolytic enzyme may be isolated by separating the cell(s) from the broth during or after fermentation so that the lipolytic enzyme remains in the broth.. The lipolytic enzyme may be isolated by subjecting the fermentation broth to cell separation by vacuum filtration.
PURIFIED
In one aspect, preferably the lipolytic enzyme according to the present invention is in a purified form. The term "purified" means that the given component is present at a high level. The component is desirably the predominant component present in a composition. Preferably, it is present at a level of at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80% said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration.. For some embodiments the amount is at least about 85% said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration..
CONCENTRATE
In one aspect, preferably the lipolytic enzyme according to the present invention is used as a concentrate. The concentrate may be a concentrated form of the medium into which the enzyme has been excreted. Preferably, the concentrate may be a concentrated form of the medium into which the enzyme has been secreted and wherein the cell(s) have been removed NUCLEOTIDE SEQUENCE
The scope of the present invention encompasses nucleotide sequences encoding proteins having the specific properties and/or parameters as defined herein, The term "nucleotide sequence" as used herein refers to an oligonucleotide sequence or polynucleotide sequence, and variant, homologues, fragments and derivatives thereof (such as portions thereof).. The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or anti-sense strand.
The term "nucleotide sequence" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA sequence coding for the present invention.
in a preferred embodiment, the nucleotide sequence when relating to and when encompassed by the per se scope of the present invention does not include the native nucleotide sequence according to the present invention when in its natural 5 environment and when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment. For ease of reference, we shall call this preferred embodiment the "non-native nucleotide sequence", In this regard, the term "native nucleotide sequence" means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is
10 naturally associated, which promoter is also in its native environment.
However, the amino acid sequence encompassed by scope the present invention can be isolated and/or purified post expression of a nucleotide sequence in its native organism.
Preferably, however, the amino acid sequence encompassed by scope of the present invention may be expressed by a nucleotide sequence in its native organism but 15 wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.
Typically, the nucleotide sequence encompassed by the scope of the present invention is prepared using recombinant DNA techniques (i.e. recombinant DNA).
20 However, in an alternative embodiment of the invention, the nucleotide sequence could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et a!.., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T
et al., (1980) Nuc Acids Res Symp Ser 225-232) A nucleotide sequence encoding either a protein which has the specific properties as defined herein or a protein which is suitable for modifidation may be identified and/or isolated and/or purified from any cell or organism producing said protein.
Various methods are well known within the art for the identification and/or isolation and/or purification of nucleotide sequences. By way of example, PCR amplification techniques to prepare more of a sequence may be used once a suitable sequence has been identified and/or isolated and/or purified.
By way of further example, a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the enzyme. If the amino acid sequence of the enzyme is known, labelled oligonucleotide probes may be synthesised and used to identify enzyme-encoding clones from the genomic library prepared from the organism. Alternatively, a labelled oligonucleotide probe containing sequences homologous to another known enzyme gene could be used to identify enzyme-encoding clones, In the latter case, hybridisation and washing conditions of lower stringency are used, Alternatively, enzyme-encoding clones could be identified by inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming enzyme-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar plates containing a substrate for enzyme (i.e, maltose), thereby allowing clones expressing the enzyme to be identified, In a yet further alternative, the nucleotide sequence encoding the enzyme may he prepared synthetically by established standard methods, e.g, the phosphoroamidite method described by Beucage S.L. et at., (1981) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al., (1984) EMBO J. 3, p 801-805.
In the phosphoroamidite method, oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors-The nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques.. Each ligated fragment corresponds to various parts of the entire nucleotide sequence, The DNA sequence may also be prepared by polymerise chain reaction (PCR) using specific primers, for instance as described in US
4,683,202 or in Saiki R K et al., (Science (1988) 239, pp 487-491), AMINO ACID SEQUENCES
The scope of the present invention also encompasses amino acid sequences of enzymes having the specific properties as defined herein.
As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme".
The amino acid sequence may be preparedlisolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA
techniques..
The protein encompassed in the present invention may be used in conjunction with other proteins, particularly enzymes. Thus the present invention also covers a combination of proteins wherein the combination comprises the protein/enzyme of the present invention and another protein/enzyme, which may be another protein/enzyme according to the present invention. This aspect is discussed in a later section.
Preferably the amino acid sequence when relating to and when encompassed by the per se scope of the present invention is not a native enzyme. In this regard, the term "native enzyme" means an entire enzyme that is in its native environment and when it has been expressed by its native nucleotide sequence..
SEQUENCE IDENTITY OR SEQUENCE HOMOLOGY
The present invention also encompasses the use of sequences having a degree of sequence identity or sequence homology with amino acid sequence(s) of a polypeptide having the specific properties defined herein or of any nucleotide sequence encoding such a polypeptide (hereinafter referred to as a "homologous sequence(s)"). Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences, Here, the term "homology" can be equated with "identity".
The homologous amino acid sequence and/or nucleotide sequence should provide and/or encode a polypeptide which retains the functional activity and/or enhances the activity of the enzyme.
In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85 or 90%
identical, preferably at least 95 or 98% identical to the subject sequence.
Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity..
In the present context, a homologous sequence is taken to include a nucleotide sequence which may be at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85 or 90%
identical, preferably at least 95 or 98% identical to a nucleotide sequence encoding a polypeptide of the present invention (the subject sequence). Typically, the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence. Although homology can also be considered in terms of similarity (i,e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity..
Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
% homology may be calculated over contiguous sequences, i.e.. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues..
Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed.. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.
However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap.
This is the most commonly used gap scoring system High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the Vector NTI AdvanceTM 11 (Invitrogen Corp.). Examples of software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al 1999 Short Protocols in Molecular Biology, 4th Ed - Chapter 18), BLAST 2 (see FEMS
Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana(c~nebi.nlm.nih.gov), FASTA (Altschul et al 1990 J Mol. Biol. 403-410) and AlignX for example. At least BLAST, BLAST 2 and FASTA are available for offline and online searching (see Ausubel et al 1999, pages 7-58 to 7-60).
Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison, Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs Vector NTI programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the default values for the Vector NTI AdvanceTM 11 package.
Alternatively, percentage homologies may be calculated using the multiple alignment feature in Vector NTI AdvanceTM 11 (lnvitrogen Corp.), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).
Once the software has produced an optimal alignment, it is possible to calculate %
homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result, Should Gap Penalties be used when determining sequence identity, then preferably the following parameters are used for pairwise alignment.
FOR BLAST
FOR CLUSTAL DNA PROTEIN
WORD SIZE 2 1 K triple GAP EXTENSION 6.66 0.1 In one embodiment, CLUSTAL may be used with the gap penalty and gap extension set as defined above.
Suitably, the degree of identity with regard to a nucleotide sequence is determined over at least 20 contiguous nucleotides, preferably over at least 30 contiguous nucleotides, preferably over at least 40 contiguous nucleotides, preferably over at least 50 contiguous nucleotides, preferably over at least 60 contiguous nucleotides, preferably over at least 100 contiguous nucleotides, Suitably, the degree of identity with regard to a nucleotide sequence may be determined over the whole sequence.
VARIANTS/HOMOLOGUESIDERIVATIVES
The present invention also encompasses the use of variants, homologues and derivatives of any amino acid sequence of a protein or of any nucleotide sequence encoding such a protein.
Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences.. Here, the term "homology" can be equated with "identity"
In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90%
identical, preferably at least 95, 96, 97, 98 or 99% identical to the subject sequence..
Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
In the present context, an homologous sequence is taken to include a nucleotide sequence which may be at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90%
identical, preferably at least 95, 96, 97, 98 or 99% identical to a nucleotide sequence encoding an enzyme of the present invention (the subject sequence). Typically, the homologues will comprise the same sequences that code for the active sites etc, as the subject sequence, Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences, % homology may be calculated over contiguous sequences, i.e one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment.. Typically, such ungapped alignments are performed only over a relatively short number of residues, Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.
However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap.
This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons, For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et al 1984 Nuc. Acids Research 12 p387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST
package (see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4th Ed -Chapter 18), FASTA (Altschul et at, 1990 J. Mol. Biol . 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al, 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program.. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbial Lett 1999 174(2).
247-50; FEMS Microbial Lett 1999 177(1): 187-8 and tatiana@ncbi.nlrnõnih gov).
Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance, An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62, Alternatively, percentage homologies may be calculated using the multiple alignment feature in DNASISTM (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244)..
Once the software has produced an optimal alignment, it is possible to calculate %
homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance, Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine;
and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
Conservative substitutions may be made, for example according to the Table below.
Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.
ALIPHATIC Non-polar G A P
I L V
Polar - uncharged CST M
NQ
Polar -- charged D E
KR
AROMATIC H F W Y
The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i e.. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as 0), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine, Replacements may also be made by unnatural amino acids include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, p-l-phenylalanine*, L-allyl-glycine*, R-alanine*, L--amino butyric acid*, L--amino butyric acid*, L--amino isobutyric acid*, L--amino caproic acid#, 7-amino heptanoic acid*, L-methionine sulfone*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline#, L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid and L-Phe (4-benzyl)*, The notation * has been utilised for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas #
has been utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.
Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or -alanine residues. A further form of variation, involves the presence of one 5 or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues wherein the -carbon substituent group is on the residue's nitrogen atom rather than the -carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ at al., PNAS (1992) 89(20), 10 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.
The nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art These include methylphosphonate and 15 phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences of the present 20 invention.
The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof, If the sequence is complementary to a fragment thereof then that 25 sequence can be used as a probe to identify similar coding sequences in other organisms etc.
Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of 30 ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations In addition, other homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences 35 may be obtained by probing cDNA libraries made from or genomic DNA
libraries from other animal species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention, Variants and strain/species homologues may also be obtained using degenerate PCR
which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG
Wisconsin PileUp program is widely used.
The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences, Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon sequence changes are required to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
Polynucleotides (nucleotide sequences) of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors, Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art.. They may also be cloned by standard techniques..
In general, primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time.
Techniques for accomplishing this using automated techniques are readily available in the art, Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerise chain reaction) cloning techniques. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
HYBRIDISATION
The present invention also encompasses sequences that are complementary to the nucleic acid sequences of the present invention or sequences that are capable of hybridising either to the sequences of the present invention or to sequences that are complementary thereto..
The term "hybridisation" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerise chain reaction (PCR) technologies.
The present invention also encompasses the use of nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof.
The term "variant" also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequences presented herein,.
Preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridising under stringent conditions (e.g..
500C and 0.2xSSC {1xSSC = 0.15 M NaCl, 0.015 M Na3citrate pH 7..0)) to the nucleotide sequences presented herein.
More preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridising under high stringent conditions (e g.
650C and 0.1 xSSC {1 xSSC = 015 M NaCl, 0..015 M Na3citrate pH 7.0)) to the nucleotide sequences presented herein.
The present invention also relates to nucleotide sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein)-The present invention also relates to nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein) Also included within the scope of the present invention are polynucleotide sequences that are capable of hybridising to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency-In a preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention, or the complement thereof, under stringent conditions (e.g. 500C and 0 2xSSC)..
In a more preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention, or the complement thereof, under high stringent conditions (e.g 650C and 0.1xSSC).
MOLECULAR EVOLUTION
As a non-limiting example, it is possible to produce numerous site directed or random mutations into a nucleotide sequence, either in vivo or in vitro, and to subsequently screen for improved functionality of the encoded polypeptide by various means In addition, mutations or natural variants of a polynucleotide sequence can be recombined with either the wildtype or other mutations or natural variants to produce new variants. Such new variants can also be screened for improved functionality of the encoded polypeptide. The production of new preferred variants can be achieved by various methods well established in the art, for example the Error Threshold Mutagenesis (WO 92/18645), oligonucleotide mediated random mutagenesis (US
5,723, 323), DNA shuffling (US 5,605,793), exo-mediated gene assembly WO00/58517. The application of these and similar random directed molecular evolution methods allows the identification and selection of variants of the enzymes of the present invention which have preferred characteristics without any prior knowledge of protein structure or function, and allows the production of non-predictable but beneficial mutations or variants. There are numerous examples of the application of molecular evolution in the art for the optimisation or alteration of enzyme activity, such examples include, but are not limited to one or more of the following.
optimised expression and/or activity in a host cell or in vitro, increased enzymatic activity, altered substrate and/or product specificity, increased or decreased enzymatic or structural stability, altered enzymatic activity/specificity in preferred environmental conditions, e.g. temperature, pH, substrate SITE-DIRECTED MUTAGENESIS
Once a protein-encoding nucleotide sequence has been isolated, or a putative protein-encoding nucleotide sequence has been identified, it may be desirable to mutate the sequence in order to prepare a protein of the present invention..
Mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites.
A suitable method is disclosed in Morinaga at al., (Biotechnology (1984) 2, p646-649)..
5 Another method of introducing mutations into enzyme-encoding nucleotide sequences is described in Nelson and Long (Analytical Biochemistry (1989), 180, p 147-151).
RECOMBINANT
10 In one aspect the sequence for use in the present invention is a recombinant sequence - i_e. a sequence that has been prepared using recombinant DNA
techniques.
These recombinant DNA techniques are within the capabilities of a person of ordinary 15 skill in the art. Such techniques are explained in the literature, for example, J.
Sambrook, E. F.. Fritsch, and T Maniatis, '1989, Molecular Cloning A
Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press., SYNTHETIC
In one aspect the sequence for use in the present invention is a synthetic sequence -i.e. a sequence that has been prepared by in vitro chemical or enzymatic synthesis. It includes, but is not limited to, sequences made with optimal codon usage for host organisms T. reesei, EXPRESSION OF ENZYMES
The nucleotide sequence for use in the present invention may be incorporated into a recombinant replicable vector. The vector may be used to replicate and express the nucleotide sequence, in protein form, in and/or from a compatible host cell Expression may be controlled using control sequences a g, regulatory sequences.
The protein produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. The coding sequences may be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane EXPRESSION VECTOR
The term "expression vector" means a construct capable of in vivo or in vitro expression, Preferably, the expression vector is incorporated into the genome of a suitable host organism. The term "incorporated" preferably covers stable incorporation into the genome.
The nucleotide sequence of the present invention may be present in a vector in which the nucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the nucleotide sequence by a suitable host organism.
The vectors for use in the present invention may be transformed into a suitable host cell as described below to provide for expression of a polypeptide of the present invention.
The choice of vector e.g. a plasmid, cosmid, or phage vector will often depend on the host cell into which it is to be introduced.
The vectors for use in the present invention may contain one or more selectable marker genes- such as a gene, which confers antibiotic resistance e..g.
ampicillin, kanamycin, chloramphenicol or tetracyclin resistance Alternatively, the selection may be accomplished by co-transformation (as described in WO91/17243) Vectors may be used in vitro, for example for the production of RNA or used to transfect, transform, transduce or infect a host cell, Thus, in a further embodiment, the invention provides a method of making nucleotide sequences of the present invention by introducing a nucleotide sequence of the present invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector, The vector may further comprise a nucleotide sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and plJ702.
REGULATORY SEQUENCES
In some applications, the nucleotide sequence for use in the present invention is operably linked to a regulatory sequence which is capable of providing for the expression of the nucleotide sequence, such as by the chosen host cell. By way of example, the present invention covers a vector comprising the nucleotide sequence of the present invention operably linked to such a regulatory sequence, i.e.. the vector is an expression vector.
The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A
regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
The term "regulatory sequences" includes promoters and enhancers and other expression regulation signals.
The term "promoter" is used in the normal sense of the art, e.g. an RNA
polymerase binding site..
Enhanced expression of the nucleotide sequence encoding the enzyme of the present invention may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and terminator regions, Preferably, the nucleotide sequence according to the present invention is operably linked to at least a promoter.
Other promoters may even be used to direct expression of the polypeptide of the present invention..
Examples of suitable promoters for directing the transcription of the nucleotide sequence in a bacterial, fungal or yeast host are well known in the art.
In one embodiment a suitable promoter may be a cellobiohydrolase promoter, In one embodiment a suitable promoter may be a cellobiohydrolase promoter obtainable (or obtained) from T. reesei.
The promoter can additionally include features to ensure or to increase expression in a suitable host. For example, the features can be conserved regions such as transcription factor binding sites or deleted repressor binding sites.
CONSTRUCTS
The term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - includes a nucleotide sequence for use according to the present invention directly or indirectly attached to a promoter..
An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Sh1-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention. The same is true for the term "fused" in relation to the present invention which includes direct or indirect attachment. In some cases, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wild type gene promoter and when they are both in their natural environment.
The construct may even contain or express a marker, which allows for the selection of the genetic construct.
For some applications, preferably the construct of the present invention comprises at least the nucleotide sequence of the present invention operably linked to a promoter..
HOST CELLS
The term "host cell" - in relation to the present invention includes any T.
reesei cell that comprises either the nucleotide sequence or an expression vector as described above and which is used in the recombinant production of a protein having the specific properties as defined herein, Thus, a further embodiment of the present invention provides T. reesei host cells transformed or transfected with a nucleotide sequence that expresses the protein of the present invention, The use of a T. reesei host cell may provide for post-translational modifications (e.g.
myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
ORGANISM
The term "organism" in relation to the present invention includes T. reesei that comprises the nucleotide sequence coding for the polypeptide according to the present invention and/or products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence according to the present invention when present in the organism, A suitable organism is T reesei.
The term "transgenic organism" in relation to the present invention includes a T
reesei that comprises the nucleotide sequence coding for the polypeptide according to the present invention and/or the products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence according to the present invention within the organism. Preferably the nucleotide sequence is incorporated in the genome of the organism.
The term "transgenic organism" does not cover native nucleotide coding sequences in their natural environment when they are under the control of their native promoter which is also in its natural environment.
5 Therefore, the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, the nucleotide sequence coding for the polypeptide according to the present invention, constructs according to the present invention, vectors according to the present invention, plasmids according to the present invention, cells according to the present invention, tissues according to the 10 present invention, or the products thereof.
For example the transgenic organism may also comprise the nucleotide sequence coding for the polypeptide of the present invention under the control of a heterologous promoter.
TRANSFORMATION OF HOST CELLSIORGANISM
Filamentous fungi cells may be transformed using various methods known in the art such as a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known.
General teachings on the transformation of fungi are presented in following sections.
TRANSFORMED FUNGUS
The host organism is T. reesei and the like.
Transforming filamentous fungi is discussed in US-A-5741665 which states that standard techniques for transformation of filamentous fungi and culturing the fungi are well known in the art. An extensive review of techniques as applied to N, crassa is found, for example in Davis and de Serres, Methods Enzyme! (1971) 17A: 79-143-Further teachings which may also be utilised in transforming filamentous fungi are reviewed in US-A-5674707.
In addition, gene expression in filamentous fungi is taught in Punt of a!..
(2002) Trends Biotechnol 2002 May;20(5):200-6, Archer & Peberdy Crit Rev Biotechnol (1997) 17(4):273-306-The present invention encompasses the production of transgenic filamentous fungi according to the present invention prepared by use of these standard techniques.
CULTURING AND PRODUCTION
T. reesei host cells transformed with the nucleotide sequence of the present invention may be cultured under conditions conducive to the production of the encoded polypeptide and which facilitate recovery of the polypeptide from the cell(s) and/or culture medium.
In one embodiment the transformed or transfected T reesei cell(s) provided in accordance with the present invention is cultured under selective conditions to allow for selection of the cell(s) transformed or transfected with the lipolytic enzyme as defined herein.
The medium used to cultivate the cell(s) may be any conventional medium suitable for growing the host cell in questions and obtaining expression of the polypeptide, The protein produced by a recombinant cell may be displayed on the surface of the cell..
The protein may be secreted from the host cells and may conveniently be recovered from the culture medium using well-known procedures.
SECRETION
Often, it is desirable for the protein to be secreted from the expression host into the culture medium from where the protein may be more easily recovered, According to the present invention, the secretion leader sequence may be selected on the basis of the desired expression host. Hybrid signal sequences may also be used with the context of the present invention.
Typical examples of heterologous secretion leader sequences are those originating from the fungal amyloglucosidase (AG) gene (glaA - both 18 and 24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces, Klu.yveromyces and Hansenula) or the -amylase gene (Bacillus).
In one embodiment preferably the signal peptide is that shown in SEQ ID NO: 1 as bold in Figure 15, In one embodiment the signal peptide shown in bold in figure 15 is cleaved off post-translationally to provide a peptide having the sequence shown in SEQ ID NO. 2..
DETECTION
A variety of protocols for detecting and measuring the expression of the amino acid sequence are known in the art.. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FAGS).
A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays.
A number of companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, WI), and US Biochemical Corp (Cleveland, OH) supply commercial kits and protocols for these procedures.
Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3,817,837; US-A-3,850,752; US-A-3,939,350; US-A-3,996,345;
US-A-4,277,437; US-A-4,275,149 and US-A-4,366,241, Also, recombinant immunoglobulins may be produced as shown in US-A-4,816,567..
FUSION PROTEINS
The amino acid sequence for use according to the present invention may be produced as a fusion protein, for example to aid in extraction and purification.
Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA
binding and/or transcriptional activation domains) and (-galactosidase). It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences Preferably, the fusion protein will not hinder the activity of the protein sequence.
ADDITIONAL POIs The sequences for use according to the present invention may also be used in conjunction with one or more additional proteins of interest (POls) or nucleotide sequences of interest (NO[s),.
Non-limiting examples of POIs include: proteins or enzymes involved in starch metabolism, proteins or enzymes involved in glycogen metabolism, acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, -galactosidases, -galactosidases, -glucanases, glucan lysases, endo--glucanases, glucoamylases, glucose oxidases, -glucosidases, -glucosidases, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, phospholipases, galactolipases, lipid acyltransferase, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, rhamno-galacturonases, ribonucleases, thaumatin, transferases, transport proteins, transglutaminases, xylanases, hexose oxidase (D-hexose: 02-oxidoreductase, EC
1.13.5) or combinations thereof. The NOI may even be an antisense sequence for any of those sequences.
The POI may even be a fusion protein, for example to aid in extraction and purification The POI may even be fused to a secretion sequence.
Other sequences can also facilitate secretion or increase the yield of secreted POI
Such sequences could code for chaperone proteins as for example the product of Aspergillus niger cyp B gene described in UK patent application 9821198.Ø
The NOI may be engineered in order to alter their activity for a number of reasons, including but not limited to, alterations which modify the processing and/or expression of the expression product thereof.. By way of further example, the NOl may also be modified to optimise expression in a particular host cell. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites.
The NOI may include within it synthetic or modified nucleotides -- such as methylphosphonate and phosphorothioate backbones.
The NOl may be modified to increase intracellular stability and half-life.
Possible modifications include, but are not limited to, the addition of flanking sequences of the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' 0-methyl rather than phosphodiesterase linkages within the backbone of the molecule LARGE SCALE PRODUCTION/APPLICATION
In one preferred embodiment of the present invention, the lipolytic enzyme is used for large scale applications and/or is produced on a large scale.
The term large scale means in a fermentor or culturing conditions of at least litres.
Preferably the lipolytic enzyme is produced in a quantity of at least 5g per litre of the total cell culture volume after cultivation of the host organism. Preferably, the lipolytic enzyme is produced in a quantity of at least 1Og per litre of the total cell culture volume after cultivation of the host organism. Preferably, the lipolytic enzyme is produced in a quantity of at least 15g per litre of the total cell culture volume after cultivation of the host organism. Preferably, the lipolytic enzyme is produced in a quantity of at least 20g per litre of the total cell culture volume after cultivation of the host organism..
FERMENTATION
The enzymes of the present invention can be produced either by solid or submerged culture, including batch, fed-batch and continuous-flow processes. Culturing is 10 accomplished in a growth medium comprising an aqueous mineral salts medium, organic growth factors, the carbon and energy source material, molecular oxygen, and, of course, a starting inoculum of one or more particular microorganism species to be employed.
15 In addition to the carbon and energy source, oxygen, assimilable nitrogen, and an inoculum of the microorganism, it is necessary to supply suitable amounts in proper proportions of mineral nutrients to assure proper microorganism growth, maximize the assimilation of the carbon and energy source by the cells in the microbial conversion process, and achieve maximum cellular yields with maximum cell density in the 20 fermentation media.
The composition of the aqueous mineral medium can vary over a wide range, depending in part on the microorganism and substrate employed, as is known in the art., The mineral media should include, in addition to nitrogen, suitable amounts of 25 phosphorus, magnesium, calcium, potassium, sulphur, and sodium, in suitable soluble assimilable ionic and combined forms, and also present preferably should be certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine, and others, again in suitable soluble assimilable form, all as known in the art.
30 The fermentation reaction is an aerobic process in which the molecular oxygen needed is supplied by a molecular oxygen-containing gas such as air, oxygen-enriched air, or even substantially pure molecular oxygen, provided to maintain the contents of the fermentation vessel with a suitable oxygen partial pressure effective in assisting the microorganism species to grow in a thriving fashion In effect, by using 35 an oxygenated hydrocarbon substrate, the oxygen requirement for growth of the microorganism is reduced, Nevertheless, molecular oxygen must be supplied for growth, since the assimilation of the substrate and corresponding growth of the microorganisms, is, in part, a combustion process.
Although the aeration rate can vary over a considerable range, aeration generally is conducted at a rate which is in the range of about 0.5 to 10, preferably about 0.5 to 7, - volumes (at the pressure employed and at 25 C.) of oxygen-containing gas per liquid volume in the fermentor per minute. This amount is based on air of normal oxygen content being supplied to the reactor, and in terms of pure oxygen the respective ranges would be about 0..1 to 17, or preferably about 0..1 to 13, volumes (at the pressure employed and at 25 C.) of oxygen per liquid volume in the fermentor per minute.
The pressure employed for the microbial conversion process can range widely.
Pressures generally are within the range of about 0 to 50 prig, presently preferably about 0 to 30 psig, more preferably at least slightly over atmospheric pressure, as a balance of equipment and operating cost versus oxygen solubility achieved.
Greater than atmospheric pressures are advantageous in that such pressures do tend to increase a dissolved oxygen concentration in the aqueous ferment, which in turn can help increase cellular growth rates. At the same time this is balanced by the fact that high atmospheric pressures do increase equipment and operating costs.
The fermentation temperature can vary somewhat, but for filamentous fungi such as Trichoderma reesei the temperature generally will be within the range of about to 40 C, generally preferably in the range of about 25 C to 34 C, depending on the strain of microorganism chosen.
The microorganisms also require a source of assimilable nitrogen. The source of assimilable nitrogen can be any nitrogen-containing compound or compounds capable of releasing nitrogen in a form suitable for metabolic utilization by the microorganism. While a variety of organic nitrogen source compounds, such as protein hydrolysates, can be employed, usually cheap nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea, and various ammonium salts such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, or various other ammonium compounds can be utilized. Ammonia gas itself is convenient for large scale operations, and can be employed by bubbling through the aqueous ferment (fermentation medium) in suitable amounts. At the same time, such ammonia can also be employed to assist in pH control The pH range in the aqueous microbial ferment (fermentation admixture) should be in the exemplary range of about 2.0 to 8.0, With filamentous fungi, the pH
normally is within the range of about 2.5 to 8.0; with Trichoderma reesei, the pH normally is within the range of about 3.0 to 7Ø pH range preferences for certain microorganisms are dependent on the media employed to some extent, as well as the particular microorganism, and thus change somewhat with change in media as can be readily determined by those skilled in the art_ While the average retention time of the fermentation admixture in the fermentor can vary considerably, depending in part on the fermentation temperature and culture employed, generally it will be within the range of about 24 to 500 hours, preferably presently about 24 to 400 hours. Preferably, the fermentation is conducted in such a manner that the carbon-containing substrate can be controlled as a limiting factor, thereby providing good conversion of the carbon-containing substrate to cells and avoiding contamination of the cells with a substantial amount of unconverted substrate, The latter is not a problem with water-soluble substrates, since any remaining traces are readily washed off. It may be a problem, however, in the case of non-water-soluble substrates, and require added product-treatment steps such as suitable washing steps. As described above, the time to reach this level is not critical and may vary with the particular microorganism and fermentation process being conducted. However, it is well known in the art how to determine the carbon source concentration in the fermentation medium and whether or not the desired level of carbon source has been achieved.
Although the fermentation can be conducted as a batch or continuous operation, fed batch operation is much to be preferred for ease of control, production of uniform quantities of products, and most economical uses of all equipment. If desired, part or all of the carbon and energy source material and/or part of the assimilable nitrogen source such as ammonia can be added to the aqueous mineral medium prior to feeding the aqueous mineral medium to the fermentor. Each of the streams introduced into the reactor preferably is controlled at a predetermined rate, or in response to a need determinable by monitoring such as concentration of the carbon and energy substrate, pH, dissolved oxygen, oxygen or carbon dioxide in the off-gases from the fermentor, cell density measurable by light transmittancy, or the like, The feed rates of the various materials can be varied so as to obtain as rapid a cell growth rate as possible, consistent with efficient utilization of the carbon and energy source, to obtain as high a yield of microorganism cells relative to substrate charge as possible In either a batch, or the preferred fed batch operation, all equipment, reactor, or fermentation means, vessel or container, piping, attendant circulating or cooling devices, and the like, are initially sterilized, usually by employing steam such as at about 121 G for at least about 15 minutes. The sterilized reactor then is inoculated with a culture of the selected microorganism in the presence of all the required nutrients, including oxygen, and the carbon-containing substrate. The type of fermentor employed is not critical, though presently preferred is operation under 15L
Biolafitte (Saint-Germain-en-Laye, France)..
The collection and purification of the enzymes of the present invention from the fermentation broth can also be done by procedures known per se in the art. The fermentation broth will generally contain cellular debris, including cells, various suspended solids and other biomass contaminants, as well as the desired enzyme product of the present invention, which are preferably removed from the fermentation broth by means known in the art. Suitable processes for such removal include conventional solid-liquid separation techniques such as, e.g., centrifugation, filtration, dialysis, microfiltration, rotary vacuum filtration, or other known processes, to produce a cell-free filtrate. It may be preferable to further concentrate the fermentation broth or the cell-free filtrate using techniques such as ultrafiltration, evaporation or precipitation. Precipitating the proteinaceous components of the supernatant or filtrate may be accomplished by means of a salt, e.g., ammonium sulfate.
Further purification may optionally be achieved by crystallization or by a variety of chromatographic procedures, e,g., ion exchange chromatography, affinity chromatography or similar art recognized procedures.
The lipase can be further formulated before use in foodstuffs.. The lipase can be in a liquid formulation, dried or granulated.
In one embodiment a carrier can be used, preferably the carrier is wheat or a wheat component In one embodiment the lipase is dried on wheat or dried on one or more wheat components, In one embodiment the lipase is in a liquid formulation suitable for consumption, preferably such liquid composition contains either buffer, salts, sorbitol and/ or glycerol.
In one embodiment the lipase is granulated or co-granulated with other enzymes.
FOOD
The enzyme of the present invention may be used as - or in the preparation of -a food.. Here, the term "food" means food intended for human consumption Also, the term "foodstuff' means a foodstuff intended for human consumption.
The food may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration-When used as, or in the preparation of, a food -- such as functional food -the enzyme of the present invention may be used in conjunction with one or more of. a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient FOOD INGREDIENT
The enzyme of the present invention may be used as a food ingredient and/or may be comprised in a food additive composition for humans.
As used herein the term "food ingredient" includes a formulation which is or can be added to functional foods or foodstuffs as a nutritional supplement and/or fiber supplement.
The food ingredient may be in the form of a solution or as a solid --depending on the use and/or the mode of application and/or the mode of administration FOOD SUPPLEMENTS
The composition of the present invention may be -- or may be added to - food supplements for humans FUNCTIONAL FOODS
The composition of the present invention may be - or may be added to -functional foods for humans.
As used herein, the term "functional food" means food which is capable of providing not only a nutritional effect and/or a taste satisfaction to a human, but is also capable of delivering a further beneficial effect to the consumer.
Accordingly, functional foods are ordinary foods that have components or ingredients (such as those described herein) incorporated into them that impart to the food a specific functional - e.g. medical or physiological benefit _ other than a purely nutritional effect to a human.
Although there is no legal definition of a functional food, most of the parties with an interest in this area agree that they are foods marketed as having specific health effects.
Some functional foods are nutraceuticals Here, the term "nutraceutical" means a food which is capable of providing not only a nutritional effect and/or a taste satisfaction, but is also capable of delivering a therapeutic (or other beneficial) effect to the consumer- Nutraceuticals cross the traditional dividing lines between foods and medicine.
Surveys have suggested that consumers place the most emphasis on functional food claims relating to heart disease. Preventing cancer is another aspect of nutrition which interests consumers a great deal, but interestingly this is the area that consumers feel they can exert least control over. In fact, according to the World Health Organization, at least 35% of cancer cases are diet-related Furthermore claims relating to osteoporosis, gut health and obesity effects are also key factors that are likely to incite functional food purchase and drive market development FOOD PRODUCTS
The composition of the present invention can be used in the preparation of food products for humans such as one or more of; jams, marmalades, jellies, dairy products (such as milk or cheese), meat products, poultry products, fish products and bakery products.
By way of example, the enzyme of the present invention can be used as ingredients to soft drinks, a fruit juice or a beverage comprising whey protein, health teas, cocoa drinks, milk drinks and lactic acid bacteria drinks, yoghurt and drinking yoghurt, cheese, ice cream, water ices and desserts, confectionery, biscuits cakes and cake mixes, snack foods, breakfast cereals, instant noodles and cup noodles, instant soups and cup soups, balanced foods and drinks, sweeteners, tacos, tortillas, texture improved snack bars, fibre bars, bake stable fruit fillings, care glaze, chocolate bakery filling, cheese cake flavoured filling, fruit flavoured cake filling, cake and doughnut icing, heat stable bakery filling, instant bakery filling creams, filing for cookies, ready-to-use bakery filling, reduced calorie filling, adult nutritional beverage, acidified soy/juice beverage, aseptic/retorted chocolate drink, bar mixes, beverage powders, calcium fortified soy/plain and chocolate milk, calcium fortified coffee beverage.
A enzyme according to the present invention can further be used as an ingredient in food products such as American cheese sauce, anti-caking agent for grated &
shredded cheese, chip dip, cream cheese, dry blended whip topping fat free sour cream, freeze/thaw dairy whipping cream, freeze/thaw stable whipped tipping, low fat & lite natural cheddar cheese, low fat Swiss style yoghurt, aerated frozen desserts, and novelty bars, hard pack ice cream, label friendly, improved economics &
indulgence of hard pack ice cream, low fat ice cream. soft serve, barbecue sauce, cheese dip sauce, cottage cheese dressing, dry mix Alfredo sauce, mix cheese sauce, dry mix tomato sauce and others.
For certain aspects, preferably the foodstuff is a beverage.
For certain aspects, preferably the foodstuff is a bakery product - such as bread, Danish pastry, biscuits or cookies.
FEED
The enzyme of the present invention may be used as - or in the preparation of -an animal feed or a component thereof. Thus, the present invention also encompasses a feed comprising or made from the enzyme of the present invention, and a process for making same.
DETERGENT
The enzyme of the present invention may be used as - or in the preparation of -- a detergent or a component thereof.. Thus, the present invention also encompasses a detergent comprising or made from the enzyme of the present invention, and a process for making same.
GENERAL RECOMBINANT DNA METHODOLOGY TECHNIQUES
The present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art.
Such techniques are explained in the literature See, for example, J.. Sambrook, E.
F.
Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Lab oratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al.
(1995 and periodic supplements; Current Protocols in Molecular Biology, ch.. 9, 13, and 16, John Wiley & Sons, New York, N.Y.), B. Roe, J. Crabtree, and A. Kahn, 1996, DNA
Isolation and Sequencing. Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis A Practical Approach, Irl Press;
and, D. M
J. Lilley and J- E. Dahlberg, 1992, Methods of Enzymology; DNA Structure Part A.
Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press Each of these general texts is herein incorporated by reference-The invention will now be described, by way of example only, with reference to the following Figures and Examples.
BRIEF DESCRIPTION OF THE FIGURES
The present invention is further illustrated by reference to the accompanying figures in which:
Figure 1 shows a model of the predicted translational modifications in a lipolytic enzyme (sometimes referred to herein as "lipase 3") and shown herein as SEQ
ID No 2; active site residues are shown in a circle - see Ser146, Asp201, His258; there are 7 cysteine residues and 4 are involved in disulfide bonds, shown as a dashed line and as a solid line above the residues, there are 2 sites for N-linked glycosylation, namely N32 & N242 which are shown in bold and are underlined (the latter, namely N242, is near to the His active site). NOTE:
the numbering in this Figure relates to the lipolytic enzyme shown as SEQ ID No. 2 without the signal peptide of 27 amino acids - therefore when referring to the lipolytic enzyme shown in SEQ ID No. 1 (i.e. with the signal peptide) the numbering in respect of the active site residues needs to be adjusted by adding 27 amino acids.
Figure 2 shows a schematic of the method of the present invention, Figure 3 shows a schematic diagram of the Aspergillus tubingensis lipolytic enzyme genomic DNA.
Figure 4 shows the expression construct "ATlipase3Trex'.
Figure 5 shows results of a Lipase Activity Assay performed on supernatant from Trichoderma reeseitransformants.
Figure 6 shows a protein profile by SDS-PAGE performed on supernatant from Trichoderma reesei transformants. The lane indicated with an arrow shows one of the transformants that expresses very high levels of the lipolytic enzyme ("lipase 3") Figure 7 shows a protein profile by SDS-PAGE of supernatant from Trichoderma reesei transformants cultivated in a 3 litre fermentor.. The arrow indicates the lipolytic enzyme ("lipase 3") protein band, Figure 8 shows the yield of the lipolytic enzyme (lipase 3) protein in the fermentation broth when the lipolytic enzyme is expressed in different expression hosts. Yield is expressed as a % increase in yield in each organism:
1 Aspergillus tubingensis;
2 Pichia pastoris, 3 Hansenula polymorpha;
4 Trichoderma reesei..
Figure 9 shows a southern blot showing transformed T. reesei strains that had been transformed with multiple copies of the "lipase 3" lipolytic enzyme gene, the lanes are labelled as follows-M - Untransformed host strain, B - strain transformed using biolistic transformation;
E - strain transformed using electroporation.
Figure 10 shows a protein profile by SDS-PAGE used to characterise the lipolytic enzyme ("lipase 3") expressed in Trichoderma reesei.. Samples from cultures grown in different broths showed lipase 3 protein as double or triple bands.
Figure 11a shows two different UFCs (ultra-filtration concentrates) showing precipitation of the lipolytic enzyme "lipase 3". Less concentrated protein in 1035 UFC shows heavy precipitation and more concentrated protein in 1036 UFC shows very heavy precipitation Precipitation is concentration dependent -the higher the protein concentration the more likely it is to precipitate..
Figure 11 b shows an SDS-PAGE showing the presence of the lipolytic enzyme "lipase 3" protein in the precipitates of Figure 11 a:
Lane I - Centrifuged filtered crude supernatant Lane 2 - Resolubilized pellet from UFC 1035 Lane 3 - Resolubilized pellet from UFC 1036 Figure 12 shows resolubilisation of precipitated lipolytic enzyme ("lipase 3") using different concentrations of sodium phosphate buffer. Samples were at pH 530.
10 Lane 1, Crude, control.
Lanes 2-6. 5 mM, 10 mM, 20 mM, 40 mM & 50 mM sodium phosphate;
Figure 13 shows characterization of recombinant lipolytic enzyme "lipase 3" by MALDI-TOF/MS analysis of glycosylated compared with de-glycosylated lipase 15 The size of N-glycan is about 1384 Da and the lipolytic enzyme (lipase 3) molecules may have glycans attached at N32 site (when considering SEQ ID
No.2);
Figure 14 shows an SDS-PAGE protein analysis of purified lipolytic enzyme 20 (lipase 3) subjected to deglycosylation. Deglycosylation enzyme: Endo-H, 2.0 mgiml Endo-H was added at a ratio of 1:50 and 1:100 (w/w) and incubated for 20 hours at room temperature, pH 5.5..
Lane 1 - Control 25 Lane 2 - 1:100 dilution of supernatant Lane 3 - 1:50 dilution of supernatant The specific activities are shown in table 1 below. N-linked glycosylation has no effect on the specific activity of lipolytic enzyme (lipase 3) protein.
30 Deglycosylating the lipolytic enzyme (lipase 3) did not affect enzyme activity;
Figure 15 shows the amino acid sequence (SEQ ID No.. 1) of a lipolytic enzyme from Aspergillus tubingensis wherein the endogenous signal peptide is shown in bold, Figure 16 shows the amino acid sequence (SEQ ID No. 2) of a lipolytic enzyme from Aspergillus tubingensis which is the same as SEQ ID No, 1 except that the endogenous signal peptide has been removed.
Figure 17 shows the nucleotide sequence encoding an Aspergillus tubingensis lipolytic enzyme (as shown in SEQ ID No, 1) including the signal sequence -the nucleotide sequence is a genomic DNA sequence (and has been designated as SEQ ID No. 3.. The signal sequence is shown in bold and the introns are shown in lower caseõ
Figure 18 shows the nucleotide sequence encoding an Aspergillus tubingensis lipolytic enzyme (as shown in SEQ ID No 2) not including the signal sequence the nucleotide sequence is a genomic DNA sequence (and has been designated as SEQ 1D No. 4, The introns are shown in lower case.
Figure 19 shows the sequence of the cellobiohydrolase 1 gene (cbhl) promoter designated SEQ ID No, 5.
EXAMPLE I
Expression of the Aspergillus tubingensis lipase 3 in Trichoderma reesei 1. Expression Construct & Strain used for Transformation pDONRTM221 plasmid DNA obtained from Invitrogen (catalogue no.12536-017) was used as the donor vector. pDONR221::lip 3 containing the Aspergillus tubingensis lipase 3 genomic DNA (Figure 3) was recombined into the T. reesei gateway destination vector pTrex3G (described in detail in WO 05/001036), resulting in the final expression construct ATlipase3Trex (Figure 4), The expression cassette contained the promoter and terminator regions of the T
reesei cellobiohydrolase 1 (cbhl) gene.. It also contained the Aspergillus nidulans acetamidase, amdS gene as a selectable marker for transformation of T reesei.
The Aspergillus tubingensis lipase 3 genomic DNA encodes a lipase 3 lipolytic enzyme having the amino acid sequence shown in SEQ ID No, 1.
The term "lipase 3" when used herein refers to a lipolytic enzyme comprising the amino acid sequence shown in SEQ ID No. 2, such as the amino acid sequence shown in SEQ ID No.. 1. Seq.. ID No. 1 contains the signal seq and seq. ID No.
2 is the mature lipase protein without the signal sequence.
The strain used for transformation was Trichoderma reesei, a derivative of the non-GMM strain RL-P37 from which the genes encoding the two secreted cellobiohydrolases, CBHI and CBHII, and two of the secreted endoglucanases, EGI
and EGII, have been deleted..
The expression vector pTrex3g.
The following describes the construction of the vector pTrex3g which may be used to express the genes of the present invention.
This vector is based on the E. coli vector pSL1180 (Pharmacia Inc.., Piscataway, NJ, USA) which is a pUC118 phagemid based vector (Brosius, J. (1989) DNA 8:759) with an extended multiple cloning site containing 64 hexamer restriction enzyme recognition sequences. It was designed as a Gateway destination vector (Hartley, J.L., Temple, G.P.. and Brasch, M A, (2000) Genome Research 10:1788-1795) to allow insertion using Gateway technology (Invitrogen) of any desired open reading frame between the promoter and terminator regions of the T. reesei cbhl gene.
It also contains the Aspergillus nidulans amdS gene for use as a selectable marker in transformation of T reesei.
The details of pTrex3g are as follows, The vector is 10.3 kb in size.
Inserted into the polylinker region of pSL1 180 are the following segments of DNA:
1.. A 2,2 bp segment of DNA from the promoter region of the T reesei cbhl gene 2. The 1.7 kb Gateway reading frame A cassette acquired from Invitrogen that includes the attRl and attR2 recombination sites at either end flanking the chloramphenicol resistance gene (CrnR) and the ccdB gene 3. A 336 bp segment of DNA from the terminator region of the T reesei cbh I
gene 4 A 2.7 kb fragment of DNA containing the Aspergillus nidulans amdS gene with its native promoter and terminator regions, 2. Transformation of T. reesei quad delete Host Strain The expression construct, ATlipase3Trex, containing the A, tubingensis lipase 3 gene was transformed into a T reesei strain using electroporation or biolistic transformation by particle bombardment using the PDS-1000 Helium system (BioRad Cat. No. 165-02257). PCR products containing only fungal DNA or the entire expression plasmid were used for generating transformants by biolistic transformation and electroporation..
A. Transformation by electroporation The T reesei host strain to be transformed was grown to full sporulation on PDA
plates for 5 days.. Spores from 2 plates were harvested with 1.2M sorbitol and filtered through miracloth to get rid of the agar. Spores were transferred to a 50 ml falcon tube and washed by repeated centrifugation 5-6 times with 50 ml water. The spores were resuspended in a small volume (less than 2x pellet volume) using 1.2M sorbitol solution. The spore suspension was then kept on ice.. 90 ul of spore suspension was aliqouted into the electroporation cuvette (E-shot, 0,1cm standard electroporation cuvette from lnvitrogen). 10-20 ul DNA construct (plasmid Figure 4 or PCR
product) were added to the spore suspension and electroporation was set at 16kV/cm, 25pF, 500. After electroporation, the spore suspension was left on ice, resuspended in 5 parts 1.OM sorbitol and 1 part YEPD, and allowed to germinate by incubation at with shaking 250 rpm overnight The next day, the germlings were plated in agar plates containing acetamide. Transformants were picked and transferred individually to acetamide agar plates.
B. Transformation by Particle Bombardment (Biolistic transformation) A suspension of spores from a quad deleted strain of T. reesei was prepared.
200u1 of spore suspension was spread onto the center of the minimal medium (MM) acetamide plates. (MM acetamide plates had the following composition. 0.6g/l acetamide;
1.68g/l CsCI; 20g/l glucose; 20g/l KH2PO4, 0..6g/l CaCl2 2H20; 1 ml/l 1000x trace elements solution ; 20g/l Noble agar, and pH5.5.. 1000x trace elements solution contained 5 0g/1 PeSO4 7H20; 1..6g/I MnSO4; -1.4g/l ZnSO4 7H20 and 1 Og/l CoCl2 6H20, The spore suspension was allowed to dry on the surface of MM acetamide medium for 1 hour in the sterile hood. Transformation followed the manufacturer's instruction. 60mg of tungsten particles were placed in a microfuge tube. 1ml of ethanol was added and allowed to stand for 15 seconds The ethanol was removed and the particles were washed three times with sterile dHZO before 250ul of 50% (v/v) sterile glycerol was added. 25u1 of tungsten particle suspension was placed onto a microfuge tube.
While continuously vortexing, the following were added. 5u1 (100-200 ng/u1) of plasmid DNA, 25ul of 2.5M CaCl2 and 10ul of 0.1M spermidine. The particles were centrifuged for 3 seconds, The supernatant was removed and the particles were washed with 200u1 of 100% ethanol and centrifuged for 3 seconds.. The supernatant was removed..
24u1 100% ethanol was added and mixed by pipetting, then 8 ul aliquots of particles were removed and placed in the centre of microcarrier disks that were held in a desiccator.
Once the tungsten/DNA solution had dried the microcarrier disk was placed in the bombardment chamber along with the plate of MM acetamide with spores and the bombardment process was carried out according to the manufacturer's instructions.
After bombardment of the plated spores with the tungsten DNA particles, the plates were incubated at 28C. Transformed colonies were transferred to fresh plates of MM
acetamide medium and incubated at 28C.
3. Growth of transformants in microtiter plates After 5 days of growth on MM acetamide plates, transformants obtained by electroporation or by biolistic transformation and displaying stable morphology were inoculated into 200ul Defined medium with glucose/sophorose in a 96-well microtiter plates. Defined medium with glucose/sophorose (per liter) consists of NH42SO4 5g, PIPPS buffer 33 g, Casamino Acids 9g, KH2PO4 4.5 g, CaC12 (Anhydrous) 1g, MgS04.7H20 1g, pH to 5 50 adjusted with 50% NaOH with milli-Q H2O bring to 966.5 mL. After sterilization, the following were added. Mazu 5 mL, Glucose/Sophrose 60%
26 mL and 400X T reesei Trace Metals 2.5 mL. The microtiter plate was incubated in an oxygen growth chamber at 28 C for 5 days.
Screening by Lipase spot assay & SDS-PAGE
10 Mycelium was removed by centrifugation and the supernatant was analysed for lipase activity using the spot assay. Lipase plate assay is based on the release of fatty acid from the substrate (tributyrin) in the presence of lipase. A pink colour is formed when fatty acid is released and forms a complex with Rhodamine B. The assay plate contained 2..Og Bacto Agar (dissolved by heating for 5 minutes in 100 ml 50mM
15 Sodium phosphate buffer pH 5.5.. The solution was kept at 70 C water bath and while stirring, 0.5m1 2% Rhodamine and 40m1 tributyrin was added. The mixture was subjected to sonication for 2 minutes and 10-15 ml was poured into petri dishes-Holes were punched and the culture supernatant was applied on to the holes..
The plates were incubated at 37 C until a pink colour was formed indicating the presence 20 of lipoly.tic activity. Supernatant (1Oul) from transformants was checked for lipase activity using the spot assay as shown in Figure 5, arrows indicate the appearance of the pink colour after 30 minutes of incubation at 37 C, showing high lipase activity.
The protein profile of those transformants exhibiting high lipase activity was 25 determined by SDS-PAGE using NuPAGE 4-12% polyacrylamide gels and MES as running buffer. Samples of the supernatant were mixed with appropriate volume of 2x sample loading buffer with reducing agent. The gels were stained with Simply blue Safestain (Invitrogen).. In Figure 6 the lane marked with an arrow shows one of the transformants expressing very high levels of lipase 3 appearing as a double band.
30 The rest of the transformants showed distinct and slightly fuzzy single bands.. The best transformant grown in 3 litre fermentor gave a total secreted protein titer of at least 20g/liter and SDS-PAGE analysis showed a broad lipase band (Figure 7).
Large scale Fermentation (14-liters) of lipase 3 Transformants Trichoderma reesei transformants were cultured in fermenters as described in WO
2004/035070. Four different transformants, generated by electroporation, were cultured. Measurement of total protein and lipase activity in culture supernatants, after cell removal, indicated that in excess of 20 grams per liter lipase was present after 160 hours of fermentation.. Two strains, generated by biolistic transformation, were also grown. These showed in excess of 20 grams per liter lipase in the culture supernatant after 160 hours of fermentation.. The amount of lipase 3 produced by these transformants was far in excess of the amount of lipase 3 produced by other microbial host species (Figure 8) Trichoderma reesei transformants were cultured in fermentors. Four different transformants, generated by electroporation, were cultured. Measurement of total protein and lipase activity in culture supernatants, after cell removal, indicated that in excess of 20 grams per liter lipase was present after 160 hours of fermentation. Two strains, generated by biolistic transformation, were also grown. These showed in excess of 20 grams per liter lipase in the culture supernatant after 160 hours of fermentation. The amount of lipase 3 produced by these transformants was far in excess of the amount of lipase 3 produced by other microbial host species (Figure 8).
Solubility of lipase 3 Surprisingly the present inventors have found that Trichoderma reesei is capable of producing lipase 3 at very high levels. Down stream processing of the lipase after fermentation requires concentration of the culture broth 4x by ultrafiltration using a membrane with a 10,000 molecular weight cut-off. Lipase 3 is prone to precipitation as shown in Figure 11 a. Heavy precipitation is observed in the more concentrated UFC. The presence of lipase 3 protein in the precipitates was confirmed by SDS-PAGE.
Figures 11 a and 11 b show that precipitation of lipase 3 is concentration dependent.
Figure 11a shows two different ultra-filtration concentrates (UFCs) showing precipitation of lipase 3. 1035 UFC contains less concentrated protein and shows heavy precipitation. 1036 UFC contains more concentrated protein and shows very heavy precipitation..
Figure 11 b shows an SDS-PAGE showing the presence of lipase 3 protein in the precipitates shown in Figure 11 a.
Lane 1 Centrifuged filtered crude supernatant Lane 2 Resolubilized pellet from UFC 1035 Lane 3 Resolubilized pellet from UFC 1036 For resolubilisation of lipase precipitates, stock buffer was used. Stock buffer consists of 1..0 M Na-Phosphate buffer, pH 8.0 Na2HP04.2H20 (177.99 g) added to 900 ml of DI water. To 10.0 g samples of the crude lipase 3 UFC 1036, pH 4.44 was added 50, 100, 200, 400, and 500 lil of buffer stock solution to a final concentration of 5, 10, 20, 40, and 50 mM Na-Phosphate respectively. All the vials were mixed and left at room temperature for a few minutes.
As shown in Figure 12, the addition of Na-phosphate above 40 mM brought the precipitated lipase 3 into solution. The resulting solution was clear and free from any solid matter. The pH of the samples was measured at pH 5.3 (at 50 mM Na-P
concentration).
Characterization of recombinant lipase 3 by MALDI-TOF/MS Analysis &
Proteolytic digestion Lipase 3 expressed by T. reesi was purified from the culture supernatant using a combination of ion exchange and hydrophobic interaction chromatography.
Deglycosylation was carried out using endoglycosidase H in reaction buffer consisting of 50 mM Sodium Citrate, pH 5.5. Endoglycosidase H (2.0 mg/mL), was added to lipase 3 at a protein ratio from 1/1000 to 11100 (w/w).. The deglycosylation reaction was performed at 37 C for 3 hours The reacted protein sample was put on a ZipTip (Micro C4 reversed-phase column) (Millipore, Bedford, MA) for sample cleanup prior to the MALDI-TOF/MS analysis.. The cleanup process was performed to desalt the samples. At least five cycles of wash solution consisting of 5% methanol in 0.1%
TFA/water wash was used. Afterwards the samples were eluted for mass spectrometry MALDI-TOF/MS Analysis of Intact Protein The desalted protein sample was prepared for MALDI-TOF/MS analysis by co-crystalizing an equal volume (1 L) of sample with Sinapinic acid matrix (saturated in 50% acetonitrile, 0.1% formic acid) using the dried droplet method. Protein mass spectra were obtained using a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA). The MS instrument settings were as follows for the 20000-80000 m/z range: linear mode of operation, delayed extraction mode, positive polarity, 25 kV acceleration voltage, 93% grid voltage, and 750 nsec extraction delay time. 300-laser shots/spectrum and BSA was used as external calibrant Proteolytic Digestion & N-glycosylation site(s) Mapping by LCIMS/MS Analysis All Endo-H treated liquid samples were precipitated with 10% TCA followed by the reduction reactions with 20 mM DTT @ 50 C for 15-20 min. The alkylation reaction was also performed with 55 mM iodoacetamide. The alkylation reaction was allowed to proceed in the dark for 45 min at room temp. Proteolytic digestions were performed by incubation with various proteases in 25 mM ammonium bicarbonate for 4 hr at 37 C (enzyme to substrate ratio was 1:20):. Peptide mapping carried out using 3 different proteolytic digestions (trypsin, chymotrypsin, Endoproteinase GIuC) on the glycosylated lipase confirmed the absence of protein modification by truncation and the presence of an intact lipase protein with authentic N- and C-terminal ends.
Lipase 3 has 2 potential N-glycosylation sites at N32 and N242. Peptide mapping was carried out for both lipase 3 (untreated) and Endo-H treated. To determine the site of N-glycosylation, MS and MS/MS data were acquired using the Surveyor""
LC
system coupled to the LCQ Advantage or LCQ Deca XP (Thermo Finnigan, San Jose, CA). The HPLC gradient was programmed from 0% to 70% B over 50 minutes, Solvent A: 0.1 % TFA in water and Solvent R. 0 08% TFA in acetonitrile.. Data Processing was performed using TurboSEQUEST and Xcalibur (ThermoFinnigan, San Jose, CA).
As shown in Figure 13, SDS-PAGE showed the recombinant lipase secreted by Trichaderma appeared as a double band prior to endoglycosidase H treatment.
MALDI-TQF/MS analysis showed that the higher molecular weight species was a glycosylated form of lipase 3 with a molecular weight of 30,236 Dalton and the lower molecular weight species was a de-glycosylated form with molecular weight of 29,210 Dalton. Experimentally deglycosylated lipase samples were generated using endoglycosidase H.. The molecular weights observed after endoglycosidase H
treatment were 29,035 Dalton (presumed to be a non-glycosylated form), 29,219 (presumed to be a form of lipase 3 with 1 N-linked N-acetylglucosamine at position N32 or N242) and 29,422 (presumed to be a form of lipase 3 with 2 N-aceyltglucosamine residues attached to the protein backbone at positions N32 and N242) The glycan chain that is present prior to de-glycosylation with endoglycosidase H has a molecular weight of approximately 1384 daltons with the majority of lipase molecules having glycan attached only to the N32 site (numbered in accordance with SEQ ID No. 2) Specific activity of the lipolytic enzyme lipase 3 produced in T. reesei using different substrates The purified lipase 3 was subjected to deglycosylation by treatment with endoglycosidase H. The untreated and the deglycosylated samples were characterised by measurement of specific activities using both short chain (C4) and long chain (C 18) substrates-As shown in Figure 14 and Table 2 the presence of glycan side chains has no effect on the specific activity of the protein.
Table 2 shows the results of the deglycosylation experiments. The sample was Purified Trichoderma lipase 3 and the deglycosylation enzyme was Endo-H..
Specific activity, %
Sample LIPU/ml LUSol/ml (Short chain, C4) Lon chain, C1$
Control lane 1) 100 100 1:100 Endo-H (lane 2) 99.4 103 1.50 Endo-H lane 3) 97.7 105 Southern Blot Analysis Figure 9 shows a southern blot showing transformed T reesei strains that had been 10 transformed with multiple copies of the "lipase 3" lipolytic enzyme gene, the lanes are labelled as follows:
M - Untransformed host strain, B - strain transformed using biolistic transformation, E - strain transformed using electroporation;
Expressions studies Lipase from Thermom,yces lanuginosus was cloned and expressed in T. reesei Transformants were isolated and the best lipase producer was tested in 14-Liter scale fermentation to investigate whether Trichoderma reesei is a suitable host strain for expression of other lipases. The total protein at the end of (200 hours) fermentation was estimated to be in excess of 20 g/L as shown in Figure 14.
SDS-PAGE showed that lipase was the dominant protein produced. Lipase activity measured at the end of fermentation was 30,000 UImL using DGGR assay. The broth was filtered and concentrated to UFC at high concentration the lipase appeared to precipitate. It was easily brought back into solution by diluting in buffer or salt Expression studies Table 3 provides the results of a series of expression studies.
The present inventors have surprising found that the lipolytic enzyme "Lipase 3"
produced in Trichoderma reesei is less glycosylated (in particular less N-linked glycosylation) compared with the same enzyme produced in other organisms..
Table 3. Comparison with other hosts used to express lipase 3 from Aspergillus tubingensis Expression Host Glycosylation at N32 Protein Quality & N242 sites Aspergillus Hyperglycosylation Reduced Activity tubigensis 3M (overglycosylation 10%) (both N32 & N242 sites) Pichia pastoris Hyperglycosylation Activity > activity seen with A.
GS 115 (both N32 & N242 tubigensis recombinant lipase strain sites) 0- 1 cans Hansenula Glycosylation (both N32 Activity > activity seen with A.
polymorpha RB1 1 & N242 sites) tubigensis recombinant lipase strain Glycan 1%
T. reesei Majority of N- Activity is same as native lipase 3 glycosylation at N32 from non-recombinant Aspergillus 1 M341 strain The expression host cells suitable for use in the present invention may be a strain of T. reesei in which the genes encoding cellobiohydrolase I (CBHI, Cel7a), cellobiohydrolase II (CBH1I, Cel6a), endoglucanase I (EGI, Cel7b), and endoglucanase II (EGII, Cel5a) have been inactivated by deletion or disruption using molecular genetic techniques. This strain (a quad delete strain) is useful as a host for over-expression of genes encoding other T. reesei secreted proteins.
Preferably host cells suitable for use in the present invention may be derived from a Trichoderma reesei cell of the strain RL-P37 using the methods described in WO
20051001036 and US28026376 Al.
A Treesei strain suitable for use in the present invention may be derived from the publicly available strain of T,reesei RL-P37. T reesei strain RL-P37 may be modified to form T reesei strain 1A52 as described in WO 051001036. T reesei strain 1A52 may be modified as described in US 20080026376 to form a T reesei cell usable in the present invention.
RL-P37 may be modified to form T reesei strain 1A52 as described below..
The T. reesei host strain used may be derived from the publicly available strain RL-P37 which has previously been used to manufacture commercial cellulase preparations by Genencor International, Inc The derivation and characterisation of this strain has been published previously (Sheir-Neiss, G. and Montenecourt, BS. (1 984) Appl. Microbiol. Biotechnol. 20.46-53; US Patent 4,797,361). It is a cellulase over-producing mutant strain which has been obtained as a result of several mutagenesis steps from the wild-type strain (QM6a).
1) Isolation of a pyr4 mutant strain.
In order to prepare strain RL-P37 for transformation with plasmid DNA it was necessary to isolate a derivative having a null mutation in the p.yr4 gene..
The pyr4 gene encodes orotidine-5'-monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. The toxic inhibitor 5-fluoroorotic acid (FOA) is incorporated into uridine by wild-type cells and thus poisons the cells.
However, cells defective in the pyr4 gene are resistant to this inhibitor but require uridine for growth.
It is, therefore, possible to select for pyr4 mutant strains using FOA. In practice, spores of T. reesei strain RL-P37 were spread on the surface of a solidified medium containing 2 mg/ml uridine and 1.2 mg/ml FOA Spontaneous FOA-resistant colonies appeared within three to four days. FOA-resistant mutants which required uridine for growth were identified In order to identify those mutants which specifically had a defective pyr4 gene protoplasts were generated and transformed with a plasmid containing a wild-type pyr4 gene (Smith, J L., Bayliss, F.T. and Ward, M..
(1991) Currõ
Genet. 19:27-33). Following transformation protoplasts were plated on medium lacking uridine. Subsequent growth of transformed colonies demonstrated complementation of a defective pyr4 gene by the plasmid-borne pyr4 gene. In this way strain GC69 was identified as a p.yr4 mutant of strain RL-P37.
2) Construction of a plasmid designed to delete the CBHI encoding gene.
The cbhl gene, encoding the CBHI protein, was cloned from the genomic DNA of strain RL-P37 by hybridization with an oligonucleotide probe designed on the basis of the published sequence for this gene (Shoemaker, S., Schweickart, V.., Ladner, M.., Gelfand, D., Kwok, S., Myambo, K.. and Innis, M. (1983) Biotechnology 1:691-696).
The cbhl gene resides on a 6.5 kb Psfl fragment and was inserted into the Pstl site of pUC4K (Pharmacia Inc., Piscataway, NJ, USA) replacing the kanamycin-resistance gene of this vector The resulting plasmid, pUC4K::cbh 1, was then cut with Hindlll and the larger fragment was isolated and religated to give pUC4K::cbhlAH/H..
This procedure removed the entire cbhl coding sequence and approximately 12 kb of 5' and 1.5 kb of 3' flanking sequences. Approximately I kb of flanking DNA
remained from either end of the original Pst/ fragment. The T, reeseipyr4 gene was cloned as a 6.5 kb Hindlll fragment of genomic DNA in pUCl8 to form pTpyr2 (Smith, J_L., Bayliss, F.T.. and Ward, M.. (1991) Curr. Genet.. 19:27-33). The plasmid pUC4K.:cbh1L1H/H
was cut with Hindlll and the ends were dephosphorylated with calf intestinal alkaline phosphatase This DNA was ligated with the 6.5 kb Hindlll fragment containing the p,yr4 gene to give pACBHlp.yr4.
Digestion of pACBHlp.yr4 with EcoRl liberated a larger fragment which consisted of flanking regions of the Ohl locus at either end with the pyr4 gene replacing the cbhl coding sequence in the centre. The only DNA on this fragment which was not derived from T. reesei was a 21 bp fragment derived from the multiple cloning site of pUC4K.
3) Deletion of the cbhl gene of T, reesei.
Protoplasts isolated from mycelium of strain GC69 were transformed with EcoRl digested plasmid p CBHlpyr4 using methods outlined by Smith et al., 1991.
Stable transformants were obtained and those from which the cbh 1 gene had been deleted were identified as described below, Total DNA was isolated from the transformants, digested with Pstl, subjected to agarose gel electrophoresis and blotted to a membrane filter The filter was then hybridised with P32 labelled pACBHlpyr4 and the pattern of hybridisation observed by autoradiography. This probe hybridised with the native cbh 1 and pyr4 genes in an untransformed strain. In one transformant (strain P37PACBHI) a pattern of hybridisation was observed which would be predicted if a double cross-over integration event had occurred. That is, the cbh 1 gene had been deleted by integration of a single copy of the larger EcoRl fragment obtained from pACBHlpyr4 at the cbhl locus of strain RL-P37.
Southern analysis was also performed as above except that the probe used was radiolabelled plntCBHI. This plasmid consists of a pUC vector containing a 2 kb BgIII
fragment from the cbhl locus within the region that was deleted in pUC4K::cbh1AH/H.
This plasmid hybridised to the cbh-f locus of strain GC69 but did not hybridise to DNA
from strain P37PACBHI.. This confirms that the cbh 1 gene had been deleted and that the pUC DNA fragment from pACBHIpyr4 had not been incorporated by the deleted strain..
Analysis of secreted proteins by separation on isoelectric focusing gels showed that the CBHI protein was not produced by strain P37PACBHI..
4) Generation of a pyr4 null mutant of P37PACBHI.
Spores of the transformant (P37PACBHI) which was deleted for the cbhl gene were spread onto medium containing 1FOA. A pyr4 deficient derivative of this transformant was subsequently obtained using the methods described in section above, This pyr4 deficient strain was designated P37PACBHIPyr 26. Southern analysis has shown that a spontaneous deletion had occurred when strain P37PACBHIPy(26 was selected. This deletion completely removed the pyr4 gene which had integrated at the cbh 1 locus in strain P37P CBHI, as well as flanking DNA from the cbh 1 locus beyond the extent of the 6.5 kb Pstl fragment of genomic DNA which was originally cloned, 5) Construction of a vector designed to delete the cbh2 gene.
The cbh2 gene of Treesei, encoding the CBHII protein, has been cloned as a 4.1 kb 5 EcoRl fragment of genomic DNA (Chen at at., 1987, Biotechnology 5:274-278).
This.4..1 kb fragment was inserted between the EcoRl sites of pUC4XL. The latter plasmid is a pUC derivative (constructed by R. M. Berka, Genencor International Inc.) which contains a multiple cloning site with a symetrical pattern of restriction endonuclease sites arranged in the order shown here. EcoRl, BamHl, Sacl, Smal, 10 Hindlll, Xhol, Bglll, Clal, Bglll, Xhol, Hindll 1, Smal, Sacl, BamHl, EcoRl. The plasmid, pPOCBHII was constructed in which a 17 kb central region of this cbh2 clone, between a Hindlll site (at 74 bp 3' of the CBHII translation initiation site) and a Clal site (at 265 bp 3' of the last codori of CBHII), has been removed and replaced by a 1.6 kb Hindlll-Clal DNA fragment containing the T, reesei pyr4 gene obtained as follows..
15 The T. reesei pyr4 gene was excised from pTpyr2 on a 1.6 kb Nhel-Sphl fragment and inserted between the Sphl and Xbal sites of pUC219 (derived from pUC119 by expanding the multiple cloning site to include restriction sites for Bglll, Clal and Xhol, Wilson et al.., 1989, Gene 77:69 78) to create p219M (Smith at al., 1991, Curr, Genet, 19:27-33) The pyr4 gene could then be removed as a Hindlll-Clal fragment having 20 seven bp of DNA at one end and six bp of DNA at the other end derived from the pUC219 multiple cloning site and inserted into the Hindlll and Clal sites of the cbh2 gene to form the plasmid pPACBHII, Digestion of this plasmid with EcoRl liberated a fragment having 0.7 kb of flanking 25 DNA from the cbh2 locus at one end, 1.7 kb of flanking DNA from the cbh2 locus at the other end and the T. reesei pyr4 gene in the middle. The only DNA in this fragment which was not derived from T, reesei was the 6 bp and 7 bp fragments of the pUC219 multiple cloning site at either end of the pyr4 gene.
30 6) Deletion of cbh2 gene from strain P37Pi CBHIPyC26 Protoplasts of strain P37P CBHIPyr-26 were generated and transformed with EcoRl digested pPACBHII according to the methods outlined in 3 above. Stable transformants were cultured in shake flasks and the protein in the culture supernatants was examined by isoelectric focussing One transformant (designated P37PAACBH67) was identified which did not produce any CBHII (nor CBHI) protein.
DNA was extracted from strain P37PAACBH67, digested with EcoRl and Asp718, and subjected to agarose gel electrophoresis.. The DNA from this gel was blotted to a membrane filter and hybridized with 32P labelled pPACBHII. The 41 kb EcoRl fragment containing the wildtype cbh2 gene was observed in the DNA from an untransformed control strain In contrast, in strain P37PAACBH67 the single 4.1 kb band was eliminated and replaced by two bands of approximately 0.9 and 3..1 kb. This is the expected pattern if a single copy of the larger EcoRl fragment from pPACBHII
had integrated precisely at the cbh2 locus and deleted the cbh2 gene, The same DNA samples were also digested with EcoRl and Southern analysis was performed as above,, In this example the probe was 32P labelled plntCBHlI.
This plasmid contains a portion of the cbh2 gene coding sequence from within that segment of cbh2 DNA which was deleted in plasmid pPACBHII. No hybridization was seen with DNA from strain P37PACBH67 confirming that the cbh2 gene was deleted and that the pUC plasmid fragment of pPACBHII had not been incorporated by this strain.
7) Selection of a pyr4 null mutant of strain P37PAACBH67.
Spores of the transformant (P37PAACBH67) which was deleted for both the cbhl and cbh2 genes were spread onto medium containing FOA. A pyr4 deficient derivative of this transfornant was subsequently obtained using the methods described in section 1 above. This pyr4 deficient strain was designated P37PAACBH67Pyrr1. Southern analysis has shown that a spontaneous deletion had occurred when strain P37PAACBH67Pyrr1 was selected.. This deletion completely removed the pyr4 gene which had integrated at the cbh2 locus in strain P37PACBH67, as well as flanking DNA from the cbh2 locus beyond the extent of the 4.1 kb EcoRl fragment of genomic DNA which was originally cloned. The short (6 bp and 7 bp) fragments of DNA
derived from the pUC219 multiple cloning site which were present at either end of the pyr4 gene would also have been removed from the genome by this deletion 8) Construction of a plasmid designed to disrupt the egI2 gene.
The eg12 gene, encoding EGII (previously referred to as EGIII by some), has been cloned from T. reesei and the DNA sequence published (Saloheimo et a/, 1988, Gene 63:11-21). We have obtained the gene from strain RL-P37 as an approximately 4 kb Pstl-Xhol fragment of genomic DNA inserted between the Pstl and Xhol sites of pUC219. The T. reeseipyr4 gene, present on a 2.7 kb Sall fragment of genomic DNA
obtained from pTpyr2, was inserted into a Sall site within the EGII coding sequence to create plasmid pEGII::P-1 This resulted in disruption of the EGII coding sequence but without deletion of any sequences. The plasmid, pEGII::P-1, can be digested with Hindlll and BamHl to yield a linear fragment of DNA derived exclusively from T. reesei except for 5 bp on one end and 16 bp on the other end both of which are derived from the multiple cloning site of pUC219.
9) Disruption of the eg12 gene of strain P37PACBH67Pyr 1.
Strain P37PAACBH67Pyr 1 was transformed with pEGII::P-1 which had been previously digested with Hindill and BamHl and stable transformants were selected, Total DNA was isolated from transformants and Southern analysis used to identify strains in which the fragment of plasmid DNA containing the pyr4 and egI2 genes had integrated at the egI2 locus and consequently disrupted the EGII coding sequence..
Southern analysis was performed using as a probe an approximately 4 kb Pstl fragment of T. reesei DNA containing the eg12 gene. When DNA isolated from strain P37POO67P-1 was digested with Pstl for Southern analysis the egI2 locus was subsequently visualised as a single 4 kb band on the autoradiograph, However, for a transformant disrupted for the eg12 gene this band was lost and was replaced by two new bands as expected. When the DNA was digested with Bglll or EcoRV the size of the band corresponding to the egI2 gene increased in size by approximately 2.7 kb (the size of the inserted pyr4 fragment) between the untransformed P37PAA67P-1 strain and the transformant disrupted for egI2. This latter transformant, now deleted for the cbhl, cbh2, and egI2 genes, was designated as strain B31. Further Southern analysis confirmed that the pUC DNA fragment of pEGII::P-1 was not incorporated in this strain.
10) Selection of a pyr4 null mutant of strain B31 Spores of the transformant (B3 1) which was deleted for the cbhl, cbh2 and eg12 genes were spread onto medium containing FOA. A pyr4 deficient derivative of this transformant was subsequently obtained using the methods described in section above. This pyr4 deficient strain was designated B31 P6. Southern analysis has shown that a spontaneous deletion had occurred when strain B31 P6 was selected.
This deletion removed the majority of the pyr4 gene which had integrated at the egl2 locus in strain B31, but did not extend into the flanking DNA of the eg12 locus.
However, the amino acid sequence encompassed by scope the present invention can be isolated and/or purified post expression of a nucleotide sequence in its native organism.
Preferably, however, the amino acid sequence encompassed by scope of the present invention may be expressed by a nucleotide sequence in its native organism but 15 wherein the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that organism.
Typically, the nucleotide sequence encompassed by the scope of the present invention is prepared using recombinant DNA techniques (i.e. recombinant DNA).
20 However, in an alternative embodiment of the invention, the nucleotide sequence could be synthesised, in whole or in part, using chemical methods well known in the art (see Caruthers MH et a!.., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T
et al., (1980) Nuc Acids Res Symp Ser 225-232) A nucleotide sequence encoding either a protein which has the specific properties as defined herein or a protein which is suitable for modifidation may be identified and/or isolated and/or purified from any cell or organism producing said protein.
Various methods are well known within the art for the identification and/or isolation and/or purification of nucleotide sequences. By way of example, PCR amplification techniques to prepare more of a sequence may be used once a suitable sequence has been identified and/or isolated and/or purified.
By way of further example, a genomic DNA and/or cDNA library may be constructed using chromosomal DNA or messenger RNA from the organism producing the enzyme. If the amino acid sequence of the enzyme is known, labelled oligonucleotide probes may be synthesised and used to identify enzyme-encoding clones from the genomic library prepared from the organism. Alternatively, a labelled oligonucleotide probe containing sequences homologous to another known enzyme gene could be used to identify enzyme-encoding clones, In the latter case, hybridisation and washing conditions of lower stringency are used, Alternatively, enzyme-encoding clones could be identified by inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming enzyme-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar plates containing a substrate for enzyme (i.e, maltose), thereby allowing clones expressing the enzyme to be identified, In a yet further alternative, the nucleotide sequence encoding the enzyme may he prepared synthetically by established standard methods, e.g, the phosphoroamidite method described by Beucage S.L. et at., (1981) Tetrahedron Letters 22, p 1859-1869, or the method described by Matthes et al., (1984) EMBO J. 3, p 801-805.
In the phosphoroamidite method, oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in appropriate vectors-The nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin, or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) in accordance with standard techniques.. Each ligated fragment corresponds to various parts of the entire nucleotide sequence, The DNA sequence may also be prepared by polymerise chain reaction (PCR) using specific primers, for instance as described in US
4,683,202 or in Saiki R K et al., (Science (1988) 239, pp 487-491), AMINO ACID SEQUENCES
The scope of the present invention also encompasses amino acid sequences of enzymes having the specific properties as defined herein.
As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme".
The amino acid sequence may be preparedlisolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA
techniques..
The protein encompassed in the present invention may be used in conjunction with other proteins, particularly enzymes. Thus the present invention also covers a combination of proteins wherein the combination comprises the protein/enzyme of the present invention and another protein/enzyme, which may be another protein/enzyme according to the present invention. This aspect is discussed in a later section.
Preferably the amino acid sequence when relating to and when encompassed by the per se scope of the present invention is not a native enzyme. In this regard, the term "native enzyme" means an entire enzyme that is in its native environment and when it has been expressed by its native nucleotide sequence..
SEQUENCE IDENTITY OR SEQUENCE HOMOLOGY
The present invention also encompasses the use of sequences having a degree of sequence identity or sequence homology with amino acid sequence(s) of a polypeptide having the specific properties defined herein or of any nucleotide sequence encoding such a polypeptide (hereinafter referred to as a "homologous sequence(s)"). Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences, Here, the term "homology" can be equated with "identity".
The homologous amino acid sequence and/or nucleotide sequence should provide and/or encode a polypeptide which retains the functional activity and/or enhances the activity of the enzyme.
In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85 or 90%
identical, preferably at least 95 or 98% identical to the subject sequence.
Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity..
In the present context, a homologous sequence is taken to include a nucleotide sequence which may be at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85 or 90%
identical, preferably at least 95 or 98% identical to a nucleotide sequence encoding a polypeptide of the present invention (the subject sequence). Typically, the homologues will comprise the same sequences that code for the active sites etc. as the subject sequence. Although homology can also be considered in terms of similarity (i,e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity..
Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
% homology may be calculated over contiguous sequences, i.e.. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues..
Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed.. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.
However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap.
This is the most commonly used gap scoring system High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the Vector NTI AdvanceTM 11 (Invitrogen Corp.). Examples of software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al 1999 Short Protocols in Molecular Biology, 4th Ed - Chapter 18), BLAST 2 (see FEMS
Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana(c~nebi.nlm.nih.gov), FASTA (Altschul et al 1990 J Mol. Biol. 403-410) and AlignX for example. At least BLAST, BLAST 2 and FASTA are available for offline and online searching (see Ausubel et al 1999, pages 7-58 to 7-60).
Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison, Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs Vector NTI programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the default values for the Vector NTI AdvanceTM 11 package.
Alternatively, percentage homologies may be calculated using the multiple alignment feature in Vector NTI AdvanceTM 11 (lnvitrogen Corp.), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).
Once the software has produced an optimal alignment, it is possible to calculate %
homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result, Should Gap Penalties be used when determining sequence identity, then preferably the following parameters are used for pairwise alignment.
FOR BLAST
FOR CLUSTAL DNA PROTEIN
WORD SIZE 2 1 K triple GAP EXTENSION 6.66 0.1 In one embodiment, CLUSTAL may be used with the gap penalty and gap extension set as defined above.
Suitably, the degree of identity with regard to a nucleotide sequence is determined over at least 20 contiguous nucleotides, preferably over at least 30 contiguous nucleotides, preferably over at least 40 contiguous nucleotides, preferably over at least 50 contiguous nucleotides, preferably over at least 60 contiguous nucleotides, preferably over at least 100 contiguous nucleotides, Suitably, the degree of identity with regard to a nucleotide sequence may be determined over the whole sequence.
VARIANTS/HOMOLOGUESIDERIVATIVES
The present invention also encompasses the use of variants, homologues and derivatives of any amino acid sequence of a protein or of any nucleotide sequence encoding such a protein.
Here, the term "homologue" means an entity having a certain homology with the subject amino acid sequences and the subject nucleotide sequences.. Here, the term "homology" can be equated with "identity"
In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90%
identical, preferably at least 95, 96, 97, 98 or 99% identical to the subject sequence..
Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
In the present context, an homologous sequence is taken to include a nucleotide sequence which may be at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90%
identical, preferably at least 95, 96, 97, 98 or 99% identical to a nucleotide sequence encoding an enzyme of the present invention (the subject sequence). Typically, the homologues will comprise the same sequences that code for the active sites etc, as the subject sequence, Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.
Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences, % homology may be calculated over contiguous sequences, i.e one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment.. Typically, such ungapped alignments are performed only over a relatively short number of residues, Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.
However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap.
This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons, For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et al 1984 Nuc. Acids Research 12 p387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST
package (see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4th Ed -Chapter 18), FASTA (Altschul et at, 1990 J. Mol. Biol . 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al, 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program.. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbial Lett 1999 174(2).
247-50; FEMS Microbial Lett 1999 177(1): 187-8 and tatiana@ncbi.nlrnõnih gov).
Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance, An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62, Alternatively, percentage homologies may be calculated using the multiple alignment feature in DNASISTM (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244)..
Once the software has produced an optimal alignment, it is possible to calculate %
homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance, Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine;
and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
Conservative substitutions may be made, for example according to the Table below.
Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.
ALIPHATIC Non-polar G A P
I L V
Polar - uncharged CST M
NQ
Polar -- charged D E
KR
AROMATIC H F W Y
The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i e.. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as 0), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine, Replacements may also be made by unnatural amino acids include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, p-l-phenylalanine*, L-allyl-glycine*, R-alanine*, L--amino butyric acid*, L--amino butyric acid*, L--amino isobutyric acid*, L--amino caproic acid#, 7-amino heptanoic acid*, L-methionine sulfone*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline#, L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid and L-Phe (4-benzyl)*, The notation * has been utilised for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas #
has been utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.
Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or -alanine residues. A further form of variation, involves the presence of one 5 or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues wherein the -carbon substituent group is on the residue's nitrogen atom rather than the -carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ at al., PNAS (1992) 89(20), 10 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.
The nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art These include methylphosphonate and 15 phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of nucleotide sequences of the present 20 invention.
The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof, If the sequence is complementary to a fragment thereof then that 25 sequence can be used as a probe to identify similar coding sequences in other organisms etc.
Polynucleotides which are not 100% homologous to the sequences of the present invention but fall within the scope of the invention can be obtained in a number of 30 ways. Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations In addition, other homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridising to the sequences shown in the sequence listing herein. Such sequences 35 may be obtained by probing cDNA libraries made from or genomic DNA
libraries from other animal species, and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention, Variants and strain/species homologues may also be obtained using degenerate PCR
which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG
Wisconsin PileUp program is widely used.
The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences, Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterised sequences. This may be useful where for example silent codon sequence changes are required to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
Polynucleotides (nucleotide sequences) of the invention may be used to produce a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors, Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.
Polynucleotides such as DNA polynucleotides and probes according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art.. They may also be cloned by standard techniques..
In general, primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time.
Techniques for accomplishing this using automated techniques are readily available in the art, Longer polynucleotides will generally be produced using recombinant means, for example using a PCR (polymerise chain reaction) cloning techniques. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
HYBRIDISATION
The present invention also encompasses sequences that are complementary to the nucleic acid sequences of the present invention or sequences that are capable of hybridising either to the sequences of the present invention or to sequences that are complementary thereto..
The term "hybridisation" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" as well as the process of amplification as carried out in polymerise chain reaction (PCR) technologies.
The present invention also encompasses the use of nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof.
The term "variant" also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequences presented herein,.
Preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridising under stringent conditions (e.g..
500C and 0.2xSSC {1xSSC = 0.15 M NaCl, 0.015 M Na3citrate pH 7..0)) to the nucleotide sequences presented herein.
More preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridising under high stringent conditions (e g.
650C and 0.1 xSSC {1 xSSC = 015 M NaCl, 0..015 M Na3citrate pH 7.0)) to the nucleotide sequences presented herein.
The present invention also relates to nucleotide sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein)-The present invention also relates to nucleotide sequences that are complementary to sequences that can hybridise to the nucleotide sequences of the present invention (including complementary sequences of those presented herein) Also included within the scope of the present invention are polynucleotide sequences that are capable of hybridising to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency-In a preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention, or the complement thereof, under stringent conditions (e.g. 500C and 0 2xSSC)..
In a more preferred aspect, the present invention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention, or the complement thereof, under high stringent conditions (e.g 650C and 0.1xSSC).
MOLECULAR EVOLUTION
As a non-limiting example, it is possible to produce numerous site directed or random mutations into a nucleotide sequence, either in vivo or in vitro, and to subsequently screen for improved functionality of the encoded polypeptide by various means In addition, mutations or natural variants of a polynucleotide sequence can be recombined with either the wildtype or other mutations or natural variants to produce new variants. Such new variants can also be screened for improved functionality of the encoded polypeptide. The production of new preferred variants can be achieved by various methods well established in the art, for example the Error Threshold Mutagenesis (WO 92/18645), oligonucleotide mediated random mutagenesis (US
5,723, 323), DNA shuffling (US 5,605,793), exo-mediated gene assembly WO00/58517. The application of these and similar random directed molecular evolution methods allows the identification and selection of variants of the enzymes of the present invention which have preferred characteristics without any prior knowledge of protein structure or function, and allows the production of non-predictable but beneficial mutations or variants. There are numerous examples of the application of molecular evolution in the art for the optimisation or alteration of enzyme activity, such examples include, but are not limited to one or more of the following.
optimised expression and/or activity in a host cell or in vitro, increased enzymatic activity, altered substrate and/or product specificity, increased or decreased enzymatic or structural stability, altered enzymatic activity/specificity in preferred environmental conditions, e.g. temperature, pH, substrate SITE-DIRECTED MUTAGENESIS
Once a protein-encoding nucleotide sequence has been isolated, or a putative protein-encoding nucleotide sequence has been identified, it may be desirable to mutate the sequence in order to prepare a protein of the present invention..
Mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites.
A suitable method is disclosed in Morinaga at al., (Biotechnology (1984) 2, p646-649)..
5 Another method of introducing mutations into enzyme-encoding nucleotide sequences is described in Nelson and Long (Analytical Biochemistry (1989), 180, p 147-151).
RECOMBINANT
10 In one aspect the sequence for use in the present invention is a recombinant sequence - i_e. a sequence that has been prepared using recombinant DNA
techniques.
These recombinant DNA techniques are within the capabilities of a person of ordinary 15 skill in the art. Such techniques are explained in the literature, for example, J.
Sambrook, E. F.. Fritsch, and T Maniatis, '1989, Molecular Cloning A
Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press., SYNTHETIC
In one aspect the sequence for use in the present invention is a synthetic sequence -i.e. a sequence that has been prepared by in vitro chemical or enzymatic synthesis. It includes, but is not limited to, sequences made with optimal codon usage for host organisms T. reesei, EXPRESSION OF ENZYMES
The nucleotide sequence for use in the present invention may be incorporated into a recombinant replicable vector. The vector may be used to replicate and express the nucleotide sequence, in protein form, in and/or from a compatible host cell Expression may be controlled using control sequences a g, regulatory sequences.
The protein produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. The coding sequences may be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane EXPRESSION VECTOR
The term "expression vector" means a construct capable of in vivo or in vitro expression, Preferably, the expression vector is incorporated into the genome of a suitable host organism. The term "incorporated" preferably covers stable incorporation into the genome.
The nucleotide sequence of the present invention may be present in a vector in which the nucleotide sequence is operably linked to regulatory sequences capable of providing for the expression of the nucleotide sequence by a suitable host organism.
The vectors for use in the present invention may be transformed into a suitable host cell as described below to provide for expression of a polypeptide of the present invention.
The choice of vector e.g. a plasmid, cosmid, or phage vector will often depend on the host cell into which it is to be introduced.
The vectors for use in the present invention may contain one or more selectable marker genes- such as a gene, which confers antibiotic resistance e..g.
ampicillin, kanamycin, chloramphenicol or tetracyclin resistance Alternatively, the selection may be accomplished by co-transformation (as described in WO91/17243) Vectors may be used in vitro, for example for the production of RNA or used to transfect, transform, transduce or infect a host cell, Thus, in a further embodiment, the invention provides a method of making nucleotide sequences of the present invention by introducing a nucleotide sequence of the present invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector, The vector may further comprise a nucleotide sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and plJ702.
REGULATORY SEQUENCES
In some applications, the nucleotide sequence for use in the present invention is operably linked to a regulatory sequence which is capable of providing for the expression of the nucleotide sequence, such as by the chosen host cell. By way of example, the present invention covers a vector comprising the nucleotide sequence of the present invention operably linked to such a regulatory sequence, i.e.. the vector is an expression vector.
The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A
regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
The term "regulatory sequences" includes promoters and enhancers and other expression regulation signals.
The term "promoter" is used in the normal sense of the art, e.g. an RNA
polymerase binding site..
Enhanced expression of the nucleotide sequence encoding the enzyme of the present invention may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and terminator regions, Preferably, the nucleotide sequence according to the present invention is operably linked to at least a promoter.
Other promoters may even be used to direct expression of the polypeptide of the present invention..
Examples of suitable promoters for directing the transcription of the nucleotide sequence in a bacterial, fungal or yeast host are well known in the art.
In one embodiment a suitable promoter may be a cellobiohydrolase promoter, In one embodiment a suitable promoter may be a cellobiohydrolase promoter obtainable (or obtained) from T. reesei.
The promoter can additionally include features to ensure or to increase expression in a suitable host. For example, the features can be conserved regions such as transcription factor binding sites or deleted repressor binding sites.
CONSTRUCTS
The term "construct" - which is synonymous with terms such as "conjugate", "cassette" and "hybrid" - includes a nucleotide sequence for use according to the present invention directly or indirectly attached to a promoter..
An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Sh1-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention. The same is true for the term "fused" in relation to the present invention which includes direct or indirect attachment. In some cases, the terms do not cover the natural combination of the nucleotide sequence coding for the protein ordinarily associated with the wild type gene promoter and when they are both in their natural environment.
The construct may even contain or express a marker, which allows for the selection of the genetic construct.
For some applications, preferably the construct of the present invention comprises at least the nucleotide sequence of the present invention operably linked to a promoter..
HOST CELLS
The term "host cell" - in relation to the present invention includes any T.
reesei cell that comprises either the nucleotide sequence or an expression vector as described above and which is used in the recombinant production of a protein having the specific properties as defined herein, Thus, a further embodiment of the present invention provides T. reesei host cells transformed or transfected with a nucleotide sequence that expresses the protein of the present invention, The use of a T. reesei host cell may provide for post-translational modifications (e.g.
myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
ORGANISM
The term "organism" in relation to the present invention includes T. reesei that comprises the nucleotide sequence coding for the polypeptide according to the present invention and/or products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence according to the present invention when present in the organism, A suitable organism is T reesei.
The term "transgenic organism" in relation to the present invention includes a T
reesei that comprises the nucleotide sequence coding for the polypeptide according to the present invention and/or the products obtained therefrom, and/or wherein a promoter can allow expression of the nucleotide sequence according to the present invention within the organism. Preferably the nucleotide sequence is incorporated in the genome of the organism.
The term "transgenic organism" does not cover native nucleotide coding sequences in their natural environment when they are under the control of their native promoter which is also in its natural environment.
5 Therefore, the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, the nucleotide sequence coding for the polypeptide according to the present invention, constructs according to the present invention, vectors according to the present invention, plasmids according to the present invention, cells according to the present invention, tissues according to the 10 present invention, or the products thereof.
For example the transgenic organism may also comprise the nucleotide sequence coding for the polypeptide of the present invention under the control of a heterologous promoter.
TRANSFORMATION OF HOST CELLSIORGANISM
Filamentous fungi cells may be transformed using various methods known in the art such as a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known.
General teachings on the transformation of fungi are presented in following sections.
TRANSFORMED FUNGUS
The host organism is T. reesei and the like.
Transforming filamentous fungi is discussed in US-A-5741665 which states that standard techniques for transformation of filamentous fungi and culturing the fungi are well known in the art. An extensive review of techniques as applied to N, crassa is found, for example in Davis and de Serres, Methods Enzyme! (1971) 17A: 79-143-Further teachings which may also be utilised in transforming filamentous fungi are reviewed in US-A-5674707.
In addition, gene expression in filamentous fungi is taught in Punt of a!..
(2002) Trends Biotechnol 2002 May;20(5):200-6, Archer & Peberdy Crit Rev Biotechnol (1997) 17(4):273-306-The present invention encompasses the production of transgenic filamentous fungi according to the present invention prepared by use of these standard techniques.
CULTURING AND PRODUCTION
T. reesei host cells transformed with the nucleotide sequence of the present invention may be cultured under conditions conducive to the production of the encoded polypeptide and which facilitate recovery of the polypeptide from the cell(s) and/or culture medium.
In one embodiment the transformed or transfected T reesei cell(s) provided in accordance with the present invention is cultured under selective conditions to allow for selection of the cell(s) transformed or transfected with the lipolytic enzyme as defined herein.
The medium used to cultivate the cell(s) may be any conventional medium suitable for growing the host cell in questions and obtaining expression of the polypeptide, The protein produced by a recombinant cell may be displayed on the surface of the cell..
The protein may be secreted from the host cells and may conveniently be recovered from the culture medium using well-known procedures.
SECRETION
Often, it is desirable for the protein to be secreted from the expression host into the culture medium from where the protein may be more easily recovered, According to the present invention, the secretion leader sequence may be selected on the basis of the desired expression host. Hybrid signal sequences may also be used with the context of the present invention.
Typical examples of heterologous secretion leader sequences are those originating from the fungal amyloglucosidase (AG) gene (glaA - both 18 and 24 amino acid versions e.g. from Aspergillus), the a-factor gene (yeasts e.g. Saccharomyces, Klu.yveromyces and Hansenula) or the -amylase gene (Bacillus).
In one embodiment preferably the signal peptide is that shown in SEQ ID NO: 1 as bold in Figure 15, In one embodiment the signal peptide shown in bold in figure 15 is cleaved off post-translationally to provide a peptide having the sequence shown in SEQ ID NO. 2..
DETECTION
A variety of protocols for detecting and measuring the expression of the amino acid sequence are known in the art.. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FAGS).
A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays.
A number of companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, WI), and US Biochemical Corp (Cleveland, OH) supply commercial kits and protocols for these procedures.
Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3,817,837; US-A-3,850,752; US-A-3,939,350; US-A-3,996,345;
US-A-4,277,437; US-A-4,275,149 and US-A-4,366,241, Also, recombinant immunoglobulins may be produced as shown in US-A-4,816,567..
FUSION PROTEINS
The amino acid sequence for use according to the present invention may be produced as a fusion protein, for example to aid in extraction and purification.
Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA
binding and/or transcriptional activation domains) and (-galactosidase). It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences Preferably, the fusion protein will not hinder the activity of the protein sequence.
ADDITIONAL POIs The sequences for use according to the present invention may also be used in conjunction with one or more additional proteins of interest (POls) or nucleotide sequences of interest (NO[s),.
Non-limiting examples of POIs include: proteins or enzymes involved in starch metabolism, proteins or enzymes involved in glycogen metabolism, acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, -galactosidases, -galactosidases, -glucanases, glucan lysases, endo--glucanases, glucoamylases, glucose oxidases, -glucosidases, -glucosidases, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, phospholipases, galactolipases, lipid acyltransferase, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, rhamno-galacturonases, ribonucleases, thaumatin, transferases, transport proteins, transglutaminases, xylanases, hexose oxidase (D-hexose: 02-oxidoreductase, EC
1.13.5) or combinations thereof. The NOI may even be an antisense sequence for any of those sequences.
The POI may even be a fusion protein, for example to aid in extraction and purification The POI may even be fused to a secretion sequence.
Other sequences can also facilitate secretion or increase the yield of secreted POI
Such sequences could code for chaperone proteins as for example the product of Aspergillus niger cyp B gene described in UK patent application 9821198.Ø
The NOI may be engineered in order to alter their activity for a number of reasons, including but not limited to, alterations which modify the processing and/or expression of the expression product thereof.. By way of further example, the NOl may also be modified to optimise expression in a particular host cell. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites.
The NOI may include within it synthetic or modified nucleotides -- such as methylphosphonate and phosphorothioate backbones.
The NOl may be modified to increase intracellular stability and half-life.
Possible modifications include, but are not limited to, the addition of flanking sequences of the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' 0-methyl rather than phosphodiesterase linkages within the backbone of the molecule LARGE SCALE PRODUCTION/APPLICATION
In one preferred embodiment of the present invention, the lipolytic enzyme is used for large scale applications and/or is produced on a large scale.
The term large scale means in a fermentor or culturing conditions of at least litres.
Preferably the lipolytic enzyme is produced in a quantity of at least 5g per litre of the total cell culture volume after cultivation of the host organism. Preferably, the lipolytic enzyme is produced in a quantity of at least 1Og per litre of the total cell culture volume after cultivation of the host organism. Preferably, the lipolytic enzyme is produced in a quantity of at least 15g per litre of the total cell culture volume after cultivation of the host organism. Preferably, the lipolytic enzyme is produced in a quantity of at least 20g per litre of the total cell culture volume after cultivation of the host organism..
FERMENTATION
The enzymes of the present invention can be produced either by solid or submerged culture, including batch, fed-batch and continuous-flow processes. Culturing is 10 accomplished in a growth medium comprising an aqueous mineral salts medium, organic growth factors, the carbon and energy source material, molecular oxygen, and, of course, a starting inoculum of one or more particular microorganism species to be employed.
15 In addition to the carbon and energy source, oxygen, assimilable nitrogen, and an inoculum of the microorganism, it is necessary to supply suitable amounts in proper proportions of mineral nutrients to assure proper microorganism growth, maximize the assimilation of the carbon and energy source by the cells in the microbial conversion process, and achieve maximum cellular yields with maximum cell density in the 20 fermentation media.
The composition of the aqueous mineral medium can vary over a wide range, depending in part on the microorganism and substrate employed, as is known in the art., The mineral media should include, in addition to nitrogen, suitable amounts of 25 phosphorus, magnesium, calcium, potassium, sulphur, and sodium, in suitable soluble assimilable ionic and combined forms, and also present preferably should be certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine, and others, again in suitable soluble assimilable form, all as known in the art.
30 The fermentation reaction is an aerobic process in which the molecular oxygen needed is supplied by a molecular oxygen-containing gas such as air, oxygen-enriched air, or even substantially pure molecular oxygen, provided to maintain the contents of the fermentation vessel with a suitable oxygen partial pressure effective in assisting the microorganism species to grow in a thriving fashion In effect, by using 35 an oxygenated hydrocarbon substrate, the oxygen requirement for growth of the microorganism is reduced, Nevertheless, molecular oxygen must be supplied for growth, since the assimilation of the substrate and corresponding growth of the microorganisms, is, in part, a combustion process.
Although the aeration rate can vary over a considerable range, aeration generally is conducted at a rate which is in the range of about 0.5 to 10, preferably about 0.5 to 7, - volumes (at the pressure employed and at 25 C.) of oxygen-containing gas per liquid volume in the fermentor per minute. This amount is based on air of normal oxygen content being supplied to the reactor, and in terms of pure oxygen the respective ranges would be about 0..1 to 17, or preferably about 0..1 to 13, volumes (at the pressure employed and at 25 C.) of oxygen per liquid volume in the fermentor per minute.
The pressure employed for the microbial conversion process can range widely.
Pressures generally are within the range of about 0 to 50 prig, presently preferably about 0 to 30 psig, more preferably at least slightly over atmospheric pressure, as a balance of equipment and operating cost versus oxygen solubility achieved.
Greater than atmospheric pressures are advantageous in that such pressures do tend to increase a dissolved oxygen concentration in the aqueous ferment, which in turn can help increase cellular growth rates. At the same time this is balanced by the fact that high atmospheric pressures do increase equipment and operating costs.
The fermentation temperature can vary somewhat, but for filamentous fungi such as Trichoderma reesei the temperature generally will be within the range of about to 40 C, generally preferably in the range of about 25 C to 34 C, depending on the strain of microorganism chosen.
The microorganisms also require a source of assimilable nitrogen. The source of assimilable nitrogen can be any nitrogen-containing compound or compounds capable of releasing nitrogen in a form suitable for metabolic utilization by the microorganism. While a variety of organic nitrogen source compounds, such as protein hydrolysates, can be employed, usually cheap nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea, and various ammonium salts such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, or various other ammonium compounds can be utilized. Ammonia gas itself is convenient for large scale operations, and can be employed by bubbling through the aqueous ferment (fermentation medium) in suitable amounts. At the same time, such ammonia can also be employed to assist in pH control The pH range in the aqueous microbial ferment (fermentation admixture) should be in the exemplary range of about 2.0 to 8.0, With filamentous fungi, the pH
normally is within the range of about 2.5 to 8.0; with Trichoderma reesei, the pH normally is within the range of about 3.0 to 7Ø pH range preferences for certain microorganisms are dependent on the media employed to some extent, as well as the particular microorganism, and thus change somewhat with change in media as can be readily determined by those skilled in the art_ While the average retention time of the fermentation admixture in the fermentor can vary considerably, depending in part on the fermentation temperature and culture employed, generally it will be within the range of about 24 to 500 hours, preferably presently about 24 to 400 hours. Preferably, the fermentation is conducted in such a manner that the carbon-containing substrate can be controlled as a limiting factor, thereby providing good conversion of the carbon-containing substrate to cells and avoiding contamination of the cells with a substantial amount of unconverted substrate, The latter is not a problem with water-soluble substrates, since any remaining traces are readily washed off. It may be a problem, however, in the case of non-water-soluble substrates, and require added product-treatment steps such as suitable washing steps. As described above, the time to reach this level is not critical and may vary with the particular microorganism and fermentation process being conducted. However, it is well known in the art how to determine the carbon source concentration in the fermentation medium and whether or not the desired level of carbon source has been achieved.
Although the fermentation can be conducted as a batch or continuous operation, fed batch operation is much to be preferred for ease of control, production of uniform quantities of products, and most economical uses of all equipment. If desired, part or all of the carbon and energy source material and/or part of the assimilable nitrogen source such as ammonia can be added to the aqueous mineral medium prior to feeding the aqueous mineral medium to the fermentor. Each of the streams introduced into the reactor preferably is controlled at a predetermined rate, or in response to a need determinable by monitoring such as concentration of the carbon and energy substrate, pH, dissolved oxygen, oxygen or carbon dioxide in the off-gases from the fermentor, cell density measurable by light transmittancy, or the like, The feed rates of the various materials can be varied so as to obtain as rapid a cell growth rate as possible, consistent with efficient utilization of the carbon and energy source, to obtain as high a yield of microorganism cells relative to substrate charge as possible In either a batch, or the preferred fed batch operation, all equipment, reactor, or fermentation means, vessel or container, piping, attendant circulating or cooling devices, and the like, are initially sterilized, usually by employing steam such as at about 121 G for at least about 15 minutes. The sterilized reactor then is inoculated with a culture of the selected microorganism in the presence of all the required nutrients, including oxygen, and the carbon-containing substrate. The type of fermentor employed is not critical, though presently preferred is operation under 15L
Biolafitte (Saint-Germain-en-Laye, France)..
The collection and purification of the enzymes of the present invention from the fermentation broth can also be done by procedures known per se in the art. The fermentation broth will generally contain cellular debris, including cells, various suspended solids and other biomass contaminants, as well as the desired enzyme product of the present invention, which are preferably removed from the fermentation broth by means known in the art. Suitable processes for such removal include conventional solid-liquid separation techniques such as, e.g., centrifugation, filtration, dialysis, microfiltration, rotary vacuum filtration, or other known processes, to produce a cell-free filtrate. It may be preferable to further concentrate the fermentation broth or the cell-free filtrate using techniques such as ultrafiltration, evaporation or precipitation. Precipitating the proteinaceous components of the supernatant or filtrate may be accomplished by means of a salt, e.g., ammonium sulfate.
Further purification may optionally be achieved by crystallization or by a variety of chromatographic procedures, e,g., ion exchange chromatography, affinity chromatography or similar art recognized procedures.
The lipase can be further formulated before use in foodstuffs.. The lipase can be in a liquid formulation, dried or granulated.
In one embodiment a carrier can be used, preferably the carrier is wheat or a wheat component In one embodiment the lipase is dried on wheat or dried on one or more wheat components, In one embodiment the lipase is in a liquid formulation suitable for consumption, preferably such liquid composition contains either buffer, salts, sorbitol and/ or glycerol.
In one embodiment the lipase is granulated or co-granulated with other enzymes.
FOOD
The enzyme of the present invention may be used as - or in the preparation of -a food.. Here, the term "food" means food intended for human consumption Also, the term "foodstuff' means a foodstuff intended for human consumption.
The food may be in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration-When used as, or in the preparation of, a food -- such as functional food -the enzyme of the present invention may be used in conjunction with one or more of. a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient FOOD INGREDIENT
The enzyme of the present invention may be used as a food ingredient and/or may be comprised in a food additive composition for humans.
As used herein the term "food ingredient" includes a formulation which is or can be added to functional foods or foodstuffs as a nutritional supplement and/or fiber supplement.
The food ingredient may be in the form of a solution or as a solid --depending on the use and/or the mode of application and/or the mode of administration FOOD SUPPLEMENTS
The composition of the present invention may be -- or may be added to - food supplements for humans FUNCTIONAL FOODS
The composition of the present invention may be - or may be added to -functional foods for humans.
As used herein, the term "functional food" means food which is capable of providing not only a nutritional effect and/or a taste satisfaction to a human, but is also capable of delivering a further beneficial effect to the consumer.
Accordingly, functional foods are ordinary foods that have components or ingredients (such as those described herein) incorporated into them that impart to the food a specific functional - e.g. medical or physiological benefit _ other than a purely nutritional effect to a human.
Although there is no legal definition of a functional food, most of the parties with an interest in this area agree that they are foods marketed as having specific health effects.
Some functional foods are nutraceuticals Here, the term "nutraceutical" means a food which is capable of providing not only a nutritional effect and/or a taste satisfaction, but is also capable of delivering a therapeutic (or other beneficial) effect to the consumer- Nutraceuticals cross the traditional dividing lines between foods and medicine.
Surveys have suggested that consumers place the most emphasis on functional food claims relating to heart disease. Preventing cancer is another aspect of nutrition which interests consumers a great deal, but interestingly this is the area that consumers feel they can exert least control over. In fact, according to the World Health Organization, at least 35% of cancer cases are diet-related Furthermore claims relating to osteoporosis, gut health and obesity effects are also key factors that are likely to incite functional food purchase and drive market development FOOD PRODUCTS
The composition of the present invention can be used in the preparation of food products for humans such as one or more of; jams, marmalades, jellies, dairy products (such as milk or cheese), meat products, poultry products, fish products and bakery products.
By way of example, the enzyme of the present invention can be used as ingredients to soft drinks, a fruit juice or a beverage comprising whey protein, health teas, cocoa drinks, milk drinks and lactic acid bacteria drinks, yoghurt and drinking yoghurt, cheese, ice cream, water ices and desserts, confectionery, biscuits cakes and cake mixes, snack foods, breakfast cereals, instant noodles and cup noodles, instant soups and cup soups, balanced foods and drinks, sweeteners, tacos, tortillas, texture improved snack bars, fibre bars, bake stable fruit fillings, care glaze, chocolate bakery filling, cheese cake flavoured filling, fruit flavoured cake filling, cake and doughnut icing, heat stable bakery filling, instant bakery filling creams, filing for cookies, ready-to-use bakery filling, reduced calorie filling, adult nutritional beverage, acidified soy/juice beverage, aseptic/retorted chocolate drink, bar mixes, beverage powders, calcium fortified soy/plain and chocolate milk, calcium fortified coffee beverage.
A enzyme according to the present invention can further be used as an ingredient in food products such as American cheese sauce, anti-caking agent for grated &
shredded cheese, chip dip, cream cheese, dry blended whip topping fat free sour cream, freeze/thaw dairy whipping cream, freeze/thaw stable whipped tipping, low fat & lite natural cheddar cheese, low fat Swiss style yoghurt, aerated frozen desserts, and novelty bars, hard pack ice cream, label friendly, improved economics &
indulgence of hard pack ice cream, low fat ice cream. soft serve, barbecue sauce, cheese dip sauce, cottage cheese dressing, dry mix Alfredo sauce, mix cheese sauce, dry mix tomato sauce and others.
For certain aspects, preferably the foodstuff is a beverage.
For certain aspects, preferably the foodstuff is a bakery product - such as bread, Danish pastry, biscuits or cookies.
FEED
The enzyme of the present invention may be used as - or in the preparation of -an animal feed or a component thereof. Thus, the present invention also encompasses a feed comprising or made from the enzyme of the present invention, and a process for making same.
DETERGENT
The enzyme of the present invention may be used as - or in the preparation of -- a detergent or a component thereof.. Thus, the present invention also encompasses a detergent comprising or made from the enzyme of the present invention, and a process for making same.
GENERAL RECOMBINANT DNA METHODOLOGY TECHNIQUES
The present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art.
Such techniques are explained in the literature See, for example, J.. Sambrook, E.
F.
Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Lab oratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al.
(1995 and periodic supplements; Current Protocols in Molecular Biology, ch.. 9, 13, and 16, John Wiley & Sons, New York, N.Y.), B. Roe, J. Crabtree, and A. Kahn, 1996, DNA
Isolation and Sequencing. Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis A Practical Approach, Irl Press;
and, D. M
J. Lilley and J- E. Dahlberg, 1992, Methods of Enzymology; DNA Structure Part A.
Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press Each of these general texts is herein incorporated by reference-The invention will now be described, by way of example only, with reference to the following Figures and Examples.
BRIEF DESCRIPTION OF THE FIGURES
The present invention is further illustrated by reference to the accompanying figures in which:
Figure 1 shows a model of the predicted translational modifications in a lipolytic enzyme (sometimes referred to herein as "lipase 3") and shown herein as SEQ
ID No 2; active site residues are shown in a circle - see Ser146, Asp201, His258; there are 7 cysteine residues and 4 are involved in disulfide bonds, shown as a dashed line and as a solid line above the residues, there are 2 sites for N-linked glycosylation, namely N32 & N242 which are shown in bold and are underlined (the latter, namely N242, is near to the His active site). NOTE:
the numbering in this Figure relates to the lipolytic enzyme shown as SEQ ID No. 2 without the signal peptide of 27 amino acids - therefore when referring to the lipolytic enzyme shown in SEQ ID No. 1 (i.e. with the signal peptide) the numbering in respect of the active site residues needs to be adjusted by adding 27 amino acids.
Figure 2 shows a schematic of the method of the present invention, Figure 3 shows a schematic diagram of the Aspergillus tubingensis lipolytic enzyme genomic DNA.
Figure 4 shows the expression construct "ATlipase3Trex'.
Figure 5 shows results of a Lipase Activity Assay performed on supernatant from Trichoderma reeseitransformants.
Figure 6 shows a protein profile by SDS-PAGE performed on supernatant from Trichoderma reesei transformants. The lane indicated with an arrow shows one of the transformants that expresses very high levels of the lipolytic enzyme ("lipase 3") Figure 7 shows a protein profile by SDS-PAGE of supernatant from Trichoderma reesei transformants cultivated in a 3 litre fermentor.. The arrow indicates the lipolytic enzyme ("lipase 3") protein band, Figure 8 shows the yield of the lipolytic enzyme (lipase 3) protein in the fermentation broth when the lipolytic enzyme is expressed in different expression hosts. Yield is expressed as a % increase in yield in each organism:
1 Aspergillus tubingensis;
2 Pichia pastoris, 3 Hansenula polymorpha;
4 Trichoderma reesei..
Figure 9 shows a southern blot showing transformed T. reesei strains that had been transformed with multiple copies of the "lipase 3" lipolytic enzyme gene, the lanes are labelled as follows-M - Untransformed host strain, B - strain transformed using biolistic transformation;
E - strain transformed using electroporation.
Figure 10 shows a protein profile by SDS-PAGE used to characterise the lipolytic enzyme ("lipase 3") expressed in Trichoderma reesei.. Samples from cultures grown in different broths showed lipase 3 protein as double or triple bands.
Figure 11a shows two different UFCs (ultra-filtration concentrates) showing precipitation of the lipolytic enzyme "lipase 3". Less concentrated protein in 1035 UFC shows heavy precipitation and more concentrated protein in 1036 UFC shows very heavy precipitation Precipitation is concentration dependent -the higher the protein concentration the more likely it is to precipitate..
Figure 11 b shows an SDS-PAGE showing the presence of the lipolytic enzyme "lipase 3" protein in the precipitates of Figure 11 a:
Lane I - Centrifuged filtered crude supernatant Lane 2 - Resolubilized pellet from UFC 1035 Lane 3 - Resolubilized pellet from UFC 1036 Figure 12 shows resolubilisation of precipitated lipolytic enzyme ("lipase 3") using different concentrations of sodium phosphate buffer. Samples were at pH 530.
10 Lane 1, Crude, control.
Lanes 2-6. 5 mM, 10 mM, 20 mM, 40 mM & 50 mM sodium phosphate;
Figure 13 shows characterization of recombinant lipolytic enzyme "lipase 3" by MALDI-TOF/MS analysis of glycosylated compared with de-glycosylated lipase 15 The size of N-glycan is about 1384 Da and the lipolytic enzyme (lipase 3) molecules may have glycans attached at N32 site (when considering SEQ ID
No.2);
Figure 14 shows an SDS-PAGE protein analysis of purified lipolytic enzyme 20 (lipase 3) subjected to deglycosylation. Deglycosylation enzyme: Endo-H, 2.0 mgiml Endo-H was added at a ratio of 1:50 and 1:100 (w/w) and incubated for 20 hours at room temperature, pH 5.5..
Lane 1 - Control 25 Lane 2 - 1:100 dilution of supernatant Lane 3 - 1:50 dilution of supernatant The specific activities are shown in table 1 below. N-linked glycosylation has no effect on the specific activity of lipolytic enzyme (lipase 3) protein.
30 Deglycosylating the lipolytic enzyme (lipase 3) did not affect enzyme activity;
Figure 15 shows the amino acid sequence (SEQ ID No.. 1) of a lipolytic enzyme from Aspergillus tubingensis wherein the endogenous signal peptide is shown in bold, Figure 16 shows the amino acid sequence (SEQ ID No. 2) of a lipolytic enzyme from Aspergillus tubingensis which is the same as SEQ ID No, 1 except that the endogenous signal peptide has been removed.
Figure 17 shows the nucleotide sequence encoding an Aspergillus tubingensis lipolytic enzyme (as shown in SEQ ID No, 1) including the signal sequence -the nucleotide sequence is a genomic DNA sequence (and has been designated as SEQ ID No. 3.. The signal sequence is shown in bold and the introns are shown in lower caseõ
Figure 18 shows the nucleotide sequence encoding an Aspergillus tubingensis lipolytic enzyme (as shown in SEQ ID No 2) not including the signal sequence the nucleotide sequence is a genomic DNA sequence (and has been designated as SEQ 1D No. 4, The introns are shown in lower case.
Figure 19 shows the sequence of the cellobiohydrolase 1 gene (cbhl) promoter designated SEQ ID No, 5.
EXAMPLE I
Expression of the Aspergillus tubingensis lipase 3 in Trichoderma reesei 1. Expression Construct & Strain used for Transformation pDONRTM221 plasmid DNA obtained from Invitrogen (catalogue no.12536-017) was used as the donor vector. pDONR221::lip 3 containing the Aspergillus tubingensis lipase 3 genomic DNA (Figure 3) was recombined into the T. reesei gateway destination vector pTrex3G (described in detail in WO 05/001036), resulting in the final expression construct ATlipase3Trex (Figure 4), The expression cassette contained the promoter and terminator regions of the T
reesei cellobiohydrolase 1 (cbhl) gene.. It also contained the Aspergillus nidulans acetamidase, amdS gene as a selectable marker for transformation of T reesei.
The Aspergillus tubingensis lipase 3 genomic DNA encodes a lipase 3 lipolytic enzyme having the amino acid sequence shown in SEQ ID No, 1.
The term "lipase 3" when used herein refers to a lipolytic enzyme comprising the amino acid sequence shown in SEQ ID No. 2, such as the amino acid sequence shown in SEQ ID No.. 1. Seq.. ID No. 1 contains the signal seq and seq. ID No.
2 is the mature lipase protein without the signal sequence.
The strain used for transformation was Trichoderma reesei, a derivative of the non-GMM strain RL-P37 from which the genes encoding the two secreted cellobiohydrolases, CBHI and CBHII, and two of the secreted endoglucanases, EGI
and EGII, have been deleted..
The expression vector pTrex3g.
The following describes the construction of the vector pTrex3g which may be used to express the genes of the present invention.
This vector is based on the E. coli vector pSL1180 (Pharmacia Inc.., Piscataway, NJ, USA) which is a pUC118 phagemid based vector (Brosius, J. (1989) DNA 8:759) with an extended multiple cloning site containing 64 hexamer restriction enzyme recognition sequences. It was designed as a Gateway destination vector (Hartley, J.L., Temple, G.P.. and Brasch, M A, (2000) Genome Research 10:1788-1795) to allow insertion using Gateway technology (Invitrogen) of any desired open reading frame between the promoter and terminator regions of the T. reesei cbhl gene.
It also contains the Aspergillus nidulans amdS gene for use as a selectable marker in transformation of T reesei.
The details of pTrex3g are as follows, The vector is 10.3 kb in size.
Inserted into the polylinker region of pSL1 180 are the following segments of DNA:
1.. A 2,2 bp segment of DNA from the promoter region of the T reesei cbhl gene 2. The 1.7 kb Gateway reading frame A cassette acquired from Invitrogen that includes the attRl and attR2 recombination sites at either end flanking the chloramphenicol resistance gene (CrnR) and the ccdB gene 3. A 336 bp segment of DNA from the terminator region of the T reesei cbh I
gene 4 A 2.7 kb fragment of DNA containing the Aspergillus nidulans amdS gene with its native promoter and terminator regions, 2. Transformation of T. reesei quad delete Host Strain The expression construct, ATlipase3Trex, containing the A, tubingensis lipase 3 gene was transformed into a T reesei strain using electroporation or biolistic transformation by particle bombardment using the PDS-1000 Helium system (BioRad Cat. No. 165-02257). PCR products containing only fungal DNA or the entire expression plasmid were used for generating transformants by biolistic transformation and electroporation..
A. Transformation by electroporation The T reesei host strain to be transformed was grown to full sporulation on PDA
plates for 5 days.. Spores from 2 plates were harvested with 1.2M sorbitol and filtered through miracloth to get rid of the agar. Spores were transferred to a 50 ml falcon tube and washed by repeated centrifugation 5-6 times with 50 ml water. The spores were resuspended in a small volume (less than 2x pellet volume) using 1.2M sorbitol solution. The spore suspension was then kept on ice.. 90 ul of spore suspension was aliqouted into the electroporation cuvette (E-shot, 0,1cm standard electroporation cuvette from lnvitrogen). 10-20 ul DNA construct (plasmid Figure 4 or PCR
product) were added to the spore suspension and electroporation was set at 16kV/cm, 25pF, 500. After electroporation, the spore suspension was left on ice, resuspended in 5 parts 1.OM sorbitol and 1 part YEPD, and allowed to germinate by incubation at with shaking 250 rpm overnight The next day, the germlings were plated in agar plates containing acetamide. Transformants were picked and transferred individually to acetamide agar plates.
B. Transformation by Particle Bombardment (Biolistic transformation) A suspension of spores from a quad deleted strain of T. reesei was prepared.
200u1 of spore suspension was spread onto the center of the minimal medium (MM) acetamide plates. (MM acetamide plates had the following composition. 0.6g/l acetamide;
1.68g/l CsCI; 20g/l glucose; 20g/l KH2PO4, 0..6g/l CaCl2 2H20; 1 ml/l 1000x trace elements solution ; 20g/l Noble agar, and pH5.5.. 1000x trace elements solution contained 5 0g/1 PeSO4 7H20; 1..6g/I MnSO4; -1.4g/l ZnSO4 7H20 and 1 Og/l CoCl2 6H20, The spore suspension was allowed to dry on the surface of MM acetamide medium for 1 hour in the sterile hood. Transformation followed the manufacturer's instruction. 60mg of tungsten particles were placed in a microfuge tube. 1ml of ethanol was added and allowed to stand for 15 seconds The ethanol was removed and the particles were washed three times with sterile dHZO before 250ul of 50% (v/v) sterile glycerol was added. 25u1 of tungsten particle suspension was placed onto a microfuge tube.
While continuously vortexing, the following were added. 5u1 (100-200 ng/u1) of plasmid DNA, 25ul of 2.5M CaCl2 and 10ul of 0.1M spermidine. The particles were centrifuged for 3 seconds, The supernatant was removed and the particles were washed with 200u1 of 100% ethanol and centrifuged for 3 seconds.. The supernatant was removed..
24u1 100% ethanol was added and mixed by pipetting, then 8 ul aliquots of particles were removed and placed in the centre of microcarrier disks that were held in a desiccator.
Once the tungsten/DNA solution had dried the microcarrier disk was placed in the bombardment chamber along with the plate of MM acetamide with spores and the bombardment process was carried out according to the manufacturer's instructions.
After bombardment of the plated spores with the tungsten DNA particles, the plates were incubated at 28C. Transformed colonies were transferred to fresh plates of MM
acetamide medium and incubated at 28C.
3. Growth of transformants in microtiter plates After 5 days of growth on MM acetamide plates, transformants obtained by electroporation or by biolistic transformation and displaying stable morphology were inoculated into 200ul Defined medium with glucose/sophorose in a 96-well microtiter plates. Defined medium with glucose/sophorose (per liter) consists of NH42SO4 5g, PIPPS buffer 33 g, Casamino Acids 9g, KH2PO4 4.5 g, CaC12 (Anhydrous) 1g, MgS04.7H20 1g, pH to 5 50 adjusted with 50% NaOH with milli-Q H2O bring to 966.5 mL. After sterilization, the following were added. Mazu 5 mL, Glucose/Sophrose 60%
26 mL and 400X T reesei Trace Metals 2.5 mL. The microtiter plate was incubated in an oxygen growth chamber at 28 C for 5 days.
Screening by Lipase spot assay & SDS-PAGE
10 Mycelium was removed by centrifugation and the supernatant was analysed for lipase activity using the spot assay. Lipase plate assay is based on the release of fatty acid from the substrate (tributyrin) in the presence of lipase. A pink colour is formed when fatty acid is released and forms a complex with Rhodamine B. The assay plate contained 2..Og Bacto Agar (dissolved by heating for 5 minutes in 100 ml 50mM
15 Sodium phosphate buffer pH 5.5.. The solution was kept at 70 C water bath and while stirring, 0.5m1 2% Rhodamine and 40m1 tributyrin was added. The mixture was subjected to sonication for 2 minutes and 10-15 ml was poured into petri dishes-Holes were punched and the culture supernatant was applied on to the holes..
The plates were incubated at 37 C until a pink colour was formed indicating the presence 20 of lipoly.tic activity. Supernatant (1Oul) from transformants was checked for lipase activity using the spot assay as shown in Figure 5, arrows indicate the appearance of the pink colour after 30 minutes of incubation at 37 C, showing high lipase activity.
The protein profile of those transformants exhibiting high lipase activity was 25 determined by SDS-PAGE using NuPAGE 4-12% polyacrylamide gels and MES as running buffer. Samples of the supernatant were mixed with appropriate volume of 2x sample loading buffer with reducing agent. The gels were stained with Simply blue Safestain (Invitrogen).. In Figure 6 the lane marked with an arrow shows one of the transformants expressing very high levels of lipase 3 appearing as a double band.
30 The rest of the transformants showed distinct and slightly fuzzy single bands.. The best transformant grown in 3 litre fermentor gave a total secreted protein titer of at least 20g/liter and SDS-PAGE analysis showed a broad lipase band (Figure 7).
Large scale Fermentation (14-liters) of lipase 3 Transformants Trichoderma reesei transformants were cultured in fermenters as described in WO
2004/035070. Four different transformants, generated by electroporation, were cultured. Measurement of total protein and lipase activity in culture supernatants, after cell removal, indicated that in excess of 20 grams per liter lipase was present after 160 hours of fermentation.. Two strains, generated by biolistic transformation, were also grown. These showed in excess of 20 grams per liter lipase in the culture supernatant after 160 hours of fermentation.. The amount of lipase 3 produced by these transformants was far in excess of the amount of lipase 3 produced by other microbial host species (Figure 8) Trichoderma reesei transformants were cultured in fermentors. Four different transformants, generated by electroporation, were cultured. Measurement of total protein and lipase activity in culture supernatants, after cell removal, indicated that in excess of 20 grams per liter lipase was present after 160 hours of fermentation. Two strains, generated by biolistic transformation, were also grown. These showed in excess of 20 grams per liter lipase in the culture supernatant after 160 hours of fermentation. The amount of lipase 3 produced by these transformants was far in excess of the amount of lipase 3 produced by other microbial host species (Figure 8).
Solubility of lipase 3 Surprisingly the present inventors have found that Trichoderma reesei is capable of producing lipase 3 at very high levels. Down stream processing of the lipase after fermentation requires concentration of the culture broth 4x by ultrafiltration using a membrane with a 10,000 molecular weight cut-off. Lipase 3 is prone to precipitation as shown in Figure 11 a. Heavy precipitation is observed in the more concentrated UFC. The presence of lipase 3 protein in the precipitates was confirmed by SDS-PAGE.
Figures 11 a and 11 b show that precipitation of lipase 3 is concentration dependent.
Figure 11a shows two different ultra-filtration concentrates (UFCs) showing precipitation of lipase 3. 1035 UFC contains less concentrated protein and shows heavy precipitation. 1036 UFC contains more concentrated protein and shows very heavy precipitation..
Figure 11 b shows an SDS-PAGE showing the presence of lipase 3 protein in the precipitates shown in Figure 11 a.
Lane 1 Centrifuged filtered crude supernatant Lane 2 Resolubilized pellet from UFC 1035 Lane 3 Resolubilized pellet from UFC 1036 For resolubilisation of lipase precipitates, stock buffer was used. Stock buffer consists of 1..0 M Na-Phosphate buffer, pH 8.0 Na2HP04.2H20 (177.99 g) added to 900 ml of DI water. To 10.0 g samples of the crude lipase 3 UFC 1036, pH 4.44 was added 50, 100, 200, 400, and 500 lil of buffer stock solution to a final concentration of 5, 10, 20, 40, and 50 mM Na-Phosphate respectively. All the vials were mixed and left at room temperature for a few minutes.
As shown in Figure 12, the addition of Na-phosphate above 40 mM brought the precipitated lipase 3 into solution. The resulting solution was clear and free from any solid matter. The pH of the samples was measured at pH 5.3 (at 50 mM Na-P
concentration).
Characterization of recombinant lipase 3 by MALDI-TOF/MS Analysis &
Proteolytic digestion Lipase 3 expressed by T. reesi was purified from the culture supernatant using a combination of ion exchange and hydrophobic interaction chromatography.
Deglycosylation was carried out using endoglycosidase H in reaction buffer consisting of 50 mM Sodium Citrate, pH 5.5. Endoglycosidase H (2.0 mg/mL), was added to lipase 3 at a protein ratio from 1/1000 to 11100 (w/w).. The deglycosylation reaction was performed at 37 C for 3 hours The reacted protein sample was put on a ZipTip (Micro C4 reversed-phase column) (Millipore, Bedford, MA) for sample cleanup prior to the MALDI-TOF/MS analysis.. The cleanup process was performed to desalt the samples. At least five cycles of wash solution consisting of 5% methanol in 0.1%
TFA/water wash was used. Afterwards the samples were eluted for mass spectrometry MALDI-TOF/MS Analysis of Intact Protein The desalted protein sample was prepared for MALDI-TOF/MS analysis by co-crystalizing an equal volume (1 L) of sample with Sinapinic acid matrix (saturated in 50% acetonitrile, 0.1% formic acid) using the dried droplet method. Protein mass spectra were obtained using a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA). The MS instrument settings were as follows for the 20000-80000 m/z range: linear mode of operation, delayed extraction mode, positive polarity, 25 kV acceleration voltage, 93% grid voltage, and 750 nsec extraction delay time. 300-laser shots/spectrum and BSA was used as external calibrant Proteolytic Digestion & N-glycosylation site(s) Mapping by LCIMS/MS Analysis All Endo-H treated liquid samples were precipitated with 10% TCA followed by the reduction reactions with 20 mM DTT @ 50 C for 15-20 min. The alkylation reaction was also performed with 55 mM iodoacetamide. The alkylation reaction was allowed to proceed in the dark for 45 min at room temp. Proteolytic digestions were performed by incubation with various proteases in 25 mM ammonium bicarbonate for 4 hr at 37 C (enzyme to substrate ratio was 1:20):. Peptide mapping carried out using 3 different proteolytic digestions (trypsin, chymotrypsin, Endoproteinase GIuC) on the glycosylated lipase confirmed the absence of protein modification by truncation and the presence of an intact lipase protein with authentic N- and C-terminal ends.
Lipase 3 has 2 potential N-glycosylation sites at N32 and N242. Peptide mapping was carried out for both lipase 3 (untreated) and Endo-H treated. To determine the site of N-glycosylation, MS and MS/MS data were acquired using the Surveyor""
LC
system coupled to the LCQ Advantage or LCQ Deca XP (Thermo Finnigan, San Jose, CA). The HPLC gradient was programmed from 0% to 70% B over 50 minutes, Solvent A: 0.1 % TFA in water and Solvent R. 0 08% TFA in acetonitrile.. Data Processing was performed using TurboSEQUEST and Xcalibur (ThermoFinnigan, San Jose, CA).
As shown in Figure 13, SDS-PAGE showed the recombinant lipase secreted by Trichaderma appeared as a double band prior to endoglycosidase H treatment.
MALDI-TQF/MS analysis showed that the higher molecular weight species was a glycosylated form of lipase 3 with a molecular weight of 30,236 Dalton and the lower molecular weight species was a de-glycosylated form with molecular weight of 29,210 Dalton. Experimentally deglycosylated lipase samples were generated using endoglycosidase H.. The molecular weights observed after endoglycosidase H
treatment were 29,035 Dalton (presumed to be a non-glycosylated form), 29,219 (presumed to be a form of lipase 3 with 1 N-linked N-acetylglucosamine at position N32 or N242) and 29,422 (presumed to be a form of lipase 3 with 2 N-aceyltglucosamine residues attached to the protein backbone at positions N32 and N242) The glycan chain that is present prior to de-glycosylation with endoglycosidase H has a molecular weight of approximately 1384 daltons with the majority of lipase molecules having glycan attached only to the N32 site (numbered in accordance with SEQ ID No. 2) Specific activity of the lipolytic enzyme lipase 3 produced in T. reesei using different substrates The purified lipase 3 was subjected to deglycosylation by treatment with endoglycosidase H. The untreated and the deglycosylated samples were characterised by measurement of specific activities using both short chain (C4) and long chain (C 18) substrates-As shown in Figure 14 and Table 2 the presence of glycan side chains has no effect on the specific activity of the protein.
Table 2 shows the results of the deglycosylation experiments. The sample was Purified Trichoderma lipase 3 and the deglycosylation enzyme was Endo-H..
Specific activity, %
Sample LIPU/ml LUSol/ml (Short chain, C4) Lon chain, C1$
Control lane 1) 100 100 1:100 Endo-H (lane 2) 99.4 103 1.50 Endo-H lane 3) 97.7 105 Southern Blot Analysis Figure 9 shows a southern blot showing transformed T reesei strains that had been 10 transformed with multiple copies of the "lipase 3" lipolytic enzyme gene, the lanes are labelled as follows:
M - Untransformed host strain, B - strain transformed using biolistic transformation, E - strain transformed using electroporation;
Expressions studies Lipase from Thermom,yces lanuginosus was cloned and expressed in T. reesei Transformants were isolated and the best lipase producer was tested in 14-Liter scale fermentation to investigate whether Trichoderma reesei is a suitable host strain for expression of other lipases. The total protein at the end of (200 hours) fermentation was estimated to be in excess of 20 g/L as shown in Figure 14.
SDS-PAGE showed that lipase was the dominant protein produced. Lipase activity measured at the end of fermentation was 30,000 UImL using DGGR assay. The broth was filtered and concentrated to UFC at high concentration the lipase appeared to precipitate. It was easily brought back into solution by diluting in buffer or salt Expression studies Table 3 provides the results of a series of expression studies.
The present inventors have surprising found that the lipolytic enzyme "Lipase 3"
produced in Trichoderma reesei is less glycosylated (in particular less N-linked glycosylation) compared with the same enzyme produced in other organisms..
Table 3. Comparison with other hosts used to express lipase 3 from Aspergillus tubingensis Expression Host Glycosylation at N32 Protein Quality & N242 sites Aspergillus Hyperglycosylation Reduced Activity tubigensis 3M (overglycosylation 10%) (both N32 & N242 sites) Pichia pastoris Hyperglycosylation Activity > activity seen with A.
GS 115 (both N32 & N242 tubigensis recombinant lipase strain sites) 0- 1 cans Hansenula Glycosylation (both N32 Activity > activity seen with A.
polymorpha RB1 1 & N242 sites) tubigensis recombinant lipase strain Glycan 1%
T. reesei Majority of N- Activity is same as native lipase 3 glycosylation at N32 from non-recombinant Aspergillus 1 M341 strain The expression host cells suitable for use in the present invention may be a strain of T. reesei in which the genes encoding cellobiohydrolase I (CBHI, Cel7a), cellobiohydrolase II (CBH1I, Cel6a), endoglucanase I (EGI, Cel7b), and endoglucanase II (EGII, Cel5a) have been inactivated by deletion or disruption using molecular genetic techniques. This strain (a quad delete strain) is useful as a host for over-expression of genes encoding other T. reesei secreted proteins.
Preferably host cells suitable for use in the present invention may be derived from a Trichoderma reesei cell of the strain RL-P37 using the methods described in WO
20051001036 and US28026376 Al.
A Treesei strain suitable for use in the present invention may be derived from the publicly available strain of T,reesei RL-P37. T reesei strain RL-P37 may be modified to form T reesei strain 1A52 as described in WO 051001036. T reesei strain 1A52 may be modified as described in US 20080026376 to form a T reesei cell usable in the present invention.
RL-P37 may be modified to form T reesei strain 1A52 as described below..
The T. reesei host strain used may be derived from the publicly available strain RL-P37 which has previously been used to manufacture commercial cellulase preparations by Genencor International, Inc The derivation and characterisation of this strain has been published previously (Sheir-Neiss, G. and Montenecourt, BS. (1 984) Appl. Microbiol. Biotechnol. 20.46-53; US Patent 4,797,361). It is a cellulase over-producing mutant strain which has been obtained as a result of several mutagenesis steps from the wild-type strain (QM6a).
1) Isolation of a pyr4 mutant strain.
In order to prepare strain RL-P37 for transformation with plasmid DNA it was necessary to isolate a derivative having a null mutation in the p.yr4 gene..
The pyr4 gene encodes orotidine-5'-monophosphate decarboxylase, an enzyme required for the biosynthesis of uridine. The toxic inhibitor 5-fluoroorotic acid (FOA) is incorporated into uridine by wild-type cells and thus poisons the cells.
However, cells defective in the pyr4 gene are resistant to this inhibitor but require uridine for growth.
It is, therefore, possible to select for pyr4 mutant strains using FOA. In practice, spores of T. reesei strain RL-P37 were spread on the surface of a solidified medium containing 2 mg/ml uridine and 1.2 mg/ml FOA Spontaneous FOA-resistant colonies appeared within three to four days. FOA-resistant mutants which required uridine for growth were identified In order to identify those mutants which specifically had a defective pyr4 gene protoplasts were generated and transformed with a plasmid containing a wild-type pyr4 gene (Smith, J L., Bayliss, F.T. and Ward, M..
(1991) Currõ
Genet. 19:27-33). Following transformation protoplasts were plated on medium lacking uridine. Subsequent growth of transformed colonies demonstrated complementation of a defective pyr4 gene by the plasmid-borne pyr4 gene. In this way strain GC69 was identified as a p.yr4 mutant of strain RL-P37.
2) Construction of a plasmid designed to delete the CBHI encoding gene.
The cbhl gene, encoding the CBHI protein, was cloned from the genomic DNA of strain RL-P37 by hybridization with an oligonucleotide probe designed on the basis of the published sequence for this gene (Shoemaker, S., Schweickart, V.., Ladner, M.., Gelfand, D., Kwok, S., Myambo, K.. and Innis, M. (1983) Biotechnology 1:691-696).
The cbhl gene resides on a 6.5 kb Psfl fragment and was inserted into the Pstl site of pUC4K (Pharmacia Inc., Piscataway, NJ, USA) replacing the kanamycin-resistance gene of this vector The resulting plasmid, pUC4K::cbh 1, was then cut with Hindlll and the larger fragment was isolated and religated to give pUC4K::cbhlAH/H..
This procedure removed the entire cbhl coding sequence and approximately 12 kb of 5' and 1.5 kb of 3' flanking sequences. Approximately I kb of flanking DNA
remained from either end of the original Pst/ fragment. The T, reeseipyr4 gene was cloned as a 6.5 kb Hindlll fragment of genomic DNA in pUCl8 to form pTpyr2 (Smith, J_L., Bayliss, F.T.. and Ward, M.. (1991) Curr. Genet.. 19:27-33). The plasmid pUC4K.:cbh1L1H/H
was cut with Hindlll and the ends were dephosphorylated with calf intestinal alkaline phosphatase This DNA was ligated with the 6.5 kb Hindlll fragment containing the p,yr4 gene to give pACBHlp.yr4.
Digestion of pACBHlp.yr4 with EcoRl liberated a larger fragment which consisted of flanking regions of the Ohl locus at either end with the pyr4 gene replacing the cbhl coding sequence in the centre. The only DNA on this fragment which was not derived from T. reesei was a 21 bp fragment derived from the multiple cloning site of pUC4K.
3) Deletion of the cbhl gene of T, reesei.
Protoplasts isolated from mycelium of strain GC69 were transformed with EcoRl digested plasmid p CBHlpyr4 using methods outlined by Smith et al., 1991.
Stable transformants were obtained and those from which the cbh 1 gene had been deleted were identified as described below, Total DNA was isolated from the transformants, digested with Pstl, subjected to agarose gel electrophoresis and blotted to a membrane filter The filter was then hybridised with P32 labelled pACBHlpyr4 and the pattern of hybridisation observed by autoradiography. This probe hybridised with the native cbh 1 and pyr4 genes in an untransformed strain. In one transformant (strain P37PACBHI) a pattern of hybridisation was observed which would be predicted if a double cross-over integration event had occurred. That is, the cbh 1 gene had been deleted by integration of a single copy of the larger EcoRl fragment obtained from pACBHlpyr4 at the cbhl locus of strain RL-P37.
Southern analysis was also performed as above except that the probe used was radiolabelled plntCBHI. This plasmid consists of a pUC vector containing a 2 kb BgIII
fragment from the cbhl locus within the region that was deleted in pUC4K::cbh1AH/H.
This plasmid hybridised to the cbh-f locus of strain GC69 but did not hybridise to DNA
from strain P37PACBHI.. This confirms that the cbh 1 gene had been deleted and that the pUC DNA fragment from pACBHIpyr4 had not been incorporated by the deleted strain..
Analysis of secreted proteins by separation on isoelectric focusing gels showed that the CBHI protein was not produced by strain P37PACBHI..
4) Generation of a pyr4 null mutant of P37PACBHI.
Spores of the transformant (P37PACBHI) which was deleted for the cbhl gene were spread onto medium containing 1FOA. A pyr4 deficient derivative of this transformant was subsequently obtained using the methods described in section above, This pyr4 deficient strain was designated P37PACBHIPyr 26. Southern analysis has shown that a spontaneous deletion had occurred when strain P37PACBHIPy(26 was selected. This deletion completely removed the pyr4 gene which had integrated at the cbh 1 locus in strain P37P CBHI, as well as flanking DNA from the cbh 1 locus beyond the extent of the 6.5 kb Pstl fragment of genomic DNA which was originally cloned, 5) Construction of a vector designed to delete the cbh2 gene.
The cbh2 gene of Treesei, encoding the CBHII protein, has been cloned as a 4.1 kb 5 EcoRl fragment of genomic DNA (Chen at at., 1987, Biotechnology 5:274-278).
This.4..1 kb fragment was inserted between the EcoRl sites of pUC4XL. The latter plasmid is a pUC derivative (constructed by R. M. Berka, Genencor International Inc.) which contains a multiple cloning site with a symetrical pattern of restriction endonuclease sites arranged in the order shown here. EcoRl, BamHl, Sacl, Smal, 10 Hindlll, Xhol, Bglll, Clal, Bglll, Xhol, Hindll 1, Smal, Sacl, BamHl, EcoRl. The plasmid, pPOCBHII was constructed in which a 17 kb central region of this cbh2 clone, between a Hindlll site (at 74 bp 3' of the CBHII translation initiation site) and a Clal site (at 265 bp 3' of the last codori of CBHII), has been removed and replaced by a 1.6 kb Hindlll-Clal DNA fragment containing the T, reesei pyr4 gene obtained as follows..
15 The T. reesei pyr4 gene was excised from pTpyr2 on a 1.6 kb Nhel-Sphl fragment and inserted between the Sphl and Xbal sites of pUC219 (derived from pUC119 by expanding the multiple cloning site to include restriction sites for Bglll, Clal and Xhol, Wilson et al.., 1989, Gene 77:69 78) to create p219M (Smith at al., 1991, Curr, Genet, 19:27-33) The pyr4 gene could then be removed as a Hindlll-Clal fragment having 20 seven bp of DNA at one end and six bp of DNA at the other end derived from the pUC219 multiple cloning site and inserted into the Hindlll and Clal sites of the cbh2 gene to form the plasmid pPACBHII, Digestion of this plasmid with EcoRl liberated a fragment having 0.7 kb of flanking 25 DNA from the cbh2 locus at one end, 1.7 kb of flanking DNA from the cbh2 locus at the other end and the T. reesei pyr4 gene in the middle. The only DNA in this fragment which was not derived from T, reesei was the 6 bp and 7 bp fragments of the pUC219 multiple cloning site at either end of the pyr4 gene.
30 6) Deletion of cbh2 gene from strain P37Pi CBHIPyC26 Protoplasts of strain P37P CBHIPyr-26 were generated and transformed with EcoRl digested pPACBHII according to the methods outlined in 3 above. Stable transformants were cultured in shake flasks and the protein in the culture supernatants was examined by isoelectric focussing One transformant (designated P37PAACBH67) was identified which did not produce any CBHII (nor CBHI) protein.
DNA was extracted from strain P37PAACBH67, digested with EcoRl and Asp718, and subjected to agarose gel electrophoresis.. The DNA from this gel was blotted to a membrane filter and hybridized with 32P labelled pPACBHII. The 41 kb EcoRl fragment containing the wildtype cbh2 gene was observed in the DNA from an untransformed control strain In contrast, in strain P37PAACBH67 the single 4.1 kb band was eliminated and replaced by two bands of approximately 0.9 and 3..1 kb. This is the expected pattern if a single copy of the larger EcoRl fragment from pPACBHII
had integrated precisely at the cbh2 locus and deleted the cbh2 gene, The same DNA samples were also digested with EcoRl and Southern analysis was performed as above,, In this example the probe was 32P labelled plntCBHlI.
This plasmid contains a portion of the cbh2 gene coding sequence from within that segment of cbh2 DNA which was deleted in plasmid pPACBHII. No hybridization was seen with DNA from strain P37PACBH67 confirming that the cbh2 gene was deleted and that the pUC plasmid fragment of pPACBHII had not been incorporated by this strain.
7) Selection of a pyr4 null mutant of strain P37PAACBH67.
Spores of the transformant (P37PAACBH67) which was deleted for both the cbhl and cbh2 genes were spread onto medium containing FOA. A pyr4 deficient derivative of this transfornant was subsequently obtained using the methods described in section 1 above. This pyr4 deficient strain was designated P37PAACBH67Pyrr1. Southern analysis has shown that a spontaneous deletion had occurred when strain P37PAACBH67Pyrr1 was selected.. This deletion completely removed the pyr4 gene which had integrated at the cbh2 locus in strain P37PACBH67, as well as flanking DNA from the cbh2 locus beyond the extent of the 4.1 kb EcoRl fragment of genomic DNA which was originally cloned. The short (6 bp and 7 bp) fragments of DNA
derived from the pUC219 multiple cloning site which were present at either end of the pyr4 gene would also have been removed from the genome by this deletion 8) Construction of a plasmid designed to disrupt the egI2 gene.
The eg12 gene, encoding EGII (previously referred to as EGIII by some), has been cloned from T. reesei and the DNA sequence published (Saloheimo et a/, 1988, Gene 63:11-21). We have obtained the gene from strain RL-P37 as an approximately 4 kb Pstl-Xhol fragment of genomic DNA inserted between the Pstl and Xhol sites of pUC219. The T. reeseipyr4 gene, present on a 2.7 kb Sall fragment of genomic DNA
obtained from pTpyr2, was inserted into a Sall site within the EGII coding sequence to create plasmid pEGII::P-1 This resulted in disruption of the EGII coding sequence but without deletion of any sequences. The plasmid, pEGII::P-1, can be digested with Hindlll and BamHl to yield a linear fragment of DNA derived exclusively from T. reesei except for 5 bp on one end and 16 bp on the other end both of which are derived from the multiple cloning site of pUC219.
9) Disruption of the eg12 gene of strain P37PACBH67Pyr 1.
Strain P37PAACBH67Pyr 1 was transformed with pEGII::P-1 which had been previously digested with Hindill and BamHl and stable transformants were selected, Total DNA was isolated from transformants and Southern analysis used to identify strains in which the fragment of plasmid DNA containing the pyr4 and egI2 genes had integrated at the egI2 locus and consequently disrupted the EGII coding sequence..
Southern analysis was performed using as a probe an approximately 4 kb Pstl fragment of T. reesei DNA containing the eg12 gene. When DNA isolated from strain P37POO67P-1 was digested with Pstl for Southern analysis the egI2 locus was subsequently visualised as a single 4 kb band on the autoradiograph, However, for a transformant disrupted for the eg12 gene this band was lost and was replaced by two new bands as expected. When the DNA was digested with Bglll or EcoRV the size of the band corresponding to the egI2 gene increased in size by approximately 2.7 kb (the size of the inserted pyr4 fragment) between the untransformed P37PAA67P-1 strain and the transformant disrupted for egI2. This latter transformant, now deleted for the cbhl, cbh2, and egI2 genes, was designated as strain B31. Further Southern analysis confirmed that the pUC DNA fragment of pEGII::P-1 was not incorporated in this strain.
10) Selection of a pyr4 null mutant of strain B31 Spores of the transformant (B3 1) which was deleted for the cbhl, cbh2 and eg12 genes were spread onto medium containing FOA. A pyr4 deficient derivative of this transformant was subsequently obtained using the methods described in section above. This pyr4 deficient strain was designated B31 P6. Southern analysis has shown that a spontaneous deletion had occurred when strain B31 P6 was selected.
This deletion removed the majority of the pyr4 gene which had integrated at the egl2 locus in strain B31, but did not extend into the flanking DNA of the eg12 locus.
11) Construction of a plasmid designed to delete the egll gene.
The egll gene of T. reese! has been cloned and the DNA sequence of the gene has been published (Penttila et a/, 1986, Gene 45;253-263; van Arsdell et al., 1987, Bioltechnology 5:60-64).. We have obtained this gene from T. reese! strain RL-P37 as a 42 kb Hindlll fragment of genomic DNA inserted at the Hindlll site of pUC100 (a derivative of pUC18 with an oligonucleotide inserted into the multiple cloning site adding restriction sites for Bglll, C/a! and Xhol) to give pUCEGI, An approximately 1 kb EcoRV fragment extending from a position close to the middle of the EGI
coding sequence to a position beyond the 3' end of the coding sequence was removed and replaced by a 3.5 kb Scal fragment of T reesei DNA containing the pyr4 gene obtained from pTpyr2. The resulting plasmid was called pPAEGI
The plasmid, pPAEGI could be digested with Hindlll to release a DNA fragment comprising only T, reesei genomic DNA having a segment of the egli gene at either end and the pyr4 gene, replacing part of the EGI coding sequence, in the centre.
The egll gene of T. reese! has been cloned and the DNA sequence of the gene has been published (Penttila et a/, 1986, Gene 45;253-263; van Arsdell et al., 1987, Bioltechnology 5:60-64).. We have obtained this gene from T. reese! strain RL-P37 as a 42 kb Hindlll fragment of genomic DNA inserted at the Hindlll site of pUC100 (a derivative of pUC18 with an oligonucleotide inserted into the multiple cloning site adding restriction sites for Bglll, C/a! and Xhol) to give pUCEGI, An approximately 1 kb EcoRV fragment extending from a position close to the middle of the EGI
coding sequence to a position beyond the 3' end of the coding sequence was removed and replaced by a 3.5 kb Scal fragment of T reesei DNA containing the pyr4 gene obtained from pTpyr2. The resulting plasmid was called pPAEGI
The plasmid, pPAEGI could be digested with Hindlll to release a DNA fragment comprising only T, reesei genomic DNA having a segment of the egli gene at either end and the pyr4 gene, replacing part of the EGI coding sequence, in the centre.
12) Deletion of the eall gene in strain B31 P6.
Two forms of pPAEGI were constructed which differed only in the orientation of the pyr4 gene with respect to the egli flanking regions. Strain B31 P6 was transformed with a mixture of both forms of the plasmid after they had been digested with Hindlll, Total DNA was extracted from stable transformants, digested with Hindlll and subjected to Southern analysis- The probe used was radio labelled pUCEGI.
Hybridisation was observed to a 4.2 kb fragment of DNA from strain B31 P6 representing the undeleted egl1 gene A transformant (strain 1A52) was identified in which this 4..2 kb was no longer present but had been replaced by a fragment of approximately 6.8 kb.. This is the pattern expected if the larger Hindlll fragment from pP EGI had integrated precisely as predicted at the egl-1 locus leading to deletion of part of the EGI coding sequence and insertion of pyr4 at this position Using a pLJC
plasmid as a probe for Southern analysis it was confirmed that the pUC DNA
fragment of pPL1EGl had not been incorporated in strain 1A52 T.reesei strain 1A52 may be modified to form a Treesei cell usable in the present invention.
Assay for triacyiglycerol hydrolysing activity (classified as E.C. 3.1.1.3) LIPU/LUSOI assays for Triacylglycerol hydrolysing activity Determination of lipase activity by LIPU is carried out by enzymation of an emulsion of tributyrylglycerol, Enzymatic hydrolysis of lipids liberates free fatty acids.. By continuous titration of the liberated free fatty acid, the lipase activity is determined from the consumption of base. 1 LIPU (lipase unit) (also called 1 unit herein) is defined as the amount of enzyme, which releases 1 pmol free fatty acid per minute at the given assay conditions..
Enzyme samples were dissolved in demineralised water. The titrant was 0,05 M
NaOH. The substrate was a homogenised emulsion of 5% (v/v) tributyrineglycerol (Merck, item no, 101958), 0.10% (wlv) gum arabic (Sigma, item no.. G9752), 7.5%
(w/v) glycerol (Merck, item no, 1.04092), 51 mM NaG! (p.a, Merck, item no.
1.06404), 0.50 mM KH2PO4 (p..a. Merck, item no, 1.04873). Reaction pH was 5,5 and reaction temperature was 30 C.. 2,00 mL sample was added to 25.0 mL substrate acclimatised to the reaction temperature. Activity was calculated from the slope of a linear titration curve with consumption of titrant plotted against reaction time.
Substrates used are Tributyrin (Lipu) and Sunflower oil (LUSol), DGGR assay for Triacylglycerol hydrolysing activity This assay was used to measure Thermomyces lanuginosus lipase expressed in Trichoderma.
5 The substrates and buffers used in this assay were the following:
3.32 mM 1,2-Di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorufin ester) dissolved in DMSO (substrate, 2.5mg/mL), A stock solution consisting of 50 mg substrate added to 20mL DMSO is 10 sonicated, aliquoted and stored at -80 deg C until use. Buffer used is 0.5 M HEPES
pH 8 + 60gpg 3:1 Ca:Mg Water hardness (see CAM300) and 4% Gum Arabic..
Lipase enzyme stock (1 mg/L) is used as standard.
For the assay, A 50mL assay buffer is prepared by adding 5mL HEPES + Hardness 15 and 25mL 4% Gum Arabic to 10mL water. The assay buffer is incubated to desired assay temperature (typically 25 deg C). 1 OuL of enzyme samples are added into well plate at assay temperature. 1-10ppm of active enzyme in sample recommended.
For best results match unknown concentration to +1- two-fold activity of standard-20 The background rate (no enzyme, i.e. assay buffer) is determined.
10mL of 3..32mM DGGR substrate in DMSO is added to assay buffer and mixed well using a vortex. 200uL substrate in assay buffer is added immediately to enzyme samples in microplate from a reagent reservoir using a multi-channel pipet.
The microliter plate is mixed well and immediately transfer ed to plate reader. OD
at 580 25 nm for up to 10 min is measured at desired assay temperature (typically 25 deg C).
Calculation of the enzyme activity is done by subtracting the background rate from unknown and standard to obtain difference rates,. Determine the ratio of the difference rates for unknown to standard and multiply by the standard concentration. All dilutions are included in the calculation:
unknown concentration = (unknown Rate * standard concentration) / standard rate..
Determination of triacyiglyceride lipase activity: assay based on triglyceride (tributyrin) as substrate (LIPU):
Lipase activity based on tributyrin is measured according to Food Chemical Codex, Forth Edition, National Academy Press, 1996, p 803, with the modifications that the sample is dissolved in deionized water instead of glycine buffer, and the pH
stat set point is 5.5 instead of 7..
1 LIPU is defined as the quantity of enzyme which can liberate 1 mol butyric acid per minute under assay conditions.
Assay for Phospholipase activity phospholipase Al activity (E,C. 3.1.1.32) phospholipase A2 activity (E C, 3,1.1.4) phospholipase B activity (E..C. 31.1.5) Substrate 1.75% L- Plant Phosphatidylcholin 95% (441601, Avanti Polar Lipids), 6.3 %
Triton X-100 (#T9284, Sigma) and 5 mM CaCl2 dissolved in 50 mM HEPES buffer pH 70..
Assay procedure Samples, calibration and control were diluted in 10 mM HEPES pH 7 .0, 01%
Triton X-100 (#T9284, Sigma). Analysis was carried out using a Konelab Autoanalyzer (Thermo, Finland). The assay was run at 30C.. 34 pL substrate was thermostated for 180 seconds, before 4 pL sample was added. Enzymation lasted for 600s. Amount of free fatty acid liberated during enzymation was measured using the NEFA C kit (999-75406, WAKO, Germany), 56 pL NEFA A was added and the mixture was incubated for 300 s. Afterwards 113 pL NEFA B was added and the mixture was incubated for 300s. Afterwards 113 lit NEFAB was added and the mixture was incubated for 300s..
OD 520 nm was then measured. Enzyme activity LATU (pmol FFA/minmL) was calculated based on a standard enzyme preparation Assay for glycolipase (galactolipase) activity.
Substrate 135% Didalactosyldiglyceride (DGDG, Purified from wheat lipids), 6.3 % Triton (#T9284, Sigma) and 5 mM CaC12 dissolved in 50 mM HEPES buffer pH 7.0 Assay procedure Samples, calibration and control were diluted in 10 mM HEPES pH 7,0, 0.1%
Triton X-100 (#T9284, Sigma). Analysis was carried out using a Konelab Autoanalyzer (Thermo, Finland). The assay was run at 30C. 34 pL substrate was thermostated for 180 seconds, before 4 pL sample was added Enzymation lasted for 600s. Amount of free fatty acid liberated during enzymation was measured using the NEFA C kit (999-75406, WAKO, Germany).. 56 pL NEFA A was added and the mixture was incubated for 300 s.. Afterwards 113 pL NEFA B was added and the mixture was incubated for 300s. Afterwards 113 pl NEFAB was added and the mixture was incubated for 300s OD 520 nm was then measured. Enzyme activity GLU-K (pmol FFA/minmL) was calculated based on a standard enzyme preparation.
All publications mentioned in the above specification are herein incorporated by reference Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.
Two forms of pPAEGI were constructed which differed only in the orientation of the pyr4 gene with respect to the egli flanking regions. Strain B31 P6 was transformed with a mixture of both forms of the plasmid after they had been digested with Hindlll, Total DNA was extracted from stable transformants, digested with Hindlll and subjected to Southern analysis- The probe used was radio labelled pUCEGI.
Hybridisation was observed to a 4.2 kb fragment of DNA from strain B31 P6 representing the undeleted egl1 gene A transformant (strain 1A52) was identified in which this 4..2 kb was no longer present but had been replaced by a fragment of approximately 6.8 kb.. This is the pattern expected if the larger Hindlll fragment from pP EGI had integrated precisely as predicted at the egl-1 locus leading to deletion of part of the EGI coding sequence and insertion of pyr4 at this position Using a pLJC
plasmid as a probe for Southern analysis it was confirmed that the pUC DNA
fragment of pPL1EGl had not been incorporated in strain 1A52 T.reesei strain 1A52 may be modified to form a Treesei cell usable in the present invention.
Assay for triacyiglycerol hydrolysing activity (classified as E.C. 3.1.1.3) LIPU/LUSOI assays for Triacylglycerol hydrolysing activity Determination of lipase activity by LIPU is carried out by enzymation of an emulsion of tributyrylglycerol, Enzymatic hydrolysis of lipids liberates free fatty acids.. By continuous titration of the liberated free fatty acid, the lipase activity is determined from the consumption of base. 1 LIPU (lipase unit) (also called 1 unit herein) is defined as the amount of enzyme, which releases 1 pmol free fatty acid per minute at the given assay conditions..
Enzyme samples were dissolved in demineralised water. The titrant was 0,05 M
NaOH. The substrate was a homogenised emulsion of 5% (v/v) tributyrineglycerol (Merck, item no, 101958), 0.10% (wlv) gum arabic (Sigma, item no.. G9752), 7.5%
(w/v) glycerol (Merck, item no, 1.04092), 51 mM NaG! (p.a, Merck, item no.
1.06404), 0.50 mM KH2PO4 (p..a. Merck, item no, 1.04873). Reaction pH was 5,5 and reaction temperature was 30 C.. 2,00 mL sample was added to 25.0 mL substrate acclimatised to the reaction temperature. Activity was calculated from the slope of a linear titration curve with consumption of titrant plotted against reaction time.
Substrates used are Tributyrin (Lipu) and Sunflower oil (LUSol), DGGR assay for Triacylglycerol hydrolysing activity This assay was used to measure Thermomyces lanuginosus lipase expressed in Trichoderma.
5 The substrates and buffers used in this assay were the following:
3.32 mM 1,2-Di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorufin ester) dissolved in DMSO (substrate, 2.5mg/mL), A stock solution consisting of 50 mg substrate added to 20mL DMSO is 10 sonicated, aliquoted and stored at -80 deg C until use. Buffer used is 0.5 M HEPES
pH 8 + 60gpg 3:1 Ca:Mg Water hardness (see CAM300) and 4% Gum Arabic..
Lipase enzyme stock (1 mg/L) is used as standard.
For the assay, A 50mL assay buffer is prepared by adding 5mL HEPES + Hardness 15 and 25mL 4% Gum Arabic to 10mL water. The assay buffer is incubated to desired assay temperature (typically 25 deg C). 1 OuL of enzyme samples are added into well plate at assay temperature. 1-10ppm of active enzyme in sample recommended.
For best results match unknown concentration to +1- two-fold activity of standard-20 The background rate (no enzyme, i.e. assay buffer) is determined.
10mL of 3..32mM DGGR substrate in DMSO is added to assay buffer and mixed well using a vortex. 200uL substrate in assay buffer is added immediately to enzyme samples in microplate from a reagent reservoir using a multi-channel pipet.
The microliter plate is mixed well and immediately transfer ed to plate reader. OD
at 580 25 nm for up to 10 min is measured at desired assay temperature (typically 25 deg C).
Calculation of the enzyme activity is done by subtracting the background rate from unknown and standard to obtain difference rates,. Determine the ratio of the difference rates for unknown to standard and multiply by the standard concentration. All dilutions are included in the calculation:
unknown concentration = (unknown Rate * standard concentration) / standard rate..
Determination of triacyiglyceride lipase activity: assay based on triglyceride (tributyrin) as substrate (LIPU):
Lipase activity based on tributyrin is measured according to Food Chemical Codex, Forth Edition, National Academy Press, 1996, p 803, with the modifications that the sample is dissolved in deionized water instead of glycine buffer, and the pH
stat set point is 5.5 instead of 7..
1 LIPU is defined as the quantity of enzyme which can liberate 1 mol butyric acid per minute under assay conditions.
Assay for Phospholipase activity phospholipase Al activity (E,C. 3.1.1.32) phospholipase A2 activity (E C, 3,1.1.4) phospholipase B activity (E..C. 31.1.5) Substrate 1.75% L- Plant Phosphatidylcholin 95% (441601, Avanti Polar Lipids), 6.3 %
Triton X-100 (#T9284, Sigma) and 5 mM CaCl2 dissolved in 50 mM HEPES buffer pH 70..
Assay procedure Samples, calibration and control were diluted in 10 mM HEPES pH 7 .0, 01%
Triton X-100 (#T9284, Sigma). Analysis was carried out using a Konelab Autoanalyzer (Thermo, Finland). The assay was run at 30C.. 34 pL substrate was thermostated for 180 seconds, before 4 pL sample was added. Enzymation lasted for 600s. Amount of free fatty acid liberated during enzymation was measured using the NEFA C kit (999-75406, WAKO, Germany), 56 pL NEFA A was added and the mixture was incubated for 300 s. Afterwards 113 pL NEFA B was added and the mixture was incubated for 300s. Afterwards 113 lit NEFAB was added and the mixture was incubated for 300s..
OD 520 nm was then measured. Enzyme activity LATU (pmol FFA/minmL) was calculated based on a standard enzyme preparation Assay for glycolipase (galactolipase) activity.
Substrate 135% Didalactosyldiglyceride (DGDG, Purified from wheat lipids), 6.3 % Triton (#T9284, Sigma) and 5 mM CaC12 dissolved in 50 mM HEPES buffer pH 7.0 Assay procedure Samples, calibration and control were diluted in 10 mM HEPES pH 7,0, 0.1%
Triton X-100 (#T9284, Sigma). Analysis was carried out using a Konelab Autoanalyzer (Thermo, Finland). The assay was run at 30C. 34 pL substrate was thermostated for 180 seconds, before 4 pL sample was added Enzymation lasted for 600s. Amount of free fatty acid liberated during enzymation was measured using the NEFA C kit (999-75406, WAKO, Germany).. 56 pL NEFA A was added and the mixture was incubated for 300 s.. Afterwards 113 pL NEFA B was added and the mixture was incubated for 300s. Afterwards 113 pl NEFAB was added and the mixture was incubated for 300s OD 520 nm was then measured. Enzyme activity GLU-K (pmol FFA/minmL) was calculated based on a standard enzyme preparation.
All publications mentioned in the above specification are herein incorporated by reference Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.
Claims (32)
1. A method of producing a lipolytic enzyme comprising the steps of.
(i) providing a transformed or transfected Trichoderma reesei cell comprising a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID
NO: 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO, 4, and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions, and (ii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme.
(i) providing a transformed or transfected Trichoderma reesei cell comprising a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID
NO: 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO, 4, and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions, and (ii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme.
2. A method of producing a lipolytic enzyme comprising the steps of:
(i) transfecting or transforming a Trichoderma reesei cell with a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID
NO: 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO. 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4 or the complement of any thereof under stringent conditions;
(ii) optionally repeating step (i) on the cell to sequentially transfect or transform the cell with at least one additional heterologous nucleotide sequence as defined in (i)(a), (i)(b) or (i)(c); and (iii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme.
(i) transfecting or transforming a Trichoderma reesei cell with a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID
NO: 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO. 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4 or the complement of any thereof under stringent conditions;
(ii) optionally repeating step (i) on the cell to sequentially transfect or transform the cell with at least one additional heterologous nucleotide sequence as defined in (i)(a), (i)(b) or (i)(c); and (iii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme.
3. A method according to claim 1 or claim 2 wherein the heterologous nucleotide sequence further comprises a promoter sequence, which promoter sequence is a cellobiohydrolase promoter sequence.
4. A method according to any one of claims 1 to 3 wherein the lipolytic enzyme is produced in an amount of at least 20g/litre of culture supernatant.
5. A method according to any one of the preceding claims, wherein said method comprises the additional step of concentrating and/or isolating and/or recovering the lipolytic enzyme.
6. A method according to any one of the preceding claims, wherein the Trichoderma reesei cell is prepared by transforming or transfecting a Ttrichoderma reesei cell with the nucleotide sequence.
7. A method according to any one of the preceding claims, wherein the Trichoderma reesei cell is provided by transforming it with or is transformed with the nucleotide sequence using biolistic transformation
8. A lipolytic enzyme obtainable by the method of any one of the preceding claims.
9. A foodstuff comprising said lipolytic enzyme according to claim 8
10. A Trichoderma reesei cell comprising:
a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme protein having at least 40 % sequence identity to SEQ ID NO: 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40%
sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises an the nucleotide sequence which hybridizes to SEQ ID NO. 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions..
a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme protein having at least 40 % sequence identity to SEQ ID NO: 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40%
sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises an the nucleotide sequence which hybridizes to SEQ ID NO. 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions..
11. A Trichoderma reesei cell according to claim 10, wherein the cell comprises at least two (such as at least three, such as at least four, such as at least five, or such as at least six) heterologous nucleotide sequences encoding said lipolytic enzyme.
12. An expression vector comprising i) at least one nucleotide sequence which nucleotide sequence:
a) encodes a lipolytic enzyme protein having at least 40% sequence identity to SEQ ID NO: 1 or 2; and/or b) encodes a lipolytic enzyme and comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40%
sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4; and/or c) encodes a lipolytic enzyme wherein the nucleotide sequence comprises an the nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO. 3 or SEQ ID NO. 4 or the complement of any thereof under stringent conditions; and ii) at least one cellobiohydrolase promoter, wherein said at least one nucleotide sequence is under the control of said at least one cellobiohydrolase promoter.
a) encodes a lipolytic enzyme protein having at least 40% sequence identity to SEQ ID NO: 1 or 2; and/or b) encodes a lipolytic enzyme and comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40%
sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4; and/or c) encodes a lipolytic enzyme wherein the nucleotide sequence comprises an the nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO. 3 or SEQ ID NO. 4 or the complement of any thereof under stringent conditions; and ii) at least one cellobiohydrolase promoter, wherein said at least one nucleotide sequence is under the control of said at least one cellobiohydrolase promoter.
13. An expression vector according to claim 12 wherein said vector further comprises a nucleotide sequence encoding a signal peptide, which nucleotide sequence encoding said signal peptide is operably linked to said nucleotide sequence encoding said lipolytic enzyme.
14. Use of a Trichoderma reesei cell in the expression of a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID NO:
1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40%
sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence which hybridizes to SEQ ID NO. 3 or SEQ ID NO. 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions;
for improving one or more of the following: expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity or yield.
1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40%
sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence which hybridizes to SEQ ID NO. 3 or SEQ ID NO. 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions;
for improving one or more of the following: expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity or yield.
15. The invention according to any one of claims 1 to 14 wherein the Trichoderma reesei cell comprises one or more suppressed gene(s) encoding non-lipolytic enzyme(s), preferably two or more suppressed gene(s) encoding non-lipolytic enzyme(s), preferably three or more suppressed gene(s) encoding non-lipolytic enzyme(s), preferably four suppressed gene(s) encoding non-lipolytic enzyme(s).
16. The invention according to claim 15 wherein at least one or each of the suppressed gene(s) encoding non-lipolytic enzyme(s) is a suppressed gene(s) encoding a cellulase
17. The invention according to any one of claims 1 to 16 wherein after expression of the nucleotide sequence the Trichoderma reesei cell is removed from the medium into which the enzyme has been secreted.
18, The invention according to any one of claims 1 to 17 wherein after expression of the nucleotide sequence the Trichoderma reesei cell is removed from the medium into which the enzyme has been secreted; and then the cell free medium is concentrated.
19. The invention according to any one of claims 1 to 16 wherein the pH of the medium into which the enzyme is secreted is raised after a period of time to yield sufficient levels of the secreted enzyme and prior to the isolation and/or purification and/or concentration of the enzyme.
20. The invention according to any one of claims 1 to 19 wherein the following steps are carried out to the medium into which the enzyme of the present invention has been secreted following culturing of the cell: adjusting the pH of the medium, diluting the medium with water; separating the cell(s) from the medium; concentrating the medium wherein said medium is cell-free; and optionally granulating said medium wherein said medium is cell-free-
21. The invention according to any one of claims 1 to 20 wherein the enzyme is used in a method to prepare a food or foodstuff intended for human consumption said method comprising admixing said enzyme with a suitable food or foodstuff ingredient.
22. The invention according to claim 21 wherein said enzyme is in the medium into which the enzyme of the present invention has been secreted following culturing of the cell.
23. The invention according to claim 21 or claim 22 wherein said medium is cell-free.
24. The invention according to any one of claims 21 to 23 wherein said medium is concentrated.
25. The invention according to any one of claims 21 to 24 wherein said medium is granulated.
26. A method as generally described herein with reference to the examples and figures..
27. A Trichoderma reesei cell as generally described herein with reference to the examples and figures
28. An expression vector as generally described herein with reference to the examples and figures.
29. A use as generally described herein with reference to the examples and figures.
30. A method of producing a lipolytic enzyme comprising the steps of:
(i) providing a transformed or transfected Trichoderma reesei cell comprising at least one heterologous nucleotide sequence encoding a lipolytic enzyme;
(ii) culturing the cell at a pH 4 to pH 5.5 under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme, (iii) isolating, purifying or concentrating the enzyme in a medium at pH 5.5 to pH
6.5.
(i) providing a transformed or transfected Trichoderma reesei cell comprising at least one heterologous nucleotide sequence encoding a lipolytic enzyme;
(ii) culturing the cell at a pH 4 to pH 5.5 under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme, (iii) isolating, purifying or concentrating the enzyme in a medium at pH 5.5 to pH
6.5.
31. A method according to claim 30 wherein the lipolytic enzyme:
a) comprises an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or comprises an amino acid sequence which has at least 40% sequence identity to SEQ ID NO: 1 or 2; and/or b) is encoded by a nucleotide comprising the sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or comprising a nucleotide sequence which has at least 40%
sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4, and/or c) is encoded by a nucleotide sequence comprising a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or comprises a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID
NO: 4 or the complement of any thereof under stringent conditions.
a) comprises an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or comprises an amino acid sequence which has at least 40% sequence identity to SEQ ID NO: 1 or 2; and/or b) is encoded by a nucleotide comprising the sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or comprising a nucleotide sequence which has at least 40%
sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4, and/or c) is encoded by a nucleotide sequence comprising a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or comprises a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID
NO: 4 or the complement of any thereof under stringent conditions.
32. A method substantially as described herein and with reference to the accompanying figures.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17227209P | 2009-04-24 | 2009-04-24 | |
US61/172,272 | 2009-04-24 | ||
GBGB0908770.1A GB0908770D0 (en) | 2009-04-24 | 2009-05-20 | Method |
GB0908770.1 | 2009-05-20 | ||
PCT/IB2010/051802 WO2010122531A2 (en) | 2009-04-24 | 2010-04-23 | Method |
Publications (2)
Publication Number | Publication Date |
---|---|
CA2759405A1 true CA2759405A1 (en) | 2010-10-28 |
CA2759405C CA2759405C (en) | 2020-07-21 |
Family
ID=40862772
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA2759405A Active CA2759405C (en) | 2009-04-24 | 2010-04-23 | Method |
Country Status (11)
Country | Link |
---|---|
US (2) | US8940519B2 (en) |
EP (1) | EP2421963B2 (en) |
CN (1) | CN102439143B (en) |
AU (1) | AU2010240473B2 (en) |
BR (1) | BRPI1006628B8 (en) |
CA (1) | CA2759405C (en) |
DK (1) | DK2421963T4 (en) |
ES (1) | ES2616057T5 (en) |
GB (1) | GB0908770D0 (en) |
NZ (1) | NZ595833A (en) |
WO (1) | WO2010122531A2 (en) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB0908770D0 (en) * | 2009-04-24 | 2009-07-01 | Danisco | Method |
US8765425B2 (en) | 2011-03-23 | 2014-07-01 | Butamax Advanced Biofuels Llc | In situ expression of lipase for enzymatic production of alcohol esters during fermentation |
US8759044B2 (en) | 2011-03-23 | 2014-06-24 | Butamax Advanced Biofuels Llc | In situ expression of lipase for enzymatic production of alcohol esters during fermentation |
WO2013043860A1 (en) | 2011-09-22 | 2013-03-28 | Danisco Us Inc. | Endogenous dnase activity to reduce dna content |
CN102604913B (en) * | 2012-04-05 | 2013-08-14 | 湖南尤特尔生化有限公司 | Preparation method and application of thermomyces lanuginosus lipase |
WO2014207667A1 (en) * | 2013-06-27 | 2014-12-31 | Sasol Technology (Proprietary) Limited | Production of biomass for use in the treatment of fischer-tropsch reaction water |
CN106434597B (en) * | 2016-11-08 | 2019-10-01 | 湖南尤特尔生化有限公司 | A kind of chaetomium thermophilum phosphatidase and its preparation method and application |
Family Cites Families (33)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NL154598B (en) | 1970-11-10 | 1977-09-15 | Organon Nv | PROCEDURE FOR DETERMINING AND DETERMINING LOW MOLECULAR COMPOUNDS AND PROTEINS THAT CAN SPECIFICALLY BIND THESE COMPOUNDS AND TEST PACKAGING. |
US3817837A (en) | 1971-05-14 | 1974-06-18 | Syva Corp | Enzyme amplification assay |
US3939350A (en) | 1974-04-29 | 1976-02-17 | Board Of Trustees Of The Leland Stanford Junior University | Fluorescent immunoassay employing total reflection for activation |
US3996345A (en) | 1974-08-12 | 1976-12-07 | Syva Company | Fluorescence quenching with immunological pairs in immunoassays |
US4275149A (en) | 1978-11-24 | 1981-06-23 | Syva Company | Macromolecular environment control in specific receptor assays |
US4277437A (en) | 1978-04-05 | 1981-07-07 | Syva Company | Kit for carrying out chemically induced fluorescence immunoassay |
US4366241A (en) | 1980-08-07 | 1982-12-28 | Syva Company | Concentrating zone method in heterogeneous immunoassays |
US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
US4797361A (en) † | 1983-10-24 | 1989-01-10 | Lehigh University | Microorganism and process |
US4683202A (en) | 1985-03-28 | 1987-07-28 | Cetus Corporation | Process for amplifying nucleic acid sequences |
DE3546807C2 (en) | 1985-03-30 | 1991-03-28 | Marc Genf/Geneve Ch Ballivet | |
US5780292A (en) | 1987-04-29 | 1998-07-14 | Alko Group Ltd. | Production of phytate degrading enzymes in trichoderma |
EP0531372B2 (en) | 1990-05-09 | 2004-04-14 | Novozymes A/S | A cellulase preparation comprising an endoglucanase enzyme |
DE4112440C1 (en) | 1991-04-16 | 1992-10-22 | Diagen Institut Fuer Molekularbiologische Diagnostik Gmbh, 4000 Duesseldorf, De | |
AU666443B2 (en) | 1992-12-10 | 1996-02-08 | Dsm N.V. | Production of heterologous proteins in filamentous fungi |
US5605793A (en) | 1994-02-17 | 1997-02-25 | Affymax Technologies N.V. | Methods for in vitro recombination |
US5741665A (en) | 1994-05-10 | 1998-04-21 | University Of Hawaii | Light-regulated promoters for production of heterologous proteins in filamentous fungi |
ATE220502T1 (en) * | 1997-04-09 | 2002-08-15 | Danisco | IMPROVED METHOD FOR PRODUCING FLOUR PASTA AND PRODUCTS MADE FROM SUCH PASTA USING GLYCERINE OXIDASE |
GB9821198D0 (en) | 1998-09-30 | 1998-11-25 | Danisco | Enzyme |
ATE323759T1 (en) † | 1999-01-25 | 2006-05-15 | Novozymes As | RECOVERY OF A PROTEIN AT HIGH PH |
MXPA00011569A (en) | 1999-03-26 | 2004-12-03 | Diversa Corp | Exonuclease-mediated nucleic acid reassembly in directed evolution. |
US20070213518A1 (en) * | 2003-05-12 | 2007-09-13 | Jones Brian E | Novel Lipolytic Enzyme Lip2 |
EP1625217B1 (en) | 2003-05-12 | 2014-12-17 | Danisco US Inc. | Novel lipolytic enzyme elip |
ATE524491T1 (en) | 2003-05-29 | 2011-09-15 | Genencor Int | NEW TRICHODERMA GENES |
EP2410048B1 (en) | 2005-01-24 | 2016-08-10 | DSM IP Assets B.V. | Method for producing a compound of interest in a filamentous fungal cell |
US7740307B2 (en) | 2006-06-14 | 2010-06-22 | Guido Benvenuto | Motor vehicle component and methods for its manufacture |
US20080026376A1 (en) | 2006-07-11 | 2008-01-31 | Huaming Wang | KEX2 cleavage regions of recombinant fusion proteins |
EP2059590B1 (en) * | 2006-07-14 | 2014-12-31 | Novozymes, Inc. | Methods for producing secreted polypeptides having biological activity |
ATE533840T1 (en) | 2007-03-14 | 2011-12-15 | Danisco Us Inc | TRICHODERMA REESEI AMYLASE AS A MALTOGENIC ENZYME |
EP2147104B1 (en) | 2007-05-21 | 2015-09-09 | Danisco US Inc. | Use of an aspartic protease (nsp24) signal sequence for heterologous protein expression |
DK2152892T3 (en) | 2007-05-21 | 2013-07-22 | Danisco Us Inc Genencor Div | Method of introducing nucleic acids into fungal cells |
FI120835B (en) † | 2007-07-10 | 2010-03-31 | Valtion Teknillinen | New esters and their use |
GB0908770D0 (en) * | 2009-04-24 | 2009-07-01 | Danisco | Method |
-
2009
- 2009-05-20 GB GBGB0908770.1A patent/GB0908770D0/en not_active Ceased
-
2010
- 2010-04-23 AU AU2010240473A patent/AU2010240473B2/en active Active
- 2010-04-23 CN CN201080018179.3A patent/CN102439143B/en active Active
- 2010-04-23 US US13/265,712 patent/US8940519B2/en active Active
- 2010-04-23 ES ES10723329T patent/ES2616057T5/en active Active
- 2010-04-23 NZ NZ595833A patent/NZ595833A/en not_active IP Right Cessation
- 2010-04-23 BR BRPI1006628A patent/BRPI1006628B8/en active IP Right Grant
- 2010-04-23 EP EP10723329.8A patent/EP2421963B2/en active Active
- 2010-04-23 DK DK10723329.8T patent/DK2421963T4/en active
- 2010-04-23 WO PCT/IB2010/051802 patent/WO2010122531A2/en active Application Filing
- 2010-04-23 CA CA2759405A patent/CA2759405C/en active Active
-
2014
- 2014-11-07 US US14/536,016 patent/US9382523B2/en active Active
Also Published As
Publication number | Publication date |
---|---|
US20120196002A1 (en) | 2012-08-02 |
GB0908770D0 (en) | 2009-07-01 |
DK2421963T3 (en) | 2017-03-06 |
AU2010240473A1 (en) | 2011-11-10 |
US9382523B2 (en) | 2016-07-05 |
ES2616057T5 (en) | 2023-05-23 |
BRPI1006628A2 (en) | 2015-08-25 |
CN102439143A (en) | 2012-05-02 |
CA2759405C (en) | 2020-07-21 |
EP2421963B2 (en) | 2022-12-28 |
BRPI1006628B1 (en) | 2019-04-16 |
EP2421963A2 (en) | 2012-02-29 |
WO2010122531A3 (en) | 2010-12-29 |
EP2421963B1 (en) | 2016-11-30 |
ES2616057T3 (en) | 2017-06-09 |
WO2010122531A2 (en) | 2010-10-28 |
DK2421963T4 (en) | 2023-03-27 |
US8940519B2 (en) | 2015-01-27 |
NZ595833A (en) | 2013-10-25 |
CN102439143B (en) | 2016-03-09 |
US20150064309A1 (en) | 2015-03-05 |
AU2010240473B2 (en) | 2014-01-30 |
BRPI1006628B8 (en) | 2019-05-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9382523B2 (en) | Method of producing a lipolytic enzyme | |
JP6027092B2 (en) | Composition | |
AU2005290934C1 (en) | Citrobacter freundii phytase and homologues | |
JP4751877B2 (en) | Fungal lipolytic enzyme | |
EP2591123B1 (en) | Method for identifying a pepsin resistant alpha amylase | |
WO2006038062A1 (en) | Microbial phytase as supplement to food or fodder | |
AU2005297167A1 (en) | Enzymes | |
CA2444960C (en) | Method of improving dough and bread quality | |
AU2010264116B2 (en) | Protein | |
US11473073B2 (en) | Aminopeptidases for protein hydrolyzates | |
WO2009136791A2 (en) | Novel phytases |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20130531 |