CN1566152A - Immunoreactive peptide Trpi possessing antibacterial action - Google Patents

Abstract

The invention utilizes trypsinase hydrolysis cow's milk caseinum to screen immunoreactive peptide Trpi possessing antibacterial action, the separation and purification process comprises the steps of enzymolysis, heating, 80% alcohol deposition, centrifuging, dialysis, double SephadexG gel chromatography. The invention analyzes the amino acid sequence and discloses the preparation process.

Description

Immune-active peptides (Trpi) with anti-microbial effect

Technical field dairy products biotechnology

The background technology breast is newborn mammiferous main food source, also is an important component part of grownup's diet.For a long time, milk-protein only is considered as a kind of nutrient protein by people, and for newborn Mammals provides competent energy and each seed amino acid, yet result of study in recent years shows that milk-protein is except that its nutritive value, or the important source of biologically active peptides.These biologically active peptidess, all the state with non-activity is present in the milk-protein precursor, has only with after the suitable enzymic hydrolysis, and the active of them just is released, thus its physiological function of competence exertion.

The research of milk-protein biologically active peptides starts from 1979.1979, the people such as Brantl. of Germany obtained some polypeptide products by the enzymolysis bovine casein, and prove that they have class morphine activity, and from then on, people have launched the research of biologically active peptides aspect.Progressively exploration through more than 20 years, up to the present, it has been found that multiple biologically active peptides, as: morphine-like peptide (Opioid peptides), it is a class biologically active peptides of studying the earliest, have class morphine receptor ligand activity, can combine, have pain relieving, antidiarrheal, inhibition gastrointestinal peristalsis, enhancing body function the absorption of amino acid and other nutritive substance with the intravital class morphine receptor of machine; Immune-active peptides (Immunopeptides) can enhancing body immunizing power, stimulates the lymphocytic propagation of body, strengthen huge cytophilic phagocytic function, improve body and resist the ability that extraneous pathogen texture is dyed, reduce the body sickness rate, and have anti-tumor function; Antithrombotic acitivity peptide (Antithromboticpeptides) can suppress Parenogen and combine with the specific receptors of platelet surface, has anticoagulation, suppresses thrombotic physiological function; Phosphopeptide caseinate (Caseinphosphopeptides, be called for short CPP), can combine the organophosphate that forms solubility with multiple mineral element, serve as the particularly carrier that transports in vivo of calcium ion of many mineral elements, promote the absorption of small intestine calcium ion and other mineral elements; Antibacterial peptide (Antimicrobial peptides), be discovered in recent years and the class biologically active peptides that begins one's study, can suppress the growth of bacterium under the conditions in vitro, have similar antibiotic activity in vivo, can suppress the morbific streptococcus aureus of mouse infection; Antithrombotic converting enzyme inhibitor (Inhibitors ofantigiotensin-converting enzyme is called for short CEI) is a kind of multifunctional polypeptide, has the activity of blood pressure regulation, immune defense and nervous system regulation function.In addition, also had antihypertensive peptide (Antihypertensive peptides), the anti-report of forgeing peptide biologically active peptidess such as (Antiamnestic peptides) in recent years.

Immune-active peptides is a class biologically active peptides that obtains and prove its physiologically active after morphine-like peptide is found first from the Ruzhong, and the research of immune-active peptides starts from 1981.1981, Jolles. wait reported first, use the trypsin hydrolyzing people lactoprotein, separate obtaining a kind of immunocompetent peptide section that has from hydrolyzate, its aminoacid sequence is Val-Glu-Pro-Ile-Pro-Tyr, is equivalent to the amino acid residue sequence of human milk beta-casein 54-59, this small peptide, can strengthen the phagolysis of Turnover of Mouse Peritoneal Macrophages to sheep red blood cell (SRBC) in vitro tests, intravenous injection then can strengthen mouse opposing klebsiella pneumoniae

(Klebsiella.Pneumoniae) ability of Gan Raning.After this, scientist has carried out the research of a large amount of immune-active peptides aspect.It is reported, obtained 39 kinds of not homotactic immune-active peptides from cow's milk, human milk and vegetable-protein, wherein, the immune-active peptides in cow's milk protein source has 9 kinds, its sequence and originate as follows:

Aminoacid sequence	Source and position
		?Pro-Gly-Pro-Ile-Pro-Asn	?β-CNf63-68
?Gly-Leu-Phe	?α-LAf51-53
		?Leu-Leu-Tyr	?β-CNf191-193
?Thr-Thr-Met-Pro-Leu-Trp	?αsl-CNf194-199
		?Tyr-Gln-Gln-Pro-Val-Leu-Gly-Pro-Val-Arg	?β-CNf193-202
?Tyr-Pro-Phe-Pro-Gly-Pro-Ile	?β-CNf60-66
		?Tyr-G1y	?α-Lactorphin
?Tyr-Gly-Gly	κ-CN nitrogen end
		?Tyr-Gln-Gln-Pro-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val	?β-CNf194-209

(remarks: CN represents casein, and LA represents whey-protein)

In broad terms, immune-active peptides not only refers to immune-active peptides (Immunopeptids), also should comprise having immunocompetent other class physiologically active peptides, morphine-like peptide for example, it is a kind of multifunctional polypeptide, except having class morphine activity, it also has immunostimulatory activity, can stimulate the lymphocytic propagation of body, the enhancing body immunologic function, in addition, biologically active peptidess such as antibacterial peptide, Lactotransferrin B, antithrombotic converting enzyme inhibitor also have stronger immunocompetence.

The concentration that immune-active peptides is brought into play its physiological function is extremely low, and for example, dipeptides Tyr-G1y is 10 ^-11Under the concentration of mol/L, just can significantly strengthen the phagolysis of Turnover of Mouse Peritoneal Macrophages, stimulate lymphocytic propagation, improve the ability that mouse opposing klebsiella pneumoniae (Klebsiella.pneumoniae) infects sheep red blood cell (SRBC); The concentration of Tyr-Gly-Gly effect is lower, only is 10 ^-12Mol/L.

Since immune-active peptides was found and obtains systematic study, because its activity test method is cumbersome, the animal experiment of the amount of doing just can be reached a conclusion greatly, is the pure product that will obtain q.s and do the zooperal most important condition.But up to the present, the research of immune-active peptides still is in experimental level, does not also have comparatively effectively preparation method of a cover.So over 20 years, the progress of immune-active peptides aspect is slower.Germany does a lot of work in this respect with the federal dairy products research centre headed by the Hans Meisel (Federal Diary ResearchCenter) materialization institute (Institute for Chemistry and Physics), found the panimmunity bioactive peptide and its part biological activity has been done certain detection, at present, further investigate.Patent utilization trypsin hydrolyzing bovine casein of the present invention, in this complex polypeptide, isolate a kind of anti-microbial effect that has through technology such as ultrafiltration, cold extraction using alcohol, chromatograms, have immunocompetent biologically active peptides simultaneously, its sequence is analyzed, belong to novel biologically active peptides through inquiry.

Summary of the invention the present invention adopts the method for protease hydrolysis bovine casein to prepare immune-active peptides.Under study for action, at first the rule of different proteolytic enzyme (trypsinase, papoid, Sumizyme MP) caseinhydrolysate is studied.By measuring under the different concentration of substrate, degree of hydrolysis is with the difference of enzymolysis time, draw out caseic enzymolysis curve under the different concentration of substrate, then by analysis to the curve drawn, seek the relation of casein enzymolysis concentration of substrate and degree of hydrolysis, thereby draw the relation curve of hydrolysis substrate concentration and degree of hydrolysis.By discovering: the hydrolysis of (1) protein-based substrate is different with the hydrolysis of the substrate of small molecules class.Because the part of protein-based substrate hydrolysis is a peptide bond.(2) when concentration of substrate is high, viscosity increases, and proteinic activity reduces.(3) Shi Bie site difference (protein has many hydrolysis sites), and and enzyme be multidigit point bonded, thereby a lot of enzyme effect and the substrate of needs, thereby concentration of substrate will hang down.(4) seek best concentration of substrate by the hydrolysis curves of enzyme, this experimental result can be hydrolysis nutrient protein, storage protein and produces biologically active peptides thus provides theoretical foundation.

Preparation at immune-active peptides, in the Separation Research, explored the multiple protein enzyme as trypsinase, Quimotrase, stomach en-, papoid, bromeline, the various combination of Sumizyme MP and these several proteolytic enzyme screens the enzymolysis solution with immunostimulation, having found the enzymolysis solution with immunostimulation is caseic trypsin digestion liquid, the trypsin digestion liquid bacteriostatic activity size that has further compared different casein concentration, filtered out only concentration of substrate, the result shows, when caseic concentration of substrate is 9% (w/v), E: S=1: 120 o'clock, the bacteriostatic action of enzymolysis solution was the strongest.Determined to have the immune-active peptides peptide separation purification method of immunostimulation: enzymolysis solution → heating enzyme → 80% ethanol sedimentation → centrifugal → dialysis → twice SephadexG gel filtration chromatography that goes out.By the separable immune-active peptides that has been purified to of this method, and proved that through electrophoresis its purity is one-component with immunostimulation.By amino acid composition analysis,, this immune-active peptides called after " is rich in tyrosine polypeptide (Try-rich polypeptide is abbreviated as Trpi) according to the higher feature of this immune-active peptides tyrosine content.Its aminoacid sequence is:

Cys-Glu-Lys-Asp-Glu-Arg-Phe-Phe-Ser-Asp-Lys-Ile-Ala-Lys-Tyr-Ile-Pro-Ile-Gln-Tyr-Val-Leu-S

er-Arg-Tyr-Pro-Ser-Tyr-Gly-Leu-Asn-Tyr-Tyr-Gln-Gln-Lys-Pro-Val-Ala-Leu-Ile-Asn-Asn-Gln-Ph

e-Leu-Pro-Tyr-Pro-Tyr-Tyr-Ala-Lys

From its aminoacid sequence and source thereof, this immune-active peptides is different with the various immunocompetence peptide sequences of the former mistake of reporting, and is a kind of new immune-active peptides.In addition, this immune-active peptides also has anti-microbial effect simultaneously, can suppress the growth of bacteriums such as intestinal bacteria under the conditions in vitro strongly.

Embodiment is found out best removal of impurities condition, and has been studied ethanol purification Trpi method by to separating the research of Trpi condition in the casein enzymolysis liquid.Its technological process is:

Casein → pulverizing → 60 orders sieve → dissolve → and adjusting the pH value and be 45 ℃ of 7.5 → heated constant temperature → add enzymic hydrolysis → hydrolyzed solution, to adjust the pH value be that 4.5 → centrifugal → supernatant liquor → adjustment pH value is 7.5 → adds dehydrated alcohol precipitation → supernatant liquor → film vacuum concentration → vacuum freezedrying → pulverizing → packing

Processing parameter: casein purity 〉=more than 90%

Grinding particle size≤60 orders

Lysate concentration 10% (w/v)

The ratio of enzyme-to-substrate 1: 120

45 ℃ of hydrolysis temperatures

Hydrolysis time 90-120min

Claims (3)

1, change immune-active peptides (Trpi) sequence with antibacterial: the sequence that Cys-Glu-Lys-Asp-Glu-Arg-Phe-Phe-Ser-Asp-Lys-Ile-Ala-Lys-Tyr-Ile-Pro-Ile-Gln-Tyr-Val-Leu-Ser-Arg-Tyr-Pro-Ser-Tyr-Gly-Leu-Asn-Tyr-Tyr-Gln-Gln-Lys-Pro-Val-Ala-Leu-Ile-Asn-Asn-Gln-Phe-Leu-Pro-Tyr-Pro-Tyr-Tyr-Ala-Lys and homology thereof reach more than 85% all should obtain protection, must not be used for commercial purpose.

2, its high purity Trpi preparation technology (being used for the production of standard substance): bovine casein → trypsin digestion → heating enzyme → 80% ethanol sedimentation → centrifugal → dialysis → twice SephadexG gel filtration chromatography that goes out

3, its production technique (suitability for industrialized production): casein → pulverizing → 60 orders sieve → dissolve → and adjusting the pH value and be 45 ℃ of 7.5 → heated constant temperature → add enzymic hydrolysis → hydrolyzed solution, to adjust the pH value be that 4.5 → centrifugal → supernatant liquor → adjustment pH value is 7.5 → adds dehydrated alcohol precipitation → supernatant liquor → film vacuum concentration → vacuum freezedrying → pulverizing → packing.