CN102660610A - Method for preparing high-activity and low-molecular-weight heparin by enzymic method - Google Patents
Method for preparing high-activity and low-molecular-weight heparin by enzymic method Download PDFInfo
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- CN102660610A CN102660610A CN2012101729306A CN201210172930A CN102660610A CN 102660610 A CN102660610 A CN 102660610A CN 2012101729306 A CN2012101729306 A CN 2012101729306A CN 201210172930 A CN201210172930 A CN 201210172930A CN 102660610 A CN102660610 A CN 102660610A
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Abstract
The invention discloses a method for preparing high-activity and low-molecular-weight heparin by an enzymic method. The method includes using heparin as substrate; degrading the heparin by HepI (histidine-tagged heparinase I) in a controlled manner; then utilizing a PAPS (3'-phosphoadenosine-5'-phosphosulfate) regeneration system; selectively decorating the heparin by heparin biological synthetase 3-O-sulfotransferase; and increasing the quantity of anticoagulation activity centers to obtain the low-molecular-weight heparin with high anticoagulation activity. The HepI, the 3-O-sulfotransferase and AST-IV which are adopted in the method can be prepared by means of high-density fermentation in high yield; the PAPS regeneration system uses PNPS (P-nitrophenol sulfonic acid potassium salt) as sulfate radical for enzyme modification reaction, and production cost is greatly reduced. The method provides a new way for enzymic method industrialization of the high-activity and low-molecular-weight heparin.
Description
Technical field
The present invention relates to the method that a kind of enzyme process prepares low molecular weight heparin with high activity.Belong to biomedicine field.
Background technology
Heparin is the mucopolysaccharide that has hexuronic acid (L-iduronic acid, D-glucuronic acid) and D-Glucosamine Sulphate alternately to form with 1 → 4 glycosidic link, and its molecular weight is between 3000-40000Da, and molecular-weight average is 15000Da; Heparin is as anti-freezing reagent and the history in existing more than 60 year of anti-bolt reagent.The anticoagulant active of heparin mainly combines antithrombin (AT-III) through the pentose active site of containing 3-position sulfate GS, induces its occurred conformation to change, thus anticoagulant active before this.The anticoagulant active of heparin comprises anti-thrombus activity (anti-factor Xa) and anticoagulating active, and (the anti-factor II a).Anticoagulant active is also relevant with the molecular weight in Gansu.But,, thereby limited heparin application clinically greatly so a large amount of uses can cause bleeding and spinoffs such as inducibility thrombopenia, osteoporosis because heparin has anticoagulant active.In addition, the heparin heparin also have anti-inflammatory, antianaphylaxis, antiviral, anticancer, transfer the vital function of aspect such as blood fat.
Low molecular weight heparin is the small molecule segment that is obtained through chemical method or enzymic degradation by heparin, and molecular weight is between 3000-8000Da, and molecular-weight average is about 5000Da.Low molecular weight heparin has higher anti-thrombus activity, and its anticoagulating active then reduces greatly, and is little to the blood coagulation system influence when showing antithrombotic formation, thereby in clinical, is widely used.Compare with unfraction heparin, it is little that low molecular weight heparin has a molecular weight, and by the body weight administration, pharmacological property is stable, does not need hospital care, and bioavailability is high, and spinoff is little, advantage such as long half time in the body.
At present, the preparation method of LMWHs (Zhang Wanzhong, Wang Yunshan, Ma Runyu, Su Zhiguo, biochemical drug magazine, 2001,22 (1): 48-51) mainly contain chemical cracking method and enzyme liberating method.Chemical degradation method is that the normal method that adopts is gone up in industry, mainly contains that nitrous acid will be exposed, β-elimination edman degradation Edman, hydrogen peroxide degradation method, Periodic acid 99, hypochlorous acid, sulfuric acid-chlorsulfonic acid and gamma-irradiation method etc.But the reaction of chemical degradation method heparin is violent, make that some functional group in the heparin molecule more or less is destroyed in the reflection process, thereby some bioactive functions is being destroyed in various degree.And enzymic degradation is because reaction conditions is gentle, environmentally friendly, and is convenient to detect, the research focus over becoming into year.USP (Nielsen, US5106734,1992) utilizes control 232nm place light absorption value to the controlled degraded of heparin, prepare molecular weight relatively all with low molecular weight product.(Yu Guangli, Wang Qun, Guan Huashi, Xu Jiamin, Robert J, Linhardt, Qingdao Marine University's journal, 2002,32 (2): 231-235) utilize heparinase that ox lung heparin is controlled enzymolysis, obtain the pure article of oligosaccharides of polymerization degree 2-20 such as Yu Guangli.Mainly research shows that also the anticoagulant active of heparin depends on the quantity in its pentose active site in addition.N-in the pentose structure, 6-O-and 3-O-position sulfate participate in combining the AT-III, and be relevant with anticoagulant active; And the effect of 2-O-position sulfate and iduronic acid it be unclear that (I.Capila, R.J.Linhardt, Angew.Chem.Int.Ed.; 2002,41:390-412.).Wherein 3-O-position sulfate is particularly important: if lack 3-O-position sulfate, the anticoagulant active that main officer of Tibet is glared at then can reduce more than 1000 times; On the contrary, increase a 3-O-position sulfate, then can the anticoagulant active of heparin be improved several times.Heparin is synthetic in plastosome by multiple biosynthetic enzyme.Can relate to multiple synthetic enzyme in the building-up process, as: 2-O-, 6-O-; (2-OST, 6-OST 3-OST) carry out part of sulfuric acidization (U.Lindahl to the hydroxyl on the main chain different positions respectively to 3-O-sulfate transferring enzyme; M.Kusche-Gullberg, L.kJellen, J.Biol.Chem; 1998,273:24979-24982.), wherein 3-O-position sulfation is accomplished by 3-OST.These sulfate transferring enzymes have multiple isomery pattern mostly, just have six kinds of isomer like 3-OST.Wherein, 3-OST-1 and 3-OST-5 can synthesize heparin pentose active site.Existing research show heparin and low molecular weight heparin all be 3-OST-1 and the good substrate of 3-OST-5 (J.Chen, M.B.Duncan, K.Carrick et., Glycobiology, 2003,13:785-794).Therefore; With 3-OST-1 and 3-OST-5 to the low molecular weight heparin selective modification; Make 80% do not have a pentose active site low molecular weight heparin have the pentose active site after through reflection; Just can increase substantially the quantity in the pentose active site of low molecular weight heparin, thereby improve its anticoagulant active (can improve anti-bolt and anticoagulating active 3-5 in theory simultaneously doubly).
Summary of the invention
The purpose of this invention is to provide the method that a kind of enzyme process prepares the active low molecular weight heparin of highly blood coagulation resistant
The method for preparing the active low molecular weight heparin of highly blood coagulation resistant provided by the present invention; Be to be substrate with the heparin; After the controlled degraded of HepI (histidine-tagged Heparinase I), adopt PAPS (3 '-adenosine phosphate-5 '-phosphoric acid sulfuric acid) regeneration system rapidly again, with heparin biosynthetic enzyme 3-O-sulfate transferring enzyme it is carried out selective modification; Increase the quantity at anticoagulating active center, obtain the low molecular weight heparin of high anticoagulating active.The HepI that the inventive method adopted, 3-O-sulfate transferring enzyme, AST-IV all can be through the high density fermentation preparations; Said PAPS regeneration system rapidly with the sulfate donor of PNPS (p-NP sulfonic acid potassium salt) as the enzyme modification reaction, has greatly reduced production cost.The present invention provides new approach for the enzyme process industriallization of low molecular weight heparin with high activity.
Said heparin is commercially available unfraction heparin.
In the said method, heparin is initially concentration 1-100mg/mL; Preferred 50mg/mL.
In the said method, the consumption 0.1-10IU/g heparin of Heparinase I, preferred 3IU/g heparin.
In the said method, heparin enzymolysis reaction solution is the 25mM ammonium acetate, 25 μ MCaCl
2, 0.25 μ g/mLBSA, the Digestion buffer damping fluid of pH7.4.
In the said PAPS regeneration system rapidly, reaction solution is for containing 1%Triton X-100,1%BSA, 1mM MgCL
2, 1mM MnCL
2, pH is the 50mM Tris-HCL damping fluid of 5.0-8.0.
In the said PAPS regeneration system rapidly, the concentration of PNPS is 0.1-10mM, and the concentration of PAP is 0.1-50uM.
In the said method, the consumption of AST-IV is 0.1-50mg/mL; Preferred 5-10mg/mL.
In the said method, the consumption of heparin biosynthetic enzyme 3-O-sulfate transferring enzyme is the 0.1-50mg/mg substrate; Preferred 1-20mg/mL substrate.
In the said method, temperature of reaction is 10-40 ℃, preferred 30-40 ℃.
In the said method, the time of enzymatic reaction is 1-20 hour, preferred 5-10 hour.
In the said method, according to following method purifying low molecular weight heparin: after the termination reaction, spinning obtains supernatant, the buffer A that this supernatant and twice are mentioned is mixed, to appearance on the DEAE post; Carry out gradient elution with buffer A and buffer B successively, collect wash-out gained elutriant for the second time, obtain low molecular weight heparin with high activity; It is that to cry out the quality percentage composition be the NaCL aqueous solution of 0.5-2.5% that said DEAE separates used buffer A, and buffer B is to contain the NaCL aqueous solution that the quality percentage composition is 2.5-5.0%.
Method of the present invention adopts Heparinase I and heparin biosynthetic enzyme 3-O-sulfate transferring enzyme with efficient enzymic activity to prepare the active low molecular weight heparin of highly blood coagulation resistant.HepI, AST-IV and the 3-OST-1 of reorganization all can obtain through the high density fermentation high yield, and all carry 6 histidine-tagged, single step purification just can obtain the proteolytic enzyme more than 90%.Utilize the AST-IV that the Double bottom thing of PNPS and PAP is carried out the sulfate enzymatic reaction, realized the regeneration system rapidly of PNPS, thereby can use very cheap PNPS, reduced the cost of the active low molecular weight heparin of highly blood coagulation resistant as the sulfate donor.The present invention obtains the narrow low molecular weight heparin of range of molecular weight distributions (molecular-weight average 5500) through control HepI reaction conditions and action time.It is carried out selective modification, obtained the active lower molecular weight liver of highly blood coagulation resistant (anticoagulant active improves 3-5 doubly than existing low molecular weight heparin) again, had huge industrial application value with 3-OST-1.
Embodiment
Following embodiment will specify working method of the present invention, but can not be as to qualification of the present invention.
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
Embodiment 1, PAPS regeneration system rapidly
PAPS regeneration system rapidly concentration of component is: the concentration of PNPS is 5mM, and the concentration of PAP is 20uM; The consumption of AST-IV is 10mg/mM PAPS in the PAPS regeneration system rapidly.Add the solution for preparing low molecular weight heparin with high activity by above composition prepared solution, as the sulfate donor of heparin biosynthetic enzyme 3-O-sulfate transferring enzyme modification reaction.
The preparation of embodiment 2, toolenzyme HepI, AST-IV and 3-OST-1
1, the bottle that shakes of recombination bacillus coli is expressed
Picking HepI, AST-IV and 3-OST-1 recombinant bacterial strain list bacterium colony add 10 μ L penbritins (50mg/mL, final concentration are 50 μ g/mL), 37 ℃, 200rpm overnight cultures in every 10mL substratum in 10mL LB substratum respectively.By 1: 100 inoculum size, get 4mL bacterium liquid and be seeded among the 400mL LB then, add 400 μ L kantlex.37℃，200rpm。Approximately 4h between its OD 600 about arrival 0.6 to 0.8, adds inductor IPTG 2mL (0.2M, making its final concentration is 1mM), and 22 ℃, 200rpm induces 12h.In 8000rpm, 4 ℃, centrifugal 10min collects thalline with above-mentioned culture, and is resuspended to 20mL with the BufferA of 4 ℃ of precoolings (the 10mM imidazoles, pH 7.4 for 25mM Tris, 0.5M NaCl), ice-bath ultrasonic, condition is: 400W, work 5s, intermittence 10s, 200 circulations.After the ultrasonication fully, the clarification that becomes of bacterium liquid, 12000rpm, 4 ℃, centrifugal 30min collects supernatant, must crude enzyme liquid.
2, the single step purification of HepI, AST-IV and 3-OST-1 toolenzyme
Behind Buffer A damping fluid balance pillar, through upper prop behind the 0.45um membrane filtration, supernatant passes through Ni-NTA Agarose Fast Flow pillar with the speed of 0.7mL/min with crude enzyme liquid; Behind 30min, Buffer A uses Buffer B (25mM Tris at last towards a pillar 5-10 column volume; 0.5M NaCl; The 250mM imidazoles, pH 7.4) wash-out collection enzymic activity part, calculate enzyme then and live.
The preparation of embodiment 3, low-molecular-weight heparin oligose
The unfraction heparin of giving birth to worker technical service company with Shanghai is a raw material, and the reflection condition is dissolved in for the 10mg unfraction heparin in the reaction solution of 1mL, 30 ℃ of vibrations (300) reaction.Reaction solution comprises the heparin of 50mg/mL, 25mM ammonium acetate, 25 μ MCaCl
2, 0.25 μ g/mL BSA, the 3IU/g heparin, pH 7.4.About A232 to 80,5 minutes termination reactions of 100 ℃ of following reacting by heating liquid, the separation and purification of P10 pillar obtains the Heparin Oligosaccharides solution of molecular weight at 3000-8000, freeze-drying behind the dialysis desalting.
The selectively sulfated modification of the 3-O-position of embodiment 4, heparin
The Heparin Oligosaccharides that obtains with enzymolysis is a raw material, and the reflection condition is dissolved in the 20mL reaction solution in room temperature vibration (300) reaction 6h for the 1mg Heparin Oligosaccharides.Reaction solution comprises the Tris-HCl (pH 7.2) of 50mM, 1%Triton X-100,1%BSA, 1mM MgCl
2, 1mM MnCl
2, 1mM PNPS, 40uM PAP, 8mg 3-OST-1 and 4mgAST-IV.5 minutes termination reactions of 100 ℃ of following reacting by heating separate to obtain supernatant, and 2.0% the NaCl aqueous solution that the supernatant rainfall is mentioned is to appearance on the DEAE post; Once carry out gradient elution, collect wash-out gained elutriant for the second time, obtain low molecular weight heparin with high activity with 2.0% the NaCL aqueous solution and 3.0% the NaCl aqueous solution.Gained solution was with deionized water dialysis 24 hours, and freeze-drying gets the low molecular weight heparin with high activity lyophilized powder.
Claims (5)
1. method for preparing the high anticoagulating active low molecular weight heparin; Be to be substrate with the heparin,, obtain low molecular weight heparin with the controlled degraded heparin of HepI (histidine-tagged Heparinase I); Adopt PAPS (3 '-adenosine phosphate-5 '-phosphoric acid sulfuric acid) regeneration system rapidly again; With heparin biosynthetic enzyme 3-O-sulfate transferring enzyme it is carried out selective modification, increase the quantity at anticoagulating active center, obtain the low molecular weight heparin of high anticoagulating active.
2. according to the said method of claim 1; Its characteristics are: said histidine-tagged Heparinase I preparation method is following: with recombinant plasmid pET-15b-HepI transformed into escherichia coli BL21; Obtain the engineering bacteria of solubility expression HepI; Culturing engineering bacterium BL21 (pET-15b-HepI), abduction delivering obtains the C end and carries 6 histidine-tagged HepI.
3. according to the said method of claim 1, its characteristics are: said heparin biosynthetic enzyme 3-O-sulfate transferring enzyme is to carry 6 histidine-tagged fusion roteins.
4. according to the said method of claim 1, its characteristics are: said heparin concentration 1-100mg/mL, preferred 10-50mg/mL; The consumption 0.1-10IU/g heparin of Heparinase I, preferred 3IU/g heparin; The reaction solution of the said heparin that is used to degrade is the 25mM ammonium acetate, 25 μ MCaCl
2, 0.25 μ g/mL BSA, the Digestion buffer damping fluid of pH7.4, temperature of reaction is 10-40 ℃, preferred 30-40 ℃.
5. according to the said method of claim 1, its characteristics are: in the said PAPS regeneration system rapidly, reaction solution is for containing 1%Triton X-100,1%BSA, 1mM MgCI
2, 1mM MnCI
2, pH is the 50mM Tris-HCI damping fluid of 5.0-8.0, and the concentration of PNPS is 0.1-10mM, and the concentration of PAP is 0.1-50uM, and the consumption of AST-IV is 0.1-50mg/mL, preferred 5-10mg/mL; In the said method, the consumption of 3-O-sulfate transferring enzyme is the 0.1-50mg/mg substrate, preferred 1-20mg/mL substrate, and temperature of reaction is 10-40 ℃, preferred 30-40 ℃, the time of enzymatic reaction is 1-20 hour, preferred 5-10 hour.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103540630A (en) * | 2013-10-22 | 2014-01-29 | 常山生化药业（江苏）有限公司 | Preparation method of low molecular weight heparin |
| CN105483187A (en) * | 2015-12-09 | 2016-04-13 | 中国药科大学 | Anti-tumor effect of sulfated polysaccharide |
| CN114616340A (en) * | 2019-07-09 | 2022-06-10 | 奥普蒂姆维亚有限公司 | Process for the synthesis of anticoagulant polysaccharides |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101531723A (en) * | 2009-02-27 | 2009-09-16 | 江南大学 | Method for preparing heparin derivatives by using biological enzyme to selectively modify heparin structure |
| CN101591401A (en) * | 2009-02-27 | 2009-12-02 | 江南大学 | A kind of method for preparing low molecular weight heparin with high activity |
| CN101671711A (en) * | 2009-09-10 | 2010-03-17 | 清华大学 | Method for preparing ultra-low molecular weight heparin |
| CN102206690A (en) * | 2011-03-13 | 2011-10-05 | 广元市海鹏生物科技有限公司 | Preparation method of heparin oligosaccharides |
| CN102277396A (en) * | 2010-06-13 | 2011-12-14 | 黄欣 | Preparation method and application of heparin oligosaccharide with specific length |
-
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- 2012-05-31 CN CN2012101729306A patent/CN102660610A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101531723A (en) * | 2009-02-27 | 2009-09-16 | 江南大学 | Method for preparing heparin derivatives by using biological enzyme to selectively modify heparin structure |
| CN101591401A (en) * | 2009-02-27 | 2009-12-02 | 江南大学 | A kind of method for preparing low molecular weight heparin with high activity |
| CN101671711A (en) * | 2009-09-10 | 2010-03-17 | 清华大学 | Method for preparing ultra-low molecular weight heparin |
| CN102277396A (en) * | 2010-06-13 | 2011-12-14 | 黄欣 | Preparation method and application of heparin oligosaccharide with specific length |
| CN102206690A (en) * | 2011-03-13 | 2011-10-05 | 广元市海鹏生物科技有限公司 | Preparation method of heparin oligosaccharides |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103540630A (en) * | 2013-10-22 | 2014-01-29 | 常山生化药业（江苏）有限公司 | Preparation method of low molecular weight heparin |
| CN103540630B (en) * | 2013-10-22 | 2016-04-06 | 常山生化药业（江苏）有限公司 | A kind of preparation method of low molecular weight heparin |
| CN105483187A (en) * | 2015-12-09 | 2016-04-13 | 中国药科大学 | Anti-tumor effect of sulfated polysaccharide |
| CN114616340A (en) * | 2019-07-09 | 2022-06-10 | 奥普蒂姆维亚有限公司 | Process for the synthesis of anticoagulant polysaccharides |
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