CN114787195A - 一种肝素酶iii的制备方法 - Google Patents
一种肝素酶iii的制备方法 Download PDFInfo
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- CN114787195A CN114787195A CN201980102778.4A CN201980102778A CN114787195A CN 114787195 A CN114787195 A CN 114787195A CN 201980102778 A CN201980102778 A CN 201980102778A CN 114787195 A CN114787195 A CN 114787195A
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- dialysis
- tris
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- heparinase
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- 108010083213 heparitinsulfate lyase Proteins 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 44
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 40
- 239000000243 solution Substances 0.000 claims abstract description 33
- 230000000694 effects Effects 0.000 claims abstract description 29
- 108010022901 Heparin Lyase Proteins 0.000 claims abstract description 25
- 239000007853 buffer solution Substances 0.000 claims abstract description 25
- 239000011780 sodium chloride Substances 0.000 claims abstract description 23
- 238000011068 loading method Methods 0.000 claims abstract description 18
- 241000605114 Pedobacter heparinus Species 0.000 claims abstract description 16
- 239000002244 precipitate Substances 0.000 claims abstract description 9
- 239000006228 supernatant Substances 0.000 claims abstract description 8
- 238000010829 isocratic elution Methods 0.000 claims abstract description 6
- 238000000502 dialysis Methods 0.000 claims description 41
- 108090000790 Enzymes Proteins 0.000 claims description 25
- 102000004190 Enzymes Human genes 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 19
- 239000000872 buffer Substances 0.000 claims description 17
- 239000001110 calcium chloride Substances 0.000 claims description 17
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 17
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 12
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 12
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 8
- 102100024025 Heparanase Human genes 0.000 claims description 6
- 108010037536 heparanase Proteins 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 abstract description 17
- 229960002897 heparin Drugs 0.000 abstract description 17
- 229920000669 heparin Polymers 0.000 abstract description 16
- 238000012870 ammonium sulfate precipitation Methods 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 description 9
- 230000015556 catabolic process Effects 0.000 description 7
- 238000006731 degradation reaction Methods 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000010828 elution Methods 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 6
- 230000004151 fermentation Effects 0.000 description 6
- 239000003480 eluent Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 229920002971 Heparan sulfate Polymers 0.000 description 3
- 108010006406 heparinase II Proteins 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 235000011148 calcium chloride Nutrition 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 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-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-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
- PRDZVHCOEWJPOB-IVMDWMLBSA-N N-sulfo-D-glucosamine Chemical compound OC[C@H]1OC(O)[C@H](NS(O)(=O)=O)[C@@H](O)[C@@H]1O PRDZVHCOEWJPOB-IVMDWMLBSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 239000003055 low molecular weight heparin Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
-
- 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
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
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- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
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- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
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- Analytical Chemistry (AREA)
- Immunology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
一种肝素酶III的制备方法，包括以下步骤：将肝素黄杆菌破碎离心后所得上清液进行硫酸铵沉淀，沉淀物溶解后透析；上样SP柱，以20‑30mM Tris‑HCl缓冲液平衡，相同缓冲液中0‑0.5M NaCl线性洗脱，收集肝素酶活性组分，透析；上样SP柱，以含0.05‑0.2M NaCl的50mMTris‑HCl缓冲液洗脱，收集肝素酶活性组分，透析；上样SP柱，含0.3％肝素的50mM Tris‑HCl缓冲液等度洗脱，收集肝素酶活性组分，透析；上样SP柱，以含0.055M NaCl的50mM Tris‑HCl溶液等度洗脱，收集肝素酶活性组分，浓缩得到肝素酶III。
Description
PCT国内申请，说明书已公开。
Claims (15)
- PCT国内申请，权利要求书已公开。
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2019/129111 WO2021128254A1 (zh) | 2019-12-27 | 2019-12-27 | 一种肝素酶iii的制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114787195A true CN114787195A (zh) | 2022-07-22 |
| CN114787195B CN114787195B (zh) | 2024-01-12 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201980102778.4A Active CN114787195B (zh) | 2019-12-27 | 2019-12-27 | 一种肝素酶iii的制备方法 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN114787195B (zh) |
| WO (1) | WO2021128254A1 (zh) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5169772A (en) * | 1988-06-06 | 1992-12-08 | Massachusetts Institute Of Technology | Large scale method for purification of high purity heparinase from flavobacterium heparinum |
| CN1429913A (zh) * | 2001-12-30 | 2003-07-16 | 中国科学院微生物研究所 | 一种用肝素酶生产肝素寡糖的方法 |
| CN1670187A (zh) * | 2005-03-28 | 2005-09-21 | 国家海洋局第三海洋研究所 | 一种碱性低温蛋白酶及其制备方法 |
| CN102286448A (zh) * | 2011-08-22 | 2011-12-21 | 深圳市海普瑞药业股份有限公司 | 一种肝素黄杆菌肝素酶i、ii、iii的制备方法 |
| CN104593347A (zh) * | 2015-03-05 | 2015-05-06 | 深圳市海普瑞药业股份有限公司 | 来自Sphingobacterium daejeonense的肝素酶及其制备和应用 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5389539A (en) * | 1992-11-30 | 1995-02-14 | Massachusetts Institute Of Technology | Purification of heparinase I, II, and III from Flavobacterium heparinum |
| US5681733A (en) * | 1994-06-10 | 1997-10-28 | Ibex Technologies | Nucleic acid sequences and expression systems for heparinase II and heparinase III derived from Flavobacterium heparinum |
| CN101942025B (zh) * | 2010-08-20 | 2012-07-04 | 清华大学 | 一种肝素酶ⅲ融合蛋白及其编码基因与表达方法 |
| CN104630197A (zh) * | 2015-01-28 | 2015-05-20 | 深圳市海普瑞药业股份有限公司 | 一种来自脑膜脓毒性金黄杆菌的肝素酶及其制备和应用 |
-
2019
- 2019-12-27 CN CN201980102778.4A patent/CN114787195B/zh active Active
- 2019-12-27 WO PCT/CN2019/129111 patent/WO2021128254A1/zh active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5169772A (en) * | 1988-06-06 | 1992-12-08 | Massachusetts Institute Of Technology | Large scale method for purification of high purity heparinase from flavobacterium heparinum |
| CN1429913A (zh) * | 2001-12-30 | 2003-07-16 | 中国科学院微生物研究所 | 一种用肝素酶生产肝素寡糖的方法 |
| CN1670187A (zh) * | 2005-03-28 | 2005-09-21 | 国家海洋局第三海洋研究所 | 一种碱性低温蛋白酶及其制备方法 |
| CN102286448A (zh) * | 2011-08-22 | 2011-12-21 | 深圳市海普瑞药业股份有限公司 | 一种肝素黄杆菌肝素酶i、ii、iii的制备方法 |
| CN104593347A (zh) * | 2015-03-05 | 2015-05-06 | 深圳市海普瑞药业股份有限公司 | 来自Sphingobacterium daejeonense的肝素酶及其制备和应用 |
| US20180051270A1 (en) * | 2015-03-05 | 2018-02-22 | Shenzhen Hepalink Pharmaceutical Group Co., Ltd. | Heparinases obtained from sphingobacterium daejeonense, preparation therefor and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2021128254A1 (zh) | 2021-07-01 |
| CN114787195B (zh) | 2024-01-12 |
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