CN107164286B - 一种的微生物菌株及其选育方法和应用 - Google Patents
一种的微生物菌株及其选育方法和应用 Download PDFInfo
- Publication number
- CN107164286B CN107164286B CN201710566643.6A CN201710566643A CN107164286B CN 107164286 B CN107164286 B CN 107164286B CN 201710566643 A CN201710566643 A CN 201710566643A CN 107164286 B CN107164286 B CN 107164286B
- Authority
- CN
- China
- Prior art keywords
- strain
- culture medium
- mutagenesis
- bacterial suspension
- culturing
- 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.)
- Active
Links
- 230000000813 microbial effect Effects 0.000 title claims abstract description 13
- 238000009395 breeding Methods 0.000 title abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 241000186359 Mycobacterium Species 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims description 13
- 244000005700 microbiome Species 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims 1
- 239000001963 growth medium Substances 0.000 abstract description 27
- 230000001580 bacterial effect Effects 0.000 abstract description 24
- 238000002703 mutagenesis Methods 0.000 abstract description 24
- 231100000350 mutagenesis Toxicity 0.000 abstract description 24
- 239000000725 suspension Substances 0.000 abstract description 18
- 230000004913 activation Effects 0.000 abstract description 17
- 238000000855 fermentation Methods 0.000 abstract description 11
- 230000004151 fermentation Effects 0.000 abstract description 11
- 239000007787 solid Substances 0.000 abstract description 9
- 230000003213 activating effect Effects 0.000 abstract description 8
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 abstract description 7
- 238000009630 liquid culture Methods 0.000 abstract description 7
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Inorganic materials [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 abstract description 7
- 239000002131 composite material Substances 0.000 abstract description 6
- 238000012258 culturing Methods 0.000 abstract description 5
- 238000012216 screening Methods 0.000 abstract description 5
- 238000000605 extraction Methods 0.000 abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000000034 method Methods 0.000 abstract description 3
- 230000002068 genetic effect Effects 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 238000003756 stirring Methods 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 8
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 8
- 239000012043 crude product Substances 0.000 description 7
- 238000001914 filtration Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 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 6
- 239000003814 drug Substances 0.000 description 6
- 239000012065 filter cake Substances 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 238000010438 heat treatment Methods 0.000 description 6
- 238000011218 seed culture Methods 0.000 description 6
- 241000187488 Mycobacterium sp. Species 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 240000008042 Zea mays Species 0.000 description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 235000005822 corn Nutrition 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 4
- 235000019341 magnesium sulphate Nutrition 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000004451 qualitative analysis Methods 0.000 description 4
- 239000004317 sodium nitrate Substances 0.000 description 4
- 235000010344 sodium nitrate Nutrition 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 150000003431 steroids Chemical class 0.000 description 4
- AEMFNILZOJDQLW-QAGGRKNESA-N androst-4-ene-3,17-dione Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 AEMFNILZOJDQLW-QAGGRKNESA-N 0.000 description 3
- AEMFNILZOJDQLW-UHFFFAOYSA-N androstenedione Natural products O=C1CCC2(C)C3CCC(C)(C(CC4)=O)C4C3CCC2=C1 AEMFNILZOJDQLW-UHFFFAOYSA-N 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 241000186361 Actinobacteria <class> Species 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 229910013470 LiC1 Inorganic materials 0.000 description 2
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000006356 dehydrogenation reaction Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000007599 discharging Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 239000010451 perlite Substances 0.000 description 2
- 235000019362 perlite Nutrition 0.000 description 2
- 229940068065 phytosterols Drugs 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- RFDNAAHBIYPJCO-KVSHDJGKSA-N (8S,9S,10R,13R,14S)-10,13-dimethyl-3,5,6,7,8,9,11,12,14,15-decahydro-2H-cyclopenta[a]phenanthrene-1,4-dione Chemical compound C[C@@]12C=CC[C@H]1[C@@H]1CCC3C(CCC([C@]3(C)[C@H]1CC2)=O)=O RFDNAAHBIYPJCO-KVSHDJGKSA-N 0.000 description 1
- ZNWOYQVXPIEQRC-ZRFCQXGJSA-N (8s,9s,10r,13s,14s,17r)-17-(1-hydroxypropan-2-yl)-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-3-one Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(CO)C)[C@@]1(C)CC2 ZNWOYQVXPIEQRC-ZRFCQXGJSA-N 0.000 description 1
- SNMVJSSWZSJOGL-PLOWYNNNSA-N 9alpha-hydroxyandrost-4-en-3,17-dione Chemical compound O=C1CC[C@]2(C)[C@@]3(O)CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 SNMVJSSWZSJOGL-PLOWYNNNSA-N 0.000 description 1
- 241000590020 Achromobacter Species 0.000 description 1
- 241000186063 Arthrobacter Species 0.000 description 1
- 241000186146 Brevibacterium Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 241000589519 Comamonas Species 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 108020005199 Dehydrogenases Proteins 0.000 description 1
- 208000028018 Lymphocytic leukaemia Diseases 0.000 description 1
- 241001467578 Microbacterium Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000187654 Nocardia Species 0.000 description 1
- 241000586779 Protaminobacter Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000009654 indole test Methods 0.000 description 1
- VUZPPFZMUPKLLV-UHFFFAOYSA-N methane;hydrate Chemical compound C.O VUZPPFZMUPKLLV-UHFFFAOYSA-N 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- -1 nitrosoguanidine compound Chemical class 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 238000011085 pressure filtration Methods 0.000 description 1
- 239000000186 progesterone Substances 0.000 description 1
- 229960003387 progesterone Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/32—Mycobacterium
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J7/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms
- C07J7/0005—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21
- C07J7/0065—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by an OH group free esterified or etherified
- C07J7/007—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by an OH group free esterified or etherified not substituted in position 17 alfa
-
- 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
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
-
- 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
- C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/005—Degradation of the lateral chains at position 17
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明提供了一种用于微生物菌株及其选育方法和应用，包括：以分枝杆菌菌株CCTCCNO:M2012522为出发菌株，将菌株经固体活化培养基在斜面或平板培养活化，然后将活化的菌株通过液体培养基制备成菌体浓度为107～108个/mL的第一菌悬液；将第一菌悬液进行紫外‑LiCl复合诱变，分离后培养，挑取单菌落进行活化和发酵；以紫外‑LiCl复合诱变筛选出的菌株制备第二菌悬液，菌体浓度为107～108个/mL，进行亚硝基胍诱变，分离后培养，挑取单菌落进行活化和发酵，筛选后得到双降醇生产菌株。然后对双降醇提取工艺进行优化，获得高纯度的精制产品。与现有技术相比，本发明得到了遗传性状稳定的双降醇生产菌株，优化提取技术，为进一步工业化生产奠定了良好的基础。
Description
技术领域
本发明属于生物发酵技术领域，具体涉及一种用于制备双降醇的微生物菌株及其选育方法和应用。
背景技术
甾体药物是品种多和应用广泛的一大类药物，临床上需求量仅次于抗生素，主要用于治疗风湿病、心血管、胶原性病症、淋巴白血病、肿瘤、细菌性脑炎、皮肤病、内分泌失调、老年性疾病等疾病。我国在甾体药物研究开发与世界先进国家相比还有一定的差距，主要表现为甾体药物合成步骤多，反应复杂，基团的远程效应十分明显，收率低，特别是分离纯化困难。图1为植物甾醇转化双降醇的途径及关键酶系。
双降醇(21-hydroxy-20-methylpregn-4-en-3-one)分子式为C22H34O2，分子量为330.5，是甾体激素类药物的一种重要中间体，可以用作甾体药物黄体酮和氢化可的松的合成，具有重要的商业价值。微生物降解植物甾醇的的工作开始于20世纪60年代，目前已经发现许多微生物可以降解植物甾醇，包括Arthrobacter，Brevibacterium，Pseudomonas，Nocardia，BaciI/us，Streptomyces，Microbacterium，Achromobacter，Protaminobacter，Comamonas，Mycobacterium；fIJRhodococcus等菌种，微生物通过体内酶系高效降解植物甾醇侧链以及氧化母核环。研究表明，9-羟化酶KshA与KshB共同催化形成9-羟基雄烯二酮，从而进一步催化最终降解为二氧化碳和水。研究表明，C22脱氢酶是植物甾醇生成4-雄烯二酮与双降醇的关键酶系，脱氢活力较高时主要向4-雄烯二酮转化，而相对较弱时主要向双降醇途径转化。此外也有研究表明，3-甾酮-Δ1-脱氢酶(3-ketosteroiddelta1-dehydrogenase)可以促使4-雄烯二酮与双降醇分别转化为1，4-雄烯二酮与1，4-双降醇。湖北共同生物科技有限公司通过诱变方法(专利号CN103382445A)成功获得9-羟化酶缺陷菌株，保藏在武汉大学中国典型微生物保藏中心，编号为CCTCCNO:M2012522。本发明在前期的诱变株基础之上进一步采用紫外线-LiCl以及亚硝基胍复合诱变方法得到C1，2脱氢酶以及C22脱氢酶双重缺陷菌株，从而富集转化生产双降醇。
发明内容
本发明的目在于提供一种用于制备双降醇的微生物菌株及其选育方法和应用，得到遗传性状稳定的双降醇生产菌株，同时优化提取工艺，获得高纯度的双降醇精制产品。
为了实现上述的目的，本发明采用以下技术方案：
一种微生物菌株，为分枝杆菌属分支杆菌种，拉丁文为Mycobacterium sp.HBCBA-1，保藏单位为中国典型菌种保藏中心，保藏地址中国武汉大学，保藏日期为2017年6月19日，保藏号为CCTCCNO：M2017349，主要特征为细胞为杆状，革兰氏染色为阳性G+，过氧化氢酶阳性，V.P.反应、甲基红试验以及吲哚试验均为阴性。
一种上述菌株的选育方法，包括以下步骤：
1、菌株及活化：属于放线菌中的分枝杆菌属分枝杆菌种，拉丁名为Mycobacteriumsp，名称为Mycobacteriumsp.GTF-69，保藏单位为中国典型培养物保藏中心，保藏地址在中国武汉武汉大学，保藏日期2012年12月11日，保藏号CCTCCN0:M2012522。将保藏于冷冻甘油管中的分支杆菌采用固体活化培养基接种于斜面或者平板划线活化，28～30℃，2～4天。固体活化培养基配方为(质量百分比)：酵母粉1％，蛋白胨2％，葡萄糖2％，琼脂2％，pH6.8～7.0。
2、紫外-LiCl复合诱变：将上述活化好的分支杆菌挑取单菌落转入液体培养基中，28～30℃，180～220rpm，2～4d。液体培养基配方为(质量百分比)：酵母粉1％，蛋白胨2％，葡萄糖2％，pH6.8～7.0。制成第一菌悬液，浓度为107～108个/mL，对第一菌悬液采用紫外诱变，诱变条件为：紫外灯功率15W，距离为30cm，照射时间为2～8min，将诱变后的第一菌悬液避光进行梯度稀释，然后涂布在LiC1质量浓度分别为0.02％～0.1％的固体活化培养基平板培养基上，28～30℃，3～5d。分离单菌落采用活化培养基活化，28～30℃，180～220rpm，2～3d，取活化后的菌液按2％的接种量接种于种子培养基中，28～32℃，200～250rpm，24～36h。种子培养基配方为(质量百分比)：葡萄糖2％，硝酸钠0.5％，磷酸二氢钾0.05％，硫酸镁0.05％，玉米浆5％，pH7.0～7.2。然后按10～15％的接种量接种于发酵培养基，28～32℃，200～250rpm，4～8d，通过TLC定性分析筛选获得突变株。发酵培养基配方为(质量百分比)：植物甾醇3％，硝酸钠0.5％，磷酸二氢钾0.05％，硫酸镁0.05％，玉米浆5％，豆饼油14～18％，pH7.0～7.2。
3、亚硝基胍诱变：将紫外-LiCl复合诱变筛选的突变株采用液体培养基活化培养，28～30℃，180～220rpm，2～4d，制成第二菌悬液，浓度为107～108个/mL。对第二菌悬液采用亚硝基胍诱变处理，亚硝基胍浓度为40～100μg/mL，诱变时间为20～60min，诱变后将第二菌悬液稀释1000倍，终止诱变，涂布固体活化培养基的平板培养，28～30℃，3～5d。分离单菌落采用活化培养基活化，28～30℃，180～220rpm，2～3d，取活化后的菌液按2％的接种量接种于种子培养基中，28～32℃，200～250rpm，24～36h。通过TLC定性分析筛选获得本发明所述的微生物菌株。
一种上述微生物菌株的应用，将其用于双降醇的提取与精制：将上述菌株按10～15％的接种量接种于发酵培养基，28～32℃，200～250rpm，4～8d，将3L发酵液注入到一个5～8L玻璃瓶，搅拌，水浴加热至70～80℃，用稀硫酸中和至pH3～3.5，加3～6％珍珠岩助滤剂，搅拌1～2h，加20～30mL聚合氯化铝和0.5～2g聚丙烯酰胺，持续搅拌2～3h，停止加热，排出热水，用冷水浴降至45～50℃，过滤油相尽可能抽干，得到黄色粉状滤饼。将黄色粉状滤饼加至5～10L玻璃瓶，搅拌，加入备好的3～5L甲醇溶液(体积比80％)，室温搅拌2～3h，过滤抽干，重复提取2～4次，合并滤液，常压蒸馏掉甲醇，降至室温，过滤得到滤饼，抽干，烘干，得到粗品。将400～600mL甲醇水溶液(体积比80％)注入1～2L三角瓶，加入以上粗品并搅拌，用水浴加热至50～60℃，直至完全溶解，再加入2～3.5g活性炭，保持2～3h，压滤，将滤液常压蒸馏至原体积的一半，常温放置分出结晶，过滤，抽干，烘干得到成品。
附图说明
图1植物甾醇转化双降醇的途径及关键酶系；
图2双降醇成品HPLC分析图。
具体实施方式
下面结合实施例对本发明做进一步详细的描述，但本发明的实施方式不限于此。
实施例1复合诱变获得双降醇生产菌株
1、出发菌株：属于放线菌中的分枝杆菌属分枝杆菌种，拉丁名为Mycobacteriumsp，名称为Mycobacteriumsp.GTF-69，保藏单位为中国典型培养物保藏中心，保藏地址在中国武汉武汉大学，保藏日期2012年12月11日，保藏号CCTCCN0:M2012522。
2、菌株活化：将保藏于冷冻甘油管中的分支杆菌采用固体活化培养基接种于斜面或者平板划线活化，30℃，4d。固体活化培养基配方为(质量百分比)：酵母粉1％，蛋白胨2％，葡萄糖2％，琼脂2％，pH7.0。
3、紫外-LiCl复合诱变：将上述活化好的分支杆菌挑取单菌落转入液体培养基中，30℃，200rpm，3d。液体培养基配方为(质量百分比)：酵母粉1％，蛋白胨2％，葡萄糖2％，pH7.0。制成第一菌悬液，浓度为107个/mL，对第一菌悬液采用紫外诱变。诱变条件为：紫外灯功率15W，距离为30cm，照射时间为6min，将诱变后的第一菌悬液避光进行梯度稀释，然后涂布在LiC1质量浓度为0.06％的固体活化培养基平板培养基上，30℃，5d。分离单菌落采用活化培养基活化，30℃，200rpm，2d，取活化后的菌液按2％的接种量接种于种子培养基中，32℃，220rpm，24h。种子培养基配方为(质量百分比)：葡萄糖2％，硝酸钠0.5％，磷酸二氢钾0.05％，硫酸镁0.05％，玉米浆5％，pH7.0。然后按12％的接种量接种于发酵培养基，32℃，220rpm，5d，通过TLC定性分析筛选获得突变株。发酵培养基配方为(质量百分比)：植物甾醇3％，硝酸钠0.5％，磷酸二氢钾0.05％，硫酸镁0.05％，玉米浆5％，豆饼油16％，pH7.0。
3、亚硝基胍诱变：将紫外-LiCl复合诱变筛选的突变株采用液体培养基活化培养，30℃，200rpm，2d，制成第二菌悬液，浓度为108个/mL。对第二菌悬液采用亚硝基胍诱变处理，亚硝基胍浓度为60μg/mL，诱变时间为40min，诱变后将第二菌悬液稀释1000倍，终止诱变，涂布固体活化培养基的平板培养，30℃，5d。分离单菌落采用活化培养基活化，30℃，200rpm，3d，取活化后的菌液按2％的接种量接种于种子培养基中，32℃，220rpm，24h。通过TLC定性分析筛选获得本发明的微生物菌株。
本发明筛选得到的微生物菌株遗传性状稳定，经过传代20代后生理生化特性都一致。
实施例2双降醇分离纯化与精制
1、粗提：将所述菌株按12％的接种量接种于发酵培养基，32℃，220rpm，5d，将3L发酵液注入到一个5L玻璃瓶，搅拌，水浴加热至80℃，用稀硫酸中和至pH3.5，加3％珍珠岩助滤剂，搅拌1h，加20mL聚合氯化铝和1g聚丙烯酰胺，持续搅拌2h，停止加热，排出热水，用冷水浴降至50℃，过滤油相尽可能抽干，得到黄色粉状滤饼。
2、采用甲醇溶液提取粗品：将黄色粉状滤饼加至10L玻璃瓶，搅拌，加入备好的3L甲醇溶液(体积比80％)，室温搅拌2.5h，过滤抽干，重复提取3次，合并3次滤液，常压蒸馏掉甲醇，降至室温，过滤得到滤饼，抽干，烘干，得到粗品。
3、精制：将400mL甲醇水溶液(体积比80％)注入1L三角瓶，加入以上粗品并搅拌，用水浴加热至60℃，直至完全溶解，再加入3.5g活性炭，保持2h，压滤，将滤液常压蒸馏至原体积的一半，常温放置分出结晶，过滤，抽干，烘干得到成品，通过HPLC分析得纯度为98.29％。
Claims (2)
1.一种微生物菌株，其特征在于：其保藏号为CCTCC NO：M2017349，所述微生物菌株经过传代20代后生理生化特性都一致；
所述菌株为分枝杆菌（Mycobacterium sp.）HBCBA-1。
2.一种权利要求1所述微生物菌株在双降醇的制备中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710566643.6A CN107164286B (zh) | 2017-07-12 | 2017-07-12 | 一种的微生物菌株及其选育方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710566643.6A CN107164286B (zh) | 2017-07-12 | 2017-07-12 | 一种的微生物菌株及其选育方法和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107164286A CN107164286A (zh) | 2017-09-15 |
| CN107164286B true CN107164286B (zh) | 2021-01-08 |
Family
ID=59818318
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710566643.6A Active CN107164286B (zh) | 2017-07-12 | 2017-07-12 | 一种的微生物菌株及其选育方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107164286B (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995016698A1 (en) * | 1993-12-17 | 1995-06-22 | The Upjohn Company | Conversion of bisnoralcohol to bisnoraldehyde |
| CN109971694A (zh) * | 2018-12-16 | 2019-07-05 | 浙江神洲药业有限公司 | 一种在双降醇生产中基因敲除新金色分枝杆菌的方法 |
-
2017
- 2017-07-12 CN CN201710566643.6A patent/CN107164286B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995016698A1 (en) * | 1993-12-17 | 1995-06-22 | The Upjohn Company | Conversion of bisnoralcohol to bisnoraldehyde |
| CN109971694A (zh) * | 2018-12-16 | 2019-07-05 | 浙江神洲药业有限公司 | 一种在双降醇生产中基因敲除新金色分枝杆菌的方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107164286A (zh) | 2017-09-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102827913A (zh) | 微生物混菌发酵制备11α-OH-ADD的方法 | |
| US20210139938A1 (en) | Aspergillus oryzae blcy-006 strain and application thereof in preparation of galactooligosaccharide | |
| WO2007137510A1 (fr) | Souche de bacillus pumilus destinée à produire un rendement élevé de tétraméthylpyrazine | |
| CN109504725B (zh) | 一种发酵猴头菌制备高纯度猴头菌多糖的方法和发酵培养基 | |
| CN101760494A (zh) | 一种采用静息细胞的雄烯二酮生物发酵方法 | |
| CN106754411B (zh) | 一株高产β-D-呋喃果糖苷酶的黑曲霉菌株及其液态发酵产酶方法 | |
| CN110982855A (zh) | 一种高效合成γ-氨基丁酸的生物转化方法 | |
| CN110592169B (zh) | 植物甾醇微生物转化制备add的方法 | |
| CN102061320A (zh) | 11α，17α-二羟基-雄甾-4-烯-3，20-二酮的制备方法 | |
| CN110951815A (zh) | 采用静息细胞生物发酵麦角固醇醚化物制备中间体的方法 | |
| CN107164286B (zh) | 一种的微生物菌株及其选育方法和应用 | |
| CN112358986A (zh) | 一种丁酸梭菌及其在固定化发酵生产 1，3-丙二醇的应用 | |
| CN110527650A (zh) | 一种假诺卡氏菌及其应用 | |
| CN110628860A (zh) | 植物甾醇转化分离9α-OH-AD和甲酯物的方法 | |
| CN109251870B (zh) | 一种新金色分枝杆菌突变株及其在制备hip的应用 | |
| CN110904171A (zh) | 低酒精残留黄原胶产品的制备工艺 | |
| CN110846370A (zh) | 采用生长细胞生物发酵麦角固醇醚化物制备中间体的方法 | |
| WO2022148191A1 (zh) | 一种高产鞘脂类微生物菌株、它的筛选方法和它的用途 | |
| WO2019127829A1 (zh) | 氧化型辅酶q10的发酵生产方法、及由其制备而得的高含量氧化型辅酶q10 | |
| CN113249226B (zh) | 一种提高桑黄生物量及总三萜含量的发酵方法 | |
| CN108085359B (zh) | 一种11α-羟基-4-烯-3,17-雄甾二酮的生产方法 | |
| CN110713509A (zh) | 采用生长细胞生物发酵麦角固醇醚化物制备中间体的方法 | |
| CN114395494B (zh) | 一株塞伯林德纳氏酵母t52及其应用 | |
| CN110156807A (zh) | 黄曲霉oucmdz-2205次级代谢产物的用途 | |
| CN115505537B (zh) | 一种高活性11α-羟基化赭曲霉及其应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20181205 Address after: 442700 Baiguoshu Lute No. 1, Shuidu Industrial Park, Danjiangkou Economic Development Zone, Shiyan City, Hubei Province Applicant after: HUBEI GONGTONG BIOTECHNOLOGY CO.,LTD. Address before: 442700 Baiguoshu Lute No. 1, Shuidu Industrial Park, Danjiangkou Economic Development Zone, Shiyan City, Hubei Province Applicant before: HUBEI GONGTONG BIOTECHNOLOGY CO.,LTD. Applicant before: HUBEI University OF TECHNOLOGY |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A microbial strain and its breeding method and application Effective date of registration: 20231127 Granted publication date: 20210108 Pledgee: Hubei Danjiangkou Rural Commercial Bank Co.,Ltd. Pledgor: HUBEI GONGTONG BIOTECHNOLOGY CO.,LTD. Registration number: Y2023980067680 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right |