CN101709322B - Method for synthesizing betulic acid by carrying out biocatalysis on betulin - Google Patents
Method for synthesizing betulic acid by carrying out biocatalysis on betulin Download PDFInfo
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- CN101709322B CN101709322B CN2009101549015A CN200910154901A CN101709322B CN 101709322 B CN101709322 B CN 101709322B CN 2009101549015 A CN2009101549015 A CN 2009101549015A CN 200910154901 A CN200910154901 A CN 200910154901A CN 101709322 B CN101709322 B CN 101709322B
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- FVWJYYTZTCVBKE-ROUWMTJPSA-N betulin Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(CO)CC[C@@H](C(=C)C)[C@@H]5[C@H]4CC[C@@H]3[C@]21C FVWJYYTZTCVBKE-ROUWMTJPSA-N 0.000 title claims abstract description 59
- QGJZLNKBHJESQX-UHFFFAOYSA-N 3-Epi-Betulin-Saeure Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C(=C)C)C5C4CCC3C21C QGJZLNKBHJESQX-UHFFFAOYSA-N 0.000 title claims abstract description 56
- QGJZLNKBHJESQX-FZFNOLFKSA-N betulinic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C(=C)C)[C@@H]5[C@H]4CC[C@@H]3[C@]21C QGJZLNKBHJESQX-FZFNOLFKSA-N 0.000 title claims abstract description 56
- JYDNKGUBLIKNAM-UHFFFAOYSA-N Oxyallobutulin Natural products C1CC(=O)C(C)(C)C2CCC3(C)C4(C)CCC5(CO)CCC(C(=C)C)C5C4CCC3C21C JYDNKGUBLIKNAM-UHFFFAOYSA-N 0.000 title claims abstract description 53
- MVIRREHRVZLANQ-UHFFFAOYSA-N betulin Natural products CC(=O)OC1CCC2(C)C(CCC3(C)C2CC=C4C5C(CCC5(CO)CCC34C)C(=C)C)C1(C)C MVIRREHRVZLANQ-UHFFFAOYSA-N 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 25
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- CLOUCVRNYSHRCF-UHFFFAOYSA-N 3beta-Hydroxy-20(29)-Lupen-3,27-oic acid Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C(O)=O)CCC5(C)CCC(C(=C)C)C5C4CCC3C21C CLOUCVRNYSHRCF-UHFFFAOYSA-N 0.000 claims abstract description 54
- DIZWSDNSTNAYHK-XGWVBXMLSA-N Betulinic acid Natural products CC(=C)[C@@H]1C[C@H]([C@H]2CC[C@]3(C)[C@H](CC[C@@H]4[C@@]5(C)CC[C@H](O)C(C)(C)[C@@H]5CC[C@@]34C)[C@@H]12)C(=O)O DIZWSDNSTNAYHK-XGWVBXMLSA-N 0.000 claims abstract description 54
- PZXJOHSZQAEJFE-UHFFFAOYSA-N dihydrobetulinic acid Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C(O)=O)CCC(C(C)C)C5C4CCC3C21C PZXJOHSZQAEJFE-UHFFFAOYSA-N 0.000 claims abstract description 54
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- NJMWOUFKYKNWDW-UHFFFAOYSA-N 1-ethyl-3-methylimidazolium Chemical compound CCN1C=C[N+](C)=C1 NJMWOUFKYKNWDW-UHFFFAOYSA-N 0.000 description 19
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- WXMVWUBWIHZLMQ-UHFFFAOYSA-N 3-methyl-1-octylimidazolium Chemical compound CCCCCCCCN1C=C[N+](C)=C1 WXMVWUBWIHZLMQ-UHFFFAOYSA-N 0.000 description 4
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
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- PAQVSWFCADWSLB-UHFFFAOYSA-N 3-cyanobenzamide Chemical compound NC(=O)C1=CC=CC(C#N)=C1 PAQVSWFCADWSLB-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/582—Recycling of unreacted starting or intermediate materials
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种生物催化白桦脂醇合成白桦脂酸的方法,包括:(1)黄绿蜜环菌ZJUQH湿细胞的制备;(2)在离子液体或两相体系中加入黄绿蜜环菌ZJUQH湿细胞进行预培养,得到预培养体系;(3)向预培养体系中加入白桦脂醇溶液,于25℃~30℃发酵培养6小时~24小时得到转化后的培养液;(4)转化后的培养液经后处理得到白桦脂酸;其中,所述的两相体系为离子液体与正己烷的混合溶剂或者离子液体与磷酸缓冲溶液的混合溶剂。该方法与传统方法相比,提高了产物的得率,缩短了催化反应时间,且产物分离简单,同时离子液体体系不易挥发,生物相容性好,不污染环境。The invention discloses a method for biocatalyzing betulin to synthesize betulinic acid, which comprises: (1) preparation of wet cells of Armillaria chrysanthemum ZJUQH; (2) adding honeysuckle to ionic liquid or a two-phase system Pre-culture the wet cells of bacteria ZJUQH to obtain a pre-culture system; (3) add betulin solution to the pre-culture system, and ferment and cultivate at 25°C to 30°C for 6 hours to 24 hours to obtain a transformed culture solution; (4) The converted culture solution is post-treated to obtain betulinic acid; wherein, the two-phase system is a mixed solvent of ionic liquid and n-hexane or a mixed solvent of ionic liquid and phosphate buffer solution. Compared with the traditional method, the method improves the yield of the product, shortens the catalytic reaction time, and the separation of the product is simple. At the same time, the ionic liquid system is not easy to volatilize, has good biocompatibility, and does not pollute the environment.
Description
技术领域 technical field
本发明涉及生物工程和微生物发酵领域,尤其涉及一种生物催化白桦脂醇合成白桦脂酸的方法。The invention relates to the fields of bioengineering and microbial fermentation, in particular to a method for biocatalyzing betulin to synthesize betulinic acid.
背景技术 Background technique
生物催化是利用酶或微生物细胞催化进行某种化学反应的过程,具有专一性强、催化效率高、环境友好、操作条件温和等优点。与水相中的生物催化反应相比,非水相生物催化有其独特的优越性。但传统的有机溶剂通常会限制酶的活性和选择性,且会造成大量工业污染。Biocatalysis is the process of using enzymes or microbial cells to catalyze certain chemical reactions. It has the advantages of strong specificity, high catalytic efficiency, environmental friendliness, and mild operating conditions. Compared with biocatalytic reactions in aqueous phase, non-aqueous phase biocatalysis has its unique advantages. However, traditional organic solvents usually limit the activity and selectivity of enzymes, and cause a lot of industrial pollution.
离子液体(Ionic liquids,ILs)作为新型绿色溶剂,是由有机阳离子与无机阴离子或有机阴离子构成的盐类,在室温或较低温(<100℃)下呈液态,具有可忽略的蒸汽压、高热稳定性、高化学稳定性及对许多化合物具有良好的溶解性等特点,作为生物催化反应介质表现出许多优越性能,近年来引起广泛重视。Ionic liquids (ILs), as a new type of green solvent, are salts composed of organic cations and inorganic anions or organic anions, which are liquid at room temperature or lower temperature (<100°C), have negligible vapor pressure, high It has the characteristics of stability, high chemical stability and good solubility to many compounds, and has shown many superior properties as a biocatalytic reaction medium, which has attracted extensive attention in recent years.
2000年,Cull等首次报道了离子液体1-丁基-3-甲基咪唑六氟磷酸盐([bmim][PF6])/水双相体系中Rhodococcus R312细胞催化1,3-二氰基苯水解生成3-氰基苯酰胺反应,并发现[bmim][PF6]对细胞的毒性远远小于甲苯等有机溶剂(Cull SG,et al.Biotechnol Bioeng 2000,69(2):227-233)。随后,有关离子液体中生物催化反应的报道日渐增多,大部分研究表明,酶或微生物整细胞在离子液体中具有较高的活性、选择性及稳定性(Manpreet S,et al.J Mol Catal B:Enzym 2009,56:294-299;He JY,et al.Process Biochem2009,44:316-321)。In 2000, Cull et al. reported for the first time that Rhodococcus R312 cells catalyzed 1,3-dicyano in the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF 6 ])/water biphasic system Benzene was hydrolyzed to generate 3-cyanobenzamide, and it was found that [bmim][PF 6 ] was far less toxic to cells than organic solvents such as toluene (Cull SG, et al.Biotechnol Bioeng 2000, 69(2): 227-233 ). Subsequently, reports on biocatalytic reactions in ionic liquids are increasing day by day, and most studies have shown that enzymes or whole cells of microorganisms have higher activity, selectivity and stability in ionic liquids (Manpreet S, et al.J Mol Catal B : Enzym 2009, 56: 294-299; He JY, et al. Process Biochem 2009, 44: 316-321).
公开号为CN101319236A的中国专利申请中公开了一种以选择性羰基还原酶产生菌为出发菌株,在水/离子液体两相体系中,以潜手性酮为底物还原制备相应手性醇,提高了反应产物的转化率、浓度和对映体过量值,加快了反应的进程。The Chinese patent application with the publication number CN101319236A discloses a selective carbonyl reductase-producing bacterium as a starting strain, in a water/ionic liquid two-phase system, using latent chiral ketones as substrates for reduction to prepare corresponding chiral alcohols, The conversion rate, concentration and enantiomeric excess value of the reaction product are improved, and the reaction process is accelerated.
白桦脂酸(betulinic acid)是非常有价值的天然化合物,它具有抗疟性、抗炎性和抗肿瘤活性,尤其在抗艾滋病活性及抗各种肿瘤细胞系细胞毒性方面可与一些临床用药相比拟,被视为最有潜力的新型药物制剂。目前,白桦脂酸的获得多以自然界中丰富的白桦脂醇为前体,经过化学方法合成,虽然产率较高,但普遍存在操作复杂、污染大、成本高、安全性低等问题,限制了其在生产实践中的应用。微生物转化通过微生物整体细胞中复杂的特异性催化酶系对天然活性成分进行结构修饰,广泛应用于药物前体化合物的转化、生物催化的不对称合成、新药开发、药物代谢体外模型预测等领域。探索以白桦脂醇为前体通过微生物催化途径合成白桦脂酸已成为目前研究重点。Betulinic acid (betulinic acid) is a very valuable natural compound, it has anti-malarial, anti-inflammatory and anti-tumor activities, especially in anti-AIDS activity and anti-cytotoxicity of various tumor cell lines, it can be compared with some clinical drugs In comparison, it is regarded as the most potential new drug preparation. At present, betulinic acid is mostly obtained from betulin, which is abundant in nature, and synthesized by chemical methods. Although the yield is high, there are generally problems such as complicated operation, large pollution, high cost, and low safety. its application in production practice. Microbial transformation modifies the structure of natural active ingredients through the complex specific catalytic enzyme system in the whole cell of microorganisms, and is widely used in the transformation of drug prodrugs, asymmetric synthesis of biocatalysis, new drug development, in vitro model prediction of drug metabolism and other fields. Exploring the synthesis of betulinic acid by using betulin as precursor through microbial catalysis has become the focus of current research.
公开号为CN101457250A的中国专利申请中已经筛选获得一株黄绿密环菌(Armillaria luteo-virens Sacc)ZJUQH,在水相体系中能催化白桦脂醇合成白桦脂酸,并利用响应面方法进一步优化了发酵条件。由于有机相的底物溶液在水相体系中溶解度不高,不但影响酶对底物的催化作用,而且对微生物细胞存在一定毒害,导致前期实验获得的菌株和发酵条件催化白桦脂醇合成白桦脂酸的产率仍较低,且转化反应时间很长。为此,对生物转化白桦脂醇合成白桦脂酸的方法还有待进一步的改进,以期提高白桦脂酸的转化得率。In the Chinese patent application with the publication number CN101457250A, a strain of Armillaria luteo-virens Sacc (Armillaria luteo-virens Sacc) ZJUQH has been screened and obtained, which can catalyze the synthesis of betulinic acid from betulin in the aqueous phase system, and further optimize the fermentation by using the response surface method condition. Due to the low solubility of the substrate solution in the organic phase in the aqueous phase system, it not only affects the catalysis of the enzyme on the substrate, but also has certain toxicity to the microbial cells, resulting in the synthesis of betulin from betulin by the strains and fermentation conditions obtained in previous experiments. The yield of acid is still low, and the conversion reaction time is very long. For this reason, the method for biotransforming betulin to synthesize betulinic acid needs to be further improved in order to increase the conversion yield of betulinic acid.
发明内容 Contents of the invention
本发明提供了一种生物催化白桦脂醇合成白桦脂酸的方法,利用绿色溶剂离子液体或者离子液体与其它溶剂组成的两相体系替代传统的水相体系作为黄绿密环菌ZJUQH催化白桦脂醇合成白桦脂酸的反应介质,将白桦脂醇生物转化成白桦脂酸,具有操作简单、产物易于分离、转化条件温和、转化效率高、转化时间短、安全性高的特点。The invention provides a method for biocatalyzing the synthesis of betulin from betulin, which uses a green solvent ionic liquid or a two-phase system composed of ionic liquid and other solvents to replace the traditional water phase system as the method for the synthesis of betulin catalyzed by the fungus ZJUQH. The reaction medium of betulinic acid is used to bioconvert betulin into betulinic acid, which has the characteristics of simple operation, easy separation of products, mild conversion conditions, high conversion efficiency, short conversion time and high safety.
本发明所使用的黄绿蜜环菌(Armillaria luteo-virens Sacc.)ZJUQHCGMCC No.1884,于2006年12月08日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.1884,其获取、培养方法和形态在中国发明专利CN200610155189.7中有详细描述。Armillaria luteo-virens Sacc. used in the present invention (Armillaria luteo-virens Sacc.) ZJUQHCGMCC No.1884, on December 08, 2006, was preserved in the General Microorganism Center of China Committee for Microorganism Culture Collection, and the preservation number is CGMCC No.1884 , its acquisition, cultivation method and morphology are described in detail in Chinese invention patent CN200610155189.7.
一种生物催化白桦脂醇合成白桦脂酸的方法,包括以下步骤:A method for biocatalyzing betulin to synthesize betulinic acid, comprising the following steps:
(1)将黄绿蜜环菌ZJUQH经过活化和种子培养后得到种子培养液,再经冷冻离心得到黄绿蜜环菌ZJUQH湿细胞;(1) Armillaria chrysanthemum ZJUQH is activated and seed cultured to obtain a seed culture solution, and then refrigerated and centrifuged to obtain Armillaria chrysanthemum ZJUQH wet cells;
(2)在离子液体或含离子液体的两相体系中加入黄绿蜜环菌ZJUQH湿细胞进行预培养,得到预培养体系;(2) adding Armillaria chrysanthemum ZJUQH wet cells to the ionic liquid or the two-phase system containing the ionic liquid for pre-cultivation to obtain a pre-culture system;
(3)向预培养体系中加入白桦脂醇溶液,于25℃~30℃发酵培养6小时~24小时得到转化后的培养液;(3) adding betulin solution to the pre-culture system, fermenting and culturing at 25° C. to 30° C. for 6 hours to 24 hours to obtain the transformed culture solution;
(4)转化后的培养液经后处理得到白桦脂酸;(4) the transformed culture fluid is post-treated to obtain betulinic acid;
其中,所述的白桦脂醇用量以每升离子液体或含离子液体的两相体系计为15mg~150mg。Wherein, the dosage of said betulin is 15 mg-150 mg per liter of the ionic liquid or the two-phase system containing the ionic liquid.
离子液体作为生物催化反应的溶剂,应用模式通常有:纯溶剂体系、作为水的共溶剂、与其他有机溶剂组成两相体系或多相体系。针对具体的生物催化反应需要选择合适的离子液体应用模式,本发明根据白桦脂醇生物转化得到白桦脂酸的转化反应的特点,选择合适的反应体系,来提高转化效率,缩短转化时间。Ionic liquids are used as solvents for biocatalytic reactions. The application modes usually include: pure solvent system, as a co-solvent of water, and a two-phase system or a multi-phase system with other organic solvents. To select a suitable ionic liquid application mode for a specific biocatalytic reaction, the present invention selects a suitable reaction system according to the characteristics of the conversion reaction of betulin biotransformation to obtain betulinic acid, so as to improve the conversion efficiency and shorten the conversion time.
所述的含离子液体的两相体系为离子液体与正己烷的混合溶剂或者离子液体与磷酸缓冲溶液的混合溶剂,优选离子液体与正己烷的混合溶剂。The two-phase system containing ionic liquid is a mixed solvent of ionic liquid and n-hexane or a mixed solvent of ionic liquid and phosphate buffer solution, preferably a mixed solvent of ionic liquid and n-hexane.
所述的磷酸缓冲溶液采用本领域常规的配制方法进行配制即可。The phosphate buffer solution can be prepared by conventional preparation methods in the art.
两相体系中合适的离子液体浓度能增强酶的活性和选择性,因此,所述的含离子液体的两相体系中离子液体的体积百分浓度优选为3%~98%,进一步优选为40%~60%,最优选50%。Appropriate ionic liquid concentration in the two-phase system can enhance the activity and selectivity of the enzyme. Therefore, the volume percent concentration of the ionic liquid in the two-phase system containing the ionic liquid is preferably 3% to 98%, more preferably 40%. % to 60%, most preferably 50%.
氧化还原酶的催化通常需要辅酶,微生物细胞中含有可催化氧化还原反应的多种脱氢酶和辅酶,用微生物整细胞作为生物催化剂可省去酶的分离纯化步骤,同时不需要添加辅酶再生系统。然而,微生物细胞中的辅酶含量有限,添加共基质有助于辅酶的回收利用。因此,可在所述的两相体系中添加共基质,每升离子液体或含离子液体的两相体系中共基质的添加量为0.05mol~0.1mol。The catalysis of redox enzymes usually requires coenzymes. Microbial cells contain a variety of dehydrogenases and coenzymes that can catalyze redox reactions. Using whole cells of microorganisms as biocatalysts can save the separation and purification steps of enzymes, and do not need to add coenzyme regeneration systems . However, the content of coenzymes in microbial cells is limited, and the addition of co-substrates facilitates the recycling of coenzymes. Therefore, a co-substrate can be added to the two-phase system, and the amount of the co-substrate added per liter of the ionic liquid or the two-phase system containing the ionic liquid is 0.05mol˜0.1mol.
其中,所述的共基质选自葡萄糖、乙醇、正丁醇、甘油、蔗糖、异丙醇中的一种。Wherein, the co-substrate is selected from one of glucose, ethanol, n-butanol, glycerol, sucrose, and isopropanol.
所述的离子液体选自1-丁基-3-甲基咪唑四氟硼酸盐([BMIM]BF4)、1-丁基-3-甲基咪唑六氟磷酸盐([BMIM]PF6)、1-乙基-3-甲基咪唑四氟硼酸盐([EMIM]BF4)、1-辛基-3-甲基咪唑六氟磷酸盐([OMIM]PF6)中的一种。可直接采用市售产品,如上海成捷化学有限公司生产的纯度≥99%的离子液体。The ionic liquid is selected from 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BF 4 ), 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF 6 ), 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM]BF 4 ), 1-octyl-3-methylimidazolium hexafluorophosphate ([OMIM]PF 6 ) . Commercially available products can be directly used, such as ionic liquids with a purity of ≥99% produced by Shanghai Chengjie Chemical Co., Ltd.
步骤(3)中,所述的白桦脂醇溶液为白桦脂醇的二甲基亚砜溶液,即溶剂为二甲基亚砜,其中的白桦脂醇浓度优选为7.5mg/mL;白桦脂醇用量优选以每升离子液体或含离子液体的两相体系计为75mg。In step (3), described betulin solution is the dimethyl sulfoxide solution of betulin, namely solvent is dimethyl sulfoxide, and the betulin concentration wherein is preferably 7.5mg/mL; Betulin The amount used is preferably 75 mg per liter of ionic liquid or two-phase system containing ionic liquid.
所述的白桦脂醇可选用根据公开号为CN101328201A中国专利申请中记载的从白桦树皮中提取得到白桦脂醇的方法而自行提取的白桦脂醇,纯度一般为90%,相比购买高纯度白桦脂醇标准样品作为催化底物更具现实意义,不但可以降低生产成本,还可以实现资源的综合利用。The described betulin can be selected from the betulin extracted by itself according to the method of extracting betulin from birch bark recorded in the Chinese patent application whose publication number is CN101328201A, and the purity is generally 90%. Compared with purchasing high-purity The standard sample of betulin as a catalytic substrate has more practical significance, which can not only reduce the production cost, but also realize the comprehensive utilization of resources.
步骤(3)中,所述的发酵培养温度优选为28℃,发酵培养时间优选为18小时,一般可在转速180~220r/min(优选200r/min)的摇床上进行培养。In step (3), the fermentation culture temperature is preferably 28° C., and the fermentation culture time is preferably 18 hours. Generally, the culture can be carried out on a shaker with a rotation speed of 180-220 r/min (preferably 200 r/min).
步骤(1)中,活化所用的斜面培养基为马铃薯葡萄糖琼脂培养基,其原料质量百分比组成为:马铃薯20%,葡萄糖2%,琼脂3%;所述的活化温度为25℃~30℃,活化时间为4天~6天。In step (1), the slant medium used for activation is potato dextrose agar medium, and its raw material mass percentage is composed of: potato 20%, glucose 2%, agar 3%; described activation temperature is 25 ℃~30 ℃, The activation time is 4 days to 6 days.
种子培养所用的种子培养基为马铃薯葡萄糖液体培养基,其原料质量百分比组成为:马铃薯20%,葡萄糖2%,余量为水,pH自然;所述的种子培养温度为25℃~30℃,培养时间为3天,一般在转速为120r/min的摇床中进行培养。The seed medium used for seed cultivation is potato glucose liquid medium, and its raw material mass percentage is composed of: 20% of potatoes, 2% of glucose, the balance is water, and the pH is natural; the seed cultivation temperature is 25°C to 30°C, The culture time is 3 days, and the culture is generally carried out in a shaker with a rotation speed of 120r/min.
将活化后的黄绿蜜环菌ZJUQH制成黄绿蜜环菌ZJUQH孢子悬浮液接入种子培养基,黄绿蜜环菌ZJUQH孢子悬浮液中孢子浓度为1×106个/毫升~1×107个/毫升,种子培养基与接入的黄绿蜜环菌ZJUQH孢子悬浮液的体积比为10~30。The activated Armillaria chrysanthemum ZJUQH is made into the Armillaria chrysanthemum ZJUQH spore suspension and inserted into the seed medium, and the concentration of spores in the Armillaria chrysogenum ZJUQH spore suspension is 1× 106 /ml~1× 10 7 cells/ml, the volume ratio of the seed medium to the inoculated Armillaria chrysanthemum ZJUQH spore suspension is 10-30.
步骤(2)中,所述的黄绿密环菌ZJUQH湿细胞的用量以每升离子液体或含离子液体的两相体系计为50g~400g,优选以每升离子液体或含离子液体的两相体系计为300g;所述的预培养条件为在25℃~30℃培养5分钟~15分钟,优选28℃培养10min,一般可在转速180~220r/min(优选200r/min)摇床上进行培养。In step (2), the amount of the wet cells of Closesia chrysogenum ZJUQH is 50g to 400g per liter of ionic liquid or two-phase system containing ionic liquid, preferably per liter of ionic liquid or two-phase system containing ionic liquid Calculated as 300g; the pre-cultivation condition is to cultivate at 25°C to 30°C for 5 minutes to 15 minutes, preferably at 28°C for 10 minutes, generally on a shaker with a rotation speed of 180 to 220r/min (preferably 200r/min).
步骤(4)中,所述的后处理包括:将转化后的培养液进行离心处理得到上清液,调节pH至3~4,乙酸乙酯萃取后得到萃取液,经浓缩得到白桦脂酸。调节pH值时可采用通用的酸、碱,如氢氧化钠、盐酸等。In step (4), the post-treatment includes: centrifuging the converted culture solution to obtain a supernatant, adjusting the pH to 3-4, extracting with ethyl acetate to obtain an extract, and concentrating to obtain betulinic acid. Common acids and bases, such as sodium hydroxide and hydrochloric acid, can be used to adjust the pH value.
本发明方法中白桦脂醇、白桦脂酸含量同步检测方法为:步骤(4)中转化后的培养液经后处理,用甲醇定容于10ml容量瓶,采用反相高效液相色谱法(RP-HPLC)检测所得物中白桦脂醇、白桦脂酸的含量。RP-HPLC色谱条件:色谱柱为Diamonsil C18(250mm×4.6mm,5μm);流动相为乙腈/水(体积比)=91/9;流速1.0mL/min;检测波长210.1nm;柱温25℃;进样量10μl;跑样时间30~45min。In the inventive method, betulin, betulinic acid content synchronous detection method is: the nutrient solution after conversion in the step (4) is through aftertreatment, is settled in 10ml volumetric flask with methanol, adopts reversed-phase high performance liquid chromatography (RP -HPLC) detect the content of betulin and betulinic acid in the obtained product. RP-HPLC chromatographic conditions: the chromatographic column is Diamonsil C18 (250mm×4.6mm, 5μm); the mobile phase is acetonitrile/water (volume ratio)=91/9; the flow rate is 1.0mL/min; the detection wavelength is 210.1nm; the column temperature is 25°C ; Injection volume 10μl; Sample running time 30-45min.
本发明具有如下有益效果:The present invention has following beneficial effects:
本发明选择离子液体或含离子液体的两相非水相体系用于微生物整细胞催化白桦脂醇合成白桦脂酸,与采用普通水相体系相比,具有以下优点:(1)离子液体或含离子液体的两相体系有利于提高转化效率和产物得率,白桦脂酸的产率可以达到12.17%以上,相比普通水相体系提高了30.58%;(2)离子液体或含离子液体的两相体系大大缩短了催化反应时间,转化时间不超过24小时;(3)离子液体或含离子液体的两相体系中产物分离简单,离子液体易于回收循环使用;(4)离子液体或含离子液体的两相体系不易挥发,生物相容性好,不污染环境,为白桦脂醇生物催化合成白桦脂酸产率的提高提供了新思路,同时为离子液体体系在白桦脂酸催化合成中的应用提供了理论依据。The present invention selects the ionic liquid or the two-phase non-aqueous phase system containing the ionic liquid to use the microbial whole cell to catalyze betulin to synthesize betulinic acid. Compared with the common aqueous phase system, it has the following advantages: (1) the ionic liquid or the non-aqueous phase system containing the ionic liquid The two-phase system of ionic liquid is conducive to improving conversion efficiency and product yield, and the yield of betulinic acid can reach more than 12.17%, which is 30.58% higher than that of ordinary aqueous phase system; (2) ionic liquid or two-phase system containing ionic liquid The phase system greatly shortens the catalytic reaction time, and the conversion time does not exceed 24 hours; (3) the product separation in the ionic liquid or the two-phase system containing the ionic liquid is simple, and the ionic liquid is easy to recycle and reuse; (4) the ionic liquid or the ionic liquid contains The two-phase system is not volatile, has good biocompatibility, and does not pollute the environment. It provides a new idea for the improvement of the yield of betulin biocatalyzed synthesis of betulinic acid, and at the same time provides a new idea for the application of ionic liquid system in the catalytic synthesis of betulinic acid. A theoretical basis is provided.
附图说明 Description of drawings
图1为本发明方法的流程示意图。Fig. 1 is a schematic flow chart of the method of the present invention.
具体实施方式 Detailed ways
实施例1~8 不同两相体系中黄绿密环菌ZJUQH催化白桦脂醇合成白桦脂酸Examples 1-8 Synthesis of betulinic acid from betulin catalyzed by betulin in different two-phase systems in different two-phase systems
(1)将黄绿蜜环菌ZJUQH接入灭菌过的马铃薯葡萄糖琼脂培养基(其原料质量百分比组成为:马铃薯20%,葡萄糖2%和琼脂3%),在28℃,活化4天后制成黄绿蜜环菌ZJUQH孢子悬浮液,然后接入马铃薯葡萄糖液体培养基(其原料质量百分比组成为:马铃薯20%,葡萄糖2%,余量为水,pH自然),在28℃、转速为120r/min的旋转式摇床中培养3天,得到种子培养液。黄绿蜜环菌ZJUQH孢子悬浮液中孢子浓度为1×106个/毫升,马铃薯葡萄糖液体培养基与接入的黄绿蜜环菌ZJUQH孢子悬浮液的体积比为30。(1) Insert Armillaria chrysanthemum ZJUQH into sterilized potato dextrose agar medium (its raw material mass percentage is composed of: potato 20%, glucose 2% and agar 3%), at 28 ℃, after 4 days of activation, prepare Become Armillaria chrysanthemum ZJUQH spore suspension, then insert potato glucose liquid culture medium (its raw material mass percentage is made up of: potato 20%, glucose 2%, surplus is water, pH is natural), at 28 ℃, rotating speed is 120r/min rotary shaker for 3 days to obtain seed culture solution. The concentration of spores in the Armillaria chrysanthemum ZJUQH spore suspension was 1×10 6 /ml, and the volume ratio of the potato dextrose liquid medium to the inserted Armillaria chrysogenum ZJUQH spore suspension was 30.
将种子培养液经3000r/min冷冻离心30分钟,无菌水洗涤3次后除去发酵液获得黄绿蜜环菌ZJUQH湿细胞。The seed culture solution was refrigerated and centrifuged at 3000 r/min for 30 minutes, washed with sterile water for 3 times, and then the fermentation solution was removed to obtain Armillaria chrysanthemum ZJUQH wet cells.
(2)具塞试管中加入5mL离子液体与磷酸缓冲液的混合溶剂或离子液体与正己烷的混合溶剂形成两相体系,两相体系中离子液体的体积浓度为50%,无菌条件下加入上述黄绿密环菌ZJUQH湿细胞,黄绿密环菌ZJUQH湿细胞的用量以每升两相体系计为200g,于28℃,转速200r/min旋转式摇床上预培养10min,得到预培养体系。(2) Add 5 mL of a mixed solvent of ionic liquid and phosphate buffer or a mixed solvent of ionic liquid and n-hexane into a stoppered test tube to form a two-phase system. The volume concentration of the ionic liquid in the two-phase system is 50%. The above-mentioned C. chrysogenum ZJUQH wet cells, the dosage of the C. chrysogenum ZJUQH wet cells is 200 g per liter of the two-phase system, and pre-cultured on a rotary shaker at 28° C. with a rotation speed of 200 r/min for 10 minutes to obtain a pre-culture system.
(3)向上述预培养体系中加入0.1mL浓度为7.5mg/mL白桦脂醇的二甲基亚砜溶液,继续于28℃,转速200rpm旋转式摇床上发酵培养18h得到转化后的培养液。(3) Add 0.1 mL of dimethyl sulfoxide solution with a concentration of 7.5 mg/mL betulin to the above pre-culture system, and continue to ferment and culture on a rotary shaker at 28° C. with a rotation speed of 200 rpm for 18 hours to obtain a transformed culture solution.
(4)将转化后的培养液经离心、调pH至3、乙酸乙酯萃取和旋转蒸发等后处理得到白桦脂酸。(4) Centrifuging the transformed culture solution, adjusting the pH to 3, extracting with ethyl acetate and rotary evaporation to obtain betulinic acid.
以[BMIM]BF4、[BMIM]PF6、[EMIM]BF4、[OMIM]PF6四种离子液体和磷酸缓冲液、正己烷两种溶剂分别组成两相体系进行白桦脂酸的催化合成,结果见表1:Catalytic synthesis of betulinic acid in a two-phase system composed of four ionic liquids [BMIM]BF 4 , [BMIM]PF 6 , [EMIM]BF 4 , and [OMIM]PF 6 and two solvents, phosphate buffer and n-hexane , the results are shown in Table 1:
表1Table 1
由表1可以看出,[EMIM]BF4/正己烷体系中黄绿密环菌ZJUQH催化效果较好,合成白桦脂酸的产率相对较高。It can be seen from Table 1 that the catalytic effect of Clostridium chrysogenum ZJUQH in [EMIM]BF 4 /n-hexane system is better, and the yield of synthetic betulinic acid is relatively high.
实施例9~14 不同离子液体浓度的两相体系中黄绿密环菌ZJUQH催化合成白桦脂酸Examples 9-14 Synthesis of betulinic acid catalyzed by Clostridium chrysogenum ZJUQH in a two-phase system with different ionic liquid concentrations
(1)黄绿蜜环菌ZJUQH湿细胞的制备同实施例1。(1) The preparation of Armillaria chrysanthemum ZJUQH wet cells is the same as in Example 1.
(2)具塞试管中加入5mL离子液体或离子液体与正己烷的混合溶剂形成两相体系,两相体系中离子液体的体积浓度为3%~100%,无菌条件下加入上述黄绿密环菌ZJUQH湿细胞,黄绿密环菌ZJUQH湿细胞的用量以每升两相体系计为200g,于28℃,转速200r/min旋转式摇床上预培养10min,得到预培养体系。(2) Add 5 mL of ionic liquid or a mixed solvent of ionic liquid and n-hexane into a stoppered test tube to form a two-phase system. The volume concentration of the ionic liquid in the two-phase system is 3% to 100%. ZJUQH wet cells, the amount of ZJUQH wet cells of Clostridium chrysogenum chrysogenum ZJUQH is 200 g per liter of the two-phase system, pre-cultured on a rotary shaker at 28° C. with a rotation speed of 200 r/min for 10 minutes to obtain a pre-culture system.
(3)向上述预培养体系中加入0.05mL浓度为7.5mg/mL白桦脂醇的二甲基亚砜溶液,继续于28℃,转速200rpm旋转式摇床上发酵培养18h得到转化后的培养液。(3) Add 0.05 mL of dimethyl sulfoxide solution with a concentration of 7.5 mg/mL betulin to the above pre-culture system, and continue to ferment and culture on a rotary shaker at 28° C. with a rotation speed of 200 rpm for 18 hours to obtain a transformed culture solution.
(4)将转化后的培养液经离心、调pH至4、乙酸乙酯萃取和旋转蒸发等后处理得到白桦脂酸,结果见表2:(4) The converted culture solution was centrifuged, adjusted to pH 4, extracted with ethyl acetate, and rotary evaporated to obtain betulinic acid. The results are shown in Table 2:
表2Table 2
由表2可以看出,离子液体[EMIM]BF4/正己烷两相体系中,黄绿密环菌ZJUQH转化白桦脂醇的反应,在离子液体[EMIM]BF4浓度为50%时白桦脂酸产率最高。It can be seen from Table 2 that in the two-phase system of ionic liquid [EMIM]BF 4 /n-hexane, the reaction of Clostridium chrysogenum ZJUQH to convert betulin, when the concentration of ionic liquid [EMIM]BF 4 is 50%, the production of betulinic acid highest rate.
实施例15~21 不同共基质中黄绿密环菌ZJUQH催化合成白桦脂酸Examples 15-21 Synthesis of betulinic acid catalyzed by Cyclobacter chrysogenum ZJUQH in different co-substrates
(1)黄绿蜜环菌ZJUQH湿细胞的制备同实施例1。(1) The preparation of Armillaria chrysanthemum ZJUQH wet cells is the same as in Example 1.
(2)具塞试管中加入5mL离子液体[EMIM]BF4与正己烷的混合溶剂形成两相体系,两相体系中离子液体的体积浓度为50%,同时在两相体系中添加共基质,每升两相体系中共基质的添加量为0.1mol。无菌条件下加入上述黄绿密环菌ZJUQH湿细胞,黄绿密环菌ZJUQH湿细胞的用量以每升两相体系计为200g,于28℃,转速200r/min旋转式摇床上预培养10min,得到预培养体系。(2) add 5mL ionic liquid [EMIM] BF 4 and the mixed solvent of normal hexane in the stoppered test tube to form a two-phase system, the volume concentration of the ionic liquid in the two-phase system is 50%, add co-substrate in the two-phase system simultaneously, The amount of co-substrate added per liter of the two-phase system is 0.1 mol. Add the above-mentioned C. chrysogenum ZJUQH wet cells under aseptic conditions. The amount of C. chrysogenum ZJUQH wet cells is 200 g per liter of the two-phase system. Pre-cultivate on a rotary shaker at 28 ° C for 10 min at a speed of 200 r/min to obtain pre-cultured system.
(3)向上述预培养体系中加入0.05mL浓度为7.5mg/mL白桦脂醇的二甲基亚砜溶液,继续于28℃,转速200rpm旋转式摇床上发酵培养18h得到转化后的培养液。(3) Add 0.05 mL of dimethyl sulfoxide solution with a concentration of 7.5 mg/mL betulin to the above pre-culture system, and continue to ferment and culture on a rotary shaker at 28° C. with a rotation speed of 200 rpm for 18 hours to obtain a transformed culture solution.
(4)将转化后的培养液经离心、调pH至3.5、乙酸乙酯萃取和旋转蒸发等后处理得到白桦脂酸。(4) Centrifuging the transformed culture solution, adjusting the pH to 3.5, extracting with ethyl acetate and rotary evaporation to obtain betulinic acid.
共基质选取葡萄糖、乙醇、正丁醇、甘油、蔗糖或异丙醇,与未添加共基质的反应体系进行相比,实验结果见表3。Glucose, ethanol, n-butanol, glycerol, sucrose or isopropanol were selected as the co-substrate, and compared with the reaction system without co-substrate, the experimental results are shown in Table 3.
表3table 3
由表3可以看出,离子液体[EMIM]BF4/正己烷两相体系中,添加0.1mol/L正丁醇作为共基质能有效提高黄绿密环菌ZJUQH催化合成白桦脂酸的能力。It can be seen from Table 3 that in the two-phase system of ionic liquid [EMIM]BF 4 /n-hexane, adding 0.1mol/L n-butanol as a co-substrate can effectively improve the ability of Cyclobacterium chrysogenum ZJUQH to catalyze the synthesis of betulinic acid.
实施例22~24 不同底物浓度对黄绿密环菌ZJUQH催化合成白桦脂酸反应的影响Examples 22-24 Effects of different substrate concentrations on the reaction of betulinic acid catalyzed by Clostridium chrysogenum ZJUQH
(1)黄绿蜜环菌ZJUQH湿细胞的制备同实施例1。(1) The preparation of Armillaria chrysanthemum ZJUQH wet cells is the same as in Example 1.
(2)具塞试管中加入5mL离子液体[EMIM]BF4与正己烷的混合溶剂形成两相体系,两相体系中离子液体的体积浓度为50%,同时在两相体系中添加正丁醇,每升两相体系中正丁醇的添加量为0.1mol。无菌条件下加入上述黄绿密环菌ZJUQH湿细胞,黄绿密环菌ZJUQH湿细胞的用量以每升两相体系计为200g,于28℃,转速200r/min旋转式摇床上预培养10min,得到预培养体系。(2) Add 5 mL of ionic liquid [EMIM] BF 4 and a mixed solvent of n-hexane to form a two-phase system in a stoppered test tube, the volume concentration of the ionic liquid in the two-phase system is 50%, and add n-butanol in the two-phase system simultaneously , the amount of n-butanol added per liter of two-phase system is 0.1mol. Add the above-mentioned C. chrysogenum ZJUQH wet cells under aseptic conditions. The amount of C. chrysogenum ZJUQH wet cells is 200 g per liter of the two-phase system. Pre-cultivate on a rotary shaker at 28 ° C for 10 min at a speed of 200 r/min to obtain pre-cultured system.
(3)向上述预培养体系中分别加入0.01mL、0.05mL、0.1mL浓度为7.5mg/mL白桦脂醇的二甲基亚砜溶液,继续于28℃,转速200rpm旋转式摇床上发酵培养18h得到转化后的培养液。(3) Add 0.01mL, 0.05mL, and 0.1mL of dimethyl sulfoxide solution with a concentration of 7.5mg/mL betulin to the above-mentioned preculture system, and continue to ferment and cultivate on a rotary shaker at 28°C with a rotation speed of 200rpm for 18h The transformed culture fluid was obtained.
(4)将转化后的培养液经离心、调pH至3.5、乙酸乙酯萃取和旋转蒸发等后处理得到白桦脂酸,结果见表4。(4) After the converted culture solution was centrifuged, the pH was adjusted to 3.5, ethyl acetate extraction and rotary evaporation were processed to obtain betulinic acid, the results are shown in Table 4.
表4Table 4
由表4可以看出,离子液体[EMIM]BF4/正己烷两相体系中,当添加的白桦脂醇底物溶液为0.05mL时,最有利于白桦脂酸的催化合成。It can be seen from Table 4 that in the ionic liquid [EMIM]BF 4 /n-hexane two-phase system, when the added betulin substrate solution is 0.05mL, the catalytic synthesis of betulinic acid is most beneficial.
实施例25~29 不同湿细胞浓度对黄绿密环菌ZJUQH催化合成白桦脂酸反应的影响Examples 25-29 Effects of Different Wet Cell Concentrations on the Synthesis of Betulinic Acid Catalyzed by Clostridium Chrysogenum ZJUQH
(1)黄绿蜜环菌ZJUQH湿细胞的制备同实施例1。(1) The preparation of Armillaria chrysanthemum ZJUQH wet cells is the same as in Example 1.
(2)具塞试管中加入5mL离子液体[EMIM]BF4与正己烷的混合溶剂形成两相体系,两相体系中离子液体的体积浓度为50%,同时在两相体系中添加正丁醇,每升两相体系中正丁醇的添加量为0.1mol。无菌条件下加入上述黄绿密环菌ZJUQH湿细胞,黄绿密环菌ZJUQH湿细胞的用量以每升两相体系计分别为50g、100g、200g、300g、400g,于28℃,转速200r/min旋转式摇床上预培养10min,得到预培养体系。(2) Add 5 mL of ionic liquid [EMIM] BF 4 and a mixed solvent of n-hexane to form a two-phase system in a stoppered test tube, the volume concentration of the ionic liquid in the two-phase system is 50%, and add n-butanol in the two-phase system simultaneously , the amount of n-butanol added per liter of two-phase system is 0.1mol. Add the above-mentioned C. chrysogenum ZJUQH wet cells under aseptic conditions. The dosage of the ZJUQH wet cells of C. chrysogenum is 50g, 100g, 200g, 300g, 400g per liter of the two-phase system. Pre-cultivate on a shaker for 10 minutes to obtain a pre-culture system.
(3)向上述预培养体系中加入0.05mL浓度为7.5mg/mL白桦脂醇的二甲基亚砜溶液,继续于28℃,转速200rpm旋转式摇床上发酵培养18h得到转化后的培养液。(3) Add 0.05 mL of dimethyl sulfoxide solution with a concentration of 7.5 mg/mL betulin to the above pre-culture system, and continue to ferment and culture on a rotary shaker at 28° C. with a rotation speed of 200 rpm for 18 hours to obtain a transformed culture solution.
(4)将转化后的培养液经离心、调pH至3.5、乙酸乙酯萃取和旋转蒸发等后处理得到白桦脂酸,结果见表5。(4) After the converted culture solution was centrifuged, the pH was adjusted to 3.5, ethyl acetate extraction and rotary evaporation were processed to obtain betulinic acid, the results are shown in Table 5.
表5table 5
由表5可以看出,离子液体[EMIM]BF4/正己烷两相体系中,当添加的黄绿密环菌ZJUQH湿细胞量为300g/L时,白桦脂酸的产率最高。It can be seen from Table 5 that in the two-phase system of ionic liquid [EMIM]BF 4 /n-hexane, the yield of betulinic acid was the highest when the added wet cell amount of Clostridium chrysogenum ZJUQH was 300g/L.
实施例30~33 黄绿密环菌ZJUQH催化合成白桦脂酸反应的转化时间过程Examples 30-33 The conversion time course of the reaction of the synthesis of betulinic acid catalyzed by the fungus ZJUQH
(1)黄绿蜜环菌ZJUQH湿细胞的制备同实施例1。(1) The preparation of Armillaria chrysanthemum ZJUQH wet cells is the same as in Example 1.
(2)具塞试管中加入5mL离子液体[EMIM]BF4与正己烷的混合溶剂形成两相体系,两相体系中离子液体的体积浓度为50%,同时在两相体系中添加正丁醇,每升两相体系中正丁醇的添加量为0.1mol。无菌条件下加入上述黄绿密环菌ZJUQH湿细胞,黄绿密环菌ZJUQH湿细胞的用量以每升两相体系计为200g,于28℃,转速200r/min旋转式摇床上预培养10min,得到预培养体系。(2) Add 5 mL of ionic liquid [EMIM] BF 4 and a mixed solvent of n-hexane to form a two-phase system in a stoppered test tube, the volume concentration of the ionic liquid in the two-phase system is 50%, and add n-butanol in the two-phase system simultaneously , the amount of n-butanol added per liter of two-phase system is 0.1mol. Add the above-mentioned C. chrysogenum ZJUQH wet cells under aseptic conditions. The amount of C. chrysogenum ZJUQH wet cells is 200 g per liter of the two-phase system. Pre-cultivate on a rotary shaker at 28 ° C for 10 min at a speed of 200 r/min to obtain pre-cultured system.
(3)向上述预培养体系中加0.05mL浓度为7.5mg/mL白桦脂醇的二甲基亚砜溶液,继续于28℃,转速200rpm旋转式摇床上发酵培养6~24h,得到转化后的培养液,每隔6h取一次样。(3) Add 0.05 mL of dimethyl sulfoxide solution with a concentration of 7.5 mg/mL betulin to the above-mentioned pre-culture system, continue to ferment and cultivate on a rotary shaker at 28° C. with a rotation speed of 200 rpm for 6 to 24 hours, and obtain the converted The culture medium was sampled every 6 hours.
(4)将转化后的培养液经离心、调pH至3.5、乙酸乙酯萃取和旋转蒸发等后处理得到白桦脂酸,结果见表6。(4) The converted culture solution was centrifuged, adjusted to pH 3.5, extracted with ethyl acetate, and rotary evaporated to obtain betulinic acid. The results are shown in Table 6.
表6Table 6
由表6可以看出,离子液体[EMIM]BF4/正己烷两相体系中,黄绿密环菌ZJUQH催化转化白桦脂醇18h可以获得较高的白桦脂酸产率。It can be seen from Table 6 that in the two-phase system of ionic liquid [EMIM]BF 4 /n-hexane, a higher yield of betulinic acid can be obtained by the catalytic conversion of betulin by Clostridium chrysogenum ZJUQH for 18 hours.
实施例34 两相体系中黄绿密环菌ZJUQH催化合成白桦脂酸Example 34 Synthesis of betulinic acid catalyzed by Cyclobacter chrysogenum ZJUQH in a two-phase system
(1)黄绿蜜环菌ZJUQH湿细胞的制备同实施例1。(1) The preparation of Armillaria chrysanthemum ZJUQH wet cells is the same as in Example 1.
(2)具塞试管中加入5mL离子液体[EMIM]BF4与正己烷的混合溶剂形成两相体系,两相体系中离子液体的体积浓度为50%,同时在两相体系中添加正丁醇,每升两相体系中正丁醇的添加量为0.1mol。无菌条件下加入上述黄绿密环菌ZJUQH湿细胞,黄绿密环菌ZJUQH湿细胞的用量以每升两相体系计为300g,于28℃,转速200r/min旋转式摇床上预培养10min,得到预培养体系。(2) Add 5 mL of ionic liquid [EMIM] BF 4 and a mixed solvent of n-hexane to form a two-phase system in a stoppered test tube, the volume concentration of the ionic liquid in the two-phase system is 50%, and add n-butanol in the two-phase system simultaneously , the amount of n-butanol added per liter of two-phase system is 0.1mol. Add the above-mentioned C. chrysogenum ZJUQH wet cells under aseptic conditions. The amount of C. chrysogenum ZJUQH wet cells is 300 g per liter of the two-phase system. Pre-cultivate on a rotary shaker at 28 ° C for 10 minutes at a speed of 200 r/min to obtain pre-cultured system.
(3)向上述预培养体系中加0.05mL浓度为7.5mg/mL白桦脂醇的二甲基亚砜溶液,继续于28℃,转速200rpm旋转式摇床上发酵培养18h得到转化后的培养液。(3) Add 0.05 mL of dimethyl sulfoxide solution with a concentration of 7.5 mg/mL betulin to the above-mentioned pre-culture system, continue to ferment and culture on a rotary shaker at 28° C. with a rotation speed of 200 rpm for 18 hours to obtain a transformed culture solution.
(4)将转化后的培养液经离心、调pH至3.5、乙酸乙酯萃取和旋转蒸发等后处理得到白桦脂酸,结果见表7。(4) The converted culture solution was centrifuged, adjusted to pH 3.5, extracted with ethyl acetate, and rotary evaporated to obtain betulinic acid. The results are shown in Table 7.
对比例1 水相体系中黄绿密环菌ZJUQH催化合成白桦脂酸Comparative example 1 Synthesis of betulinic acid catalyzed by Cyclobacterium chrysogenum ZJUQH in aqueous phase system
(1)黄绿蜜环菌ZJUQH的活化同实施例1,将活化后的黄绿蜜环菌ZJUQH制得孢子悬浮液接入30mL马铃薯液体培养基(马铃薯质量浓度20%,pH5.0,吐温80质量浓度0.57%,余量为水),置于温度28℃,转速120r/min旋转式摇床上培养3天,获得种子培养液。(1) The activation of Armillaria chrysanthemum ZJUQH is the same as in Example 1, the spore suspension obtained by the activated Armillaria chrysanthemum ZJUQH is inserted into 30mL potato liquid medium (20% potato mass concentration, pH5.0, spit out Temperature 80 (mass concentration 0.57%, the balance is water), placed on a rotary shaker at a temperature of 28° C. and a rotation speed of 120 r/min for 3 days to obtain a seed culture solution.
(2)无菌条件下向上述种子培养液中加入浓度为7.5mg/mL白桦脂醇的二甲基亚砜溶液,白桦脂醇的二甲基亚砜溶液的用量以每升种子培养液计为15mL,继续于28℃,转速120r/min旋转式摇床上发酵培养3天得到转化后的培养液。(2) Add a dimethyl sulfoxide solution with a concentration of 7.5 mg/mL betulin to the above seed culture solution under sterile conditions, and the amount of the dimethyl sulfoxide solution of betulin is calculated per liter of seed culture solution to 15 mL, and continued to ferment at 28° C. on a rotary shaker with a rotational speed of 120 r/min for 3 days to obtain a transformed culture solution.
(3)将转化后的培养液经离心、调pH至3.5、乙酸乙酯萃取和旋转蒸发等后处理得到白桦脂酸,结果见表7。(3) The converted culture medium was centrifuged, adjusted to pH 3.5, extracted with ethyl acetate, and rotary evaporated to obtain betulinic acid. The results are shown in Table 7.
表7Table 7
由表7可以看出,与普通水相体系相比,离子液体[EMIM]BF4/正己烷两相体系更有利于黄绿密环菌ZJUQH催化合成白桦脂醇。It can be seen from Table 7 that the ionic liquid [EMIM]BF 4 /n-hexane two-phase system is more conducive to the synthesis of betulin catalyzed by Clostridium chrysogenum ZJUQH compared with the common aqueous system.
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CN107573397B (en) * | 2017-09-13 | 2019-07-30 | 江苏耐雀生物工程技术有限公司 | A method of preparing betulic acid |
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