Thanatephorus cucumeris对可托多松19位甲基羟化反应条件的优化  

Optimization of the transformation conditions for the 19-hydroxylation of cortexolone by Thanatephorus cucumeris

作　　者：王玉[1] 张凤禹[1,2] 陈曦[1] 冯进辉[1] 吴洽庆[1] 朱敦明[1] 马延和[1] 

机构地区：[1]中国科学院天津工业生物技术研究所,工业酶国家工程实验室,天津市生物催化技术工程中心,天津300308 [2]中国科学院大学,北京100049

出　　处：《应用与环境生物学报》2016年第5期860-864,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家高技术研究发展计划(863计划)项目(2011AA02A211)资助~~

摘　　要：甾体化合物19位甲基羟化反应是甾体药物合成中脱除19位甲基的关键步骤,需要多步化学反应才能实现,酶催化则一步就能完成.为寻找更多能够实现甾体化合物19位甲基羟化反应的微生物和酶并利用酶法实现这一反应,本研究重新考察丝核薄膜革菌(Thanatephorus cucumeris)催化可托多松19位甲基羟基化的反应,发现除了产生主产物19-OH-可托多松和11β-OH-可托多松外,还产生一种极性与19-OH-可托多松非常相近的产物7β-OH-可托多松,导致19-OH-可托多松分离纯化困难.通过对转化条件进行控制和优化,提高了目标产物19-OH-可托多松的比例,减少了7β-OH-可托多松的生成,在初始p H 9.0,装液量100 m L(250 m L锥形瓶),助溶剂为N,N-二甲基甲酰胺(0.5%,V/V),底物投加时间36 h,底物浓度为0.5 g/L的最适条件下,几乎不产生7β-OH-可托多松,转化60 h后,19-OH-可托多松的产量可达到117 mg/L,而且产物易于分离纯化.本研究对T.cucumeris合成19-OH-可托多松的反应条件进行了控制和优化,使产物易于分离纯化,为进一步从该菌中获得甾体化合物19位羟基化酶奠定了基础.The preparation of 19-hydroxyl steroids,the key intermediates for the synthesis of 19-nor-steroids,requires multistep chemical reactions.Compared to the chemical methods,the 19-hydroxylation of steroids may be achieved with microorganisms or enzymes in one step.The biocatalytic process avoids use of toxic reagents and solvents,and reduces discharge of waste.However,the biocatalytic 19-hydroxylation of steroids has been barely studied.Therefore,searching for biocatalysts for the 19-hydroxylation steroids is of great significance.In this work,we reexamined the biotransformation of cortexolone by Thanatephorus cucumeris through isolating and characterizing the products,and investigating the effects of the incubation and biotransformation conditions on the ratio of the products.In addition to the previously reported two products（19-OHcortexolone and 11β-OH-cortexolone）,a new product 7β-OH-cortexolone was identified.The ratio of 19-OH-cortexolone and 7β-OH-cortexolone was found to depend on the biotransformation conditions.The optimal conditions for formation of 19-OHcortexolone was as follows：initial p H 9.0,medium volume 100 m L（per 250 m L flask）,co-solvent N,N-dimethylformamide（0.5%,V/V）,adding cortexolone（final concentration 0.5 g/L） after inoculation for 36 h.Under the optimal conditions,the concentration of 19-OH-cortexolone reached 117 mg/L and only trace amount of 7β-OH-cortexolone was produced at 60 h.The product 19-OH-cortexolone was easily purified by column chromatography.This work studies optimization of transformation conditions for the 19-hydroxylation of cortexolone by T.cucumeris and lays a foundation for obtaining effective enzyme catalysts for 19-hydroxylation of steroids.

关 键 词：19位羟基化 可托多松 生物转化 丝核薄膜革菌 

分 类 号：TQ460.6[化学工程—制药化工]