赭曲霉甾体11α–羟化酶基因AOH在毕赤酵母中的表达及功能分析  

Expression and Function of Steroid 11α-Hydroxylase Gene AOH from Aspergillus ochraceus in Pichia pastoris

作　　者：郭凯 曹通 黄琳琳 刘晓光 王正祥 路福平 GUO Kai;CAO Tong;HUANG Linlin;LIU Xiaoguang;WANG Zhengxiang;LU Fuping(Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,Tianjin 300457,China;National Engineering Laboratory for Industrial Enzymes,Tianjin 300457,China;Tianjin Key Laboratory of Industrial Microbiology,Tianjin 300457,China;College of Biotechnology,Tianjin University of Science & Technology,Tianjin 300457,China)

机构地区：[1]天津科技大学生物工程学院工业发酵微生物教育部重点实验室工业酶国家工程实验室天津市工业微生物重点实验室,天津300457

出　　处：《天津科技大学学报》2017年第5期34-38,78,共6页Journal of Tianjin University of Science & Technology

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

摘　　要：11α–羟基左旋乙基甾烯双酮(11α-OH-GD)是生产高效避孕药去氧孕烯的关键中间体.丝状真菌赭曲霉(Aspergillus ochraceus)TCCC41060可以转化左旋乙基甾烯双酮(GD)形成11α-OH-GD,但该转化反应特异性低,副产物偏高,限制了其工业应用.为了研究赭曲霉转化GD特异性偏低的机理,本文克隆了赭曲霉11α–羟化酶基因AOH并构建了表达载体p PIC3.5,K-AOH,获得了重组毕赤酵母菌株.聚丙烯酰胺凝胶电泳(SDS-PAGE)与免疫印迹实验(Western blot)分析表明,AOH重组蛋白相对分子质量为6.12×104,与预测结果一致.对转化产物进行薄层色谱(TLC)和高效液相色谱(HPLC)分析结果显示,AOH重组菌株能够转化GD,形成目标产物11α-OH-GD以及3种副产物.以上结果表明:AOH基因编码的甾体11α–羟化酶对底物GD转化反应的特异性较差.11α-hydroxy-13-ethyl-gon-4-en-3,17-dione(11α-OH-GD)is a key intermediate in the synthesis of desogestrel,amajor ingredient of the third generation of oral contraceptive.Filamentous fungus Aspergillus ochraceus TCCC41060canconvert13-ethyl-gon-4-en-3,17-dione(GD)into11α-OH-GD.However,low reaction specificity and high-level side productslimit its application in large-scale preparation of11α-hydroxy-GD.In order to investigate the mechanism of low reactionspecificity of A.ochraceus TCCC41060on substrate GD,cDNA of11α-hydroxylase gene AOH was cloned and ligated toplasmid pPIC3.5,K in order to generate expression vector pPIC3.5,K-AOH;and then recombinant P.pastoris strains expressingAOH were constructed.SDS-PAGE and Western blot analyses indicated that AOH was6.12×104,which was consistentwith the predicted size.TLC and HPLC analyses showed that the AOH recombinant P.pichia strain was capable of transformingGD into the target product11α-OH-GD and three types of side products.The results indicated that the11α-hydroxylase with AOH gene has poor specificity of conversion reaction on GD.

关 键 词：赭曲霉 11α–左旋乙基甾烯双酮 毕赤酵母 

分 类 号：Q786[生物学—分子生物学]