表达KsdD重组大肠杆菌的构建及转化工艺优化  

Construction of Escherichia coli Overexpressing Recombinant KsdD and Optimization of Biotransformation Process

作　　者：汤睿 申雁冰[1] 耿宇菡 赵云秋 王敏[1] TANG Rui;SHEN Yan-bing;GENG Yu-han;ZHAO Yun-qiu;WANG Min(Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,Tianjin Key Laboratory of Industrial Microbiology,College of Biotechnology/Tianjin University of Science&Technology,Tianjin 300457,China)

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

出　　处：《山东农业大学学报（自然科学版）》2020年第1期19-24,共6页Journal of Shandong Agricultural University：Natural Science Edition

基　　金：天津市自然科学基金项目(18JCYBJC24700)。

摘　　要：3-甾酮-△1-脱氢酶(KsdD)是催化甾体化合物C1,2位脱氢反应的关键酶,在甾体药物的生产中起着重要作用。本研究首先通过对红球菌、分枝杆菌及简单节杆菌来源的KsdD蛋白进行氨基酸序列分析,并进一步通过酶活比较及底物催化能力的验证,最终筛选获得具有较高C1,2位脱氢活力的脱氢酶KsdDR2。研究了过表达KsdDR2蛋白重组大肠杆菌的转化条件对底物雄甾-4-烯-3,17-二酮(AD)转化的影响,结果表明,在30℃转化条件下,当AD:羟丙基-β-环糊精(HP-β-CD)的摩尔比为1:1.5时,可使底物AD的转化率达99%以上。本研究为利用工程菌株高效生产C1,2位脱氢甾体化合物的研究奠定了理论基础。3-Ketosteroid-Δ1-dehydrogenase(KsdD) is the key enzyme that catalyzes the C1,2 dehydrogenation of steroid compound, which plays an important role in the production of steroid drugs. In this study, the sequences of different KsdD proteins from Rhodococcus sp., Mycobacterium sp. and Arthrobacter simplex were analyzed firstly. KsdDR2 protein with higher activity of C1,2 dehydrogenation was selected after further comparison of enzyme activities and substrate catalytic capacity. Finally, the transformation conditions of Escherichia coli overexpressing recombinant KsdDR2 was optimized for the conversion of 4-androstene-3,17-dione(AD). Results indicated that conversion rate of substrate AD was over 99% with a molar ratio of 1:1.5 hydroxypropyl-beta-cyclodextrin(HP-β-CD) as cosolvent at 30 ℃. This study provides a theoretical foundation for the research on efficient production of C1,2 dehydrogenation steroid compounds by engineered strains.

关 键 词：3-甾酮-△1-脱氢酶 大肠杆菌 微生物转化 雄甾-4-烯-3 17-二酮 

分 类 号：Q815[生物学—生物工程]