摘要
9α-羟基雄甾-4-烯-3,17-二酮(9-OH-AD)是一种重要的甾体药物中间体,可以用来制备β-甾酮,地塞米松和其他类固醇化合物。3-甾酮9α-羟基化酶(KSH)是由两个亚基即末端氧化亚基(KshA)和铁氧还蛋白还原亚基(KshB)构成的。在本研究中,人工合成了来源于分枝杆菌Mycobacterium sp.Strain VKM Ac-1817D的kshA和kshB基因,通过优化表达载体促进了KshA和KshB在E.coli BL21(DE3)中的可溶性表达,并探究了催化体系中KSH还原亚基和氧化亚基的最适添加比例。此外,KSH转化雄甾-4-烯-3,17-二酮(AD)为9-OH-AD的过程中需要辅酶NADH。本研究构建了羟基化反应与利用葡萄糖脱氢酶(GDH)的NADH辅酶再生反应的偶联体系。为了进一步提高转化效率,本研究进行了转化条件的优化,并采取了分批补料的策略,最终9-OH-AD产量为4.78 g/L,转化率为96.7%。此种酶介导的转化生产9-OH-AD的方法为甾体药物生产提供了一种环境友好和经济实用型的新策略。
9α-Hydroxy-4-androstene-3,17-dione(9-OH-AD)is an important intermediates of steroid drugs,which can be used to prepareβ-methasone,dexamethasone and other steroids.3-phytosterone-9α-hydroxylase(KSH)is composed of two subunits,namely,3-phytosterone-9α-hydroxylase(KshA)and 3-phytosterone-9α-hydroxylase reductase(KshB).In this study,the synthetics of kshA and kshB genes from Mycobacterium sp.Strain VKM Ac-1817 D were carried out,and the soluble expression of kshA in E.coli BL21(DE3)was promoted by optimizing the expression vector,and we explored the ratio of 3-phytosterone-9α-hydroxylase oxidase to reductase content in the catalytic systems.In addtion,KSH transform 9α-Hydroxy-4-androstene-3,17-dione(AD)into 9-OH-AD requires Coenzyme NADH.In this paper,we constructed a coupling system of hydroxylation reaction and NADH coenzyme regeneration using glucose dehydrogenase(GDH).In order to improve the conversion efficiency,the optimization of conversion conditions and the application of batch feeding strategy were carried out in this study,and the yield of 9-OH-AD was 4.78 g/L,and the conversion rate was 96.7%.This enzyme-mediated transformation of 9-OH-AD provides an environmentally friendly and economical new strategy for the production of steroid drugs.
作者
沙宗焱
张显
邵明龙
杨套伟
徐美娟
饶志明
Sha Zongyan;Zhang Xian;Shao Minglong;Yang Taowei;Xu Meijuan;Rao Zhiming(The Key Laboratory of Industrial Biotechnology of Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi,214122)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2019年第7期3074-3081,共8页
Genomics and Applied Biology
基金
国家自然科学基金(31570085
31700041
31500065)
江苏省杰出青年科学基金(BK20150002)
中国博士后科学基金资助项目(2017M610297
2015M570407
2016T90421)
江苏省博士后基金资助项目(1701100B)
中央高校基本科研业务费专项资金(JUSRP51708A)
江苏高校优势学科建设工程资助项目共同资助
