分枝杆菌细胞裂解液催化甾体激素C_(1,2)位脱氢反应的研究

Studies on the 3-Ketosteriod-1-Dehydrogenation of Steroid Hormone by Cellular lysates of Mycobacterium

9β,11β-环氧-17α,21-二羟基-16β-甲基孕-1,4-二烯-3,20-二酮(Ⅳ)是生产9-氟甾体激素的关键前体,以9β,11β-环氧-17α,21-二羟基-16β-甲基孕-4-烯-3,20-二酮-21-醋酸酯(Ⅰ)为底物合成Ⅳ是工业化生产Ⅳ的重要方法。通过比较分枝杆菌全细胞转化法与细胞裂解液转化法,发现分枝杆菌全细胞只能将Ⅰ转化为9β,11β-环氧-17α,21-二羟基-16β-甲基孕-4-烯-3,20-二酮(Ⅱ),而细胞裂解液可以有效地将Ⅰ转化为Ⅳ,其反应机制为底物Ⅰ自发水解为中间体Ⅱ,Ⅱ在C_(1,2)位脱氢酶(KSTD)的催化作用下发生C_(1,2)位脱氢反应生成产物Ⅳ。为进一步提高产物Ⅳ的转化率,利用基因工程手段在分枝杆菌中分别过表达编码KSTD的关键基因:kst D、kst D3和kstD_M,提高脱氢反应效率,结果表明1 g/L底物Ⅰ在pH7.0的重组菌株MS136-kst D_M细胞裂解液中反应45h,Ⅳ的转化率为78.4%,比出发菌株提高了38.9%;并优化缓冲液pH,提高反应速率,结果表明1 g/L底物Ⅰ在pH7.5的重组菌株MS136-kstD_M细胞裂解液中反应45 h,Ⅳ的转化率为92.8%,比出发菌株提高了63.4%。

9β,11β-Epoxypregn-4-ene-17α,21-diol-3,20-dione 21-acetate（ Ⅰ） is a substrate for the production of 9β,11β-Epoxypregn-1,4-diene-17α,21-diol-3,20-dione（ Ⅳ）,which is a key precursor for the production of many 9-fluoro-substituted corticosteroid hormones. By comparing whole cells catalysis and cellular lysates conversion,it was found that whole cells of Mycobacterium sp. MS136 could only convert Ⅰ to 9β,11β-Epoxypregn-4-ene-17α,21-diol-3,20-dione（ Ⅱ）,and Ⅰ can be effectively converted to Ⅳ by cellular lysates.The reaction order is that Ⅰ is spontaneously hydrolyzed to Ⅱ and Ⅱ undergoes C1,2-dehydrogenation reaction to Ⅳ. In order to improve the productivity of Ⅳ,the key genes kst D,kst D3 and kst DMencoding C1,2-dehydrogenase（ KSTD） were overexpressed in Mycobacterium sp. MS136 to enhance the C1,2-dehydrogenation reaction rate,and the results showed that 1 g/L substrate Ⅰ can be converted by recombinant strain MS136-kst DMcellular lysates at pH 7. 0,the productivity of Ⅳ reached 78. 4% after 45 h,which is 38. 9% higher than original strain. The reaction rate is enhanced by optimizing the pH,and the results showed that 1 g/L substrate（ Ⅰ） can be converted by recombinant strain MS136-kst DMcellular lysates at pH 7. 5,the productivity of Ⅳreached 92. 8% after 45 h,which was 63. 4% higher than original strain.