文献类型：期刊文献

英文题名：Two-step enzymatic synthesis of ursodeoxycholic acid with a new 7β-hydroxysteroid dehydrogenase from Ruminococcus torques

作者：Zheng, Ming-Min[1];Wang, Ru-Feng[3];Li, Chun-Xiu[1];Xu, Jian-He[1,2]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]E China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, Shanghai 200237, Peoples R China;[3]China Pharmaceut Univ, Dept Pharmacognosy, Nanjing 210038, Jiangsu, Peoples R China

年份：2015

卷号：50

期号：4

起止页码：598

外文期刊名：PROCESS BIOCHEMISTRY

收录：;EI(收录号：20150800548797);WOS:【SCI-EXPANDED(收录号:WOS:000353073100011)】；

基金：This work was financially supported by the National Natural Science Foundation of China (No. 21276082), Ministry of Science and Technology, P.R. China (No. 201103710800, 2011AA02A210 and 2012AA022201), and Shanghai Commission of Science and Technology (No. 11431921600).

语种：英文

外文关键词：Ursodeoxycholic acid; Chenodeoxycholic acid; 7 alpha-Hydroxysteroid dehydrogenase; 7 beta-Hydroxysteroid dehydrogenase; Two-step reaction

摘要：7 beta-Hydroxysteroid dehydrogenase (7 beta-HSDH) is a key enzyme for the efficient biosynthesis of ursodeoxycholic acid (UDCA), an effective pharmaceutical for primary biliary cirrhosis and human cholesterol gallstones. In this work, a new 7 beta-HSDH from Ruminococcus torques ATCC 35915, designated as 7 beta-HSDHRt, was identified and heterologously overexpressed in Escherichia coli for the enzymatic synthesis of ursodeoxycholic acid from chenodeoxycholic acid (COCA). 7 beta-HSDHRt was firstly employed in one-pot mode together with 7 alpha-HSDHCa, another NADPH-dependent 7 alpha-HSDH from Clostridium absonum, to convert CDCA into UDCA without additional coenzyme regeneration. However, the final yield was limited to merely 73%, probably due to chemical equilibrium. Therefore, to enhance the UDCA yield, we alternatively adopted a two-step reaction strategy where the enzymes involved in the first reaction were simply heat-inactivated between the 1st-step reaction (dehydrogenation) and the 2nd-step reaction (hydrogenation), in order to prevent the undesired bioreduction of 7-oxo-LCA into CDCA in the 2nd step. Consequently, the analytic yield of UDCA was significantly improved up to above 98% at a substrate load of 10 mM (Co. 4 g L-1), without any detectable intermediate (7-oxo-LCA) as observed in the case of one-pot reaction. (C) 2015 Elsevier Ltd. All rights reserved.