文献类型：期刊文献

中文题名：四氢噻吩-3-酮还原酶的筛选与表征

英文题名：Screening and Characterization of Tetrahydrothiophene-3-one Reductase

作者：严阳[1];郁惠蕾[1];潘江[1];许建和[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室生物催化与合成生物技术研究室,上海200237

年份：2019

卷号：45

期号：1

起止页码：74

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：CSTPCD;;Scopus;北大核心:【北大核心2017】；CSCD:【CSCD_E2019_2020】；

基金：国家自然科学基金项目(21672063)

语种：中文

中文关键词：羟基还原酶;(R)-四氢噻吩-3-醇;硫培南;酶促不对称还原

外文关键词：ketoreductase;(R)-tetrahydrothiophene-3-ol;sulopenem;asymmetric biosynthesis

摘要：通过基因挖掘技术获得一种来源于炭疽芽胞杆菌的短链脱氢酶BaCR1。它能够催化四氢噻吩-3-酮的不对称还原反应,进而合成碳青霉烯类抗生素硫培南的重要手性中间体(R)-四氢噻吩-3-醇。对BaCR1进行了催化性能表征,结果表明最适反应pH为7.0,最适反应温度为30℃,催化剂转化数(k_(cat))与米氏常数(K_M)之比(k_(cat)/K_M)为0.25 L/(mmol·s)。在100 mL的反应体系中,偶联葡萄糖脱氢酶用于辅酶再生,在底物上载量为50 mmol/L、重组大肠杆菌整细胞(干重)上载量为20 g/L的条件下,无需额外添加辅因子NADPH,反应24 h,转化率大于99%,还原产物的e.e.值为84%,实现了产物的克级制备,体现了酶法不对称还原制备(R)-四氢噻吩-3-醇的潜力。
(R)-Tetrahydrothiophene-3-ol,a key intermediate of sulopenem(a novel 2-sulfanyl pennan compound,used for therapy of livestock bacterial diseases),has been highly desired for pharmaceutical use over the last three decades.Apart from the complicated and environmentally unfriendly chemical synthesis,the most straightforward approach for this synthesis is the biological asymmetric reduction of tetrahydrothiophene-3-one.A high enantioselective ketoreductase is needed to accomplish this reaction.However,it is difficult to screen a high enantioselective ketoreductase towards the nearly spatially symmetrical substrate,tetrahydrothiophene-3-one.Some researches have been conducted to work on this issue,where mere enantiomeric excess(e.e.)or long reaction time was observed.In this study,a new enzyme(designated as BaCR1)from Bacillus anthracis str.Ames by library screening has been explored,which can be used for asymmetric synthesis of(R)-tetrahydrothiophene-3-ol.The characteristics of this enzyme were further investigated after protein purification by affinity adsorption separation with Ni-column.After performing reaction optimization,we found that the optimal pH value and temperature of BaCR1 for catalysis reaction was 7.0 and 30 ℃,respectively.kcat/KM was 0.25 L/(mmol·s),using a substrate concentration ranging from 0.125 mmol/L to 50 mmol/L.The half-life of purified BaCR1 was 9.63 h and 3.04 h at 30 ℃ and 40 ℃,respectively,suggesting good thermostability of BaCR1 in the catalysis reaction system.Finally,(R)-tetrahydrothiophene-3-ol was prepared in 100 mL scale.Without extra addition of NADPH,the reaction conversion could reach 99% with 84% of e.e.at the substrate loading of 50 mmol/L.Considering that this is a nature parent enzyme,the result is reasonably acceptable,which also reveals the potential of enzyme-catalyzed asymmetric synthesis of(R)-tetrahydrothiophen-3-ol.