Jiangsu Key Laboratory of Advanced Catalytic Materials and Technology, Changzhou University, Changzhou, PR China.
Jiangsu Key Laboratory of Advanced Catalytic Materials and Technology, Changzhou University, Changzhou, PR China.
Bioresour Technol. 2014 Nov;172:342-348. doi: 10.1016/j.biortech.2014.09.062. Epub 2014 Sep 22.
An NADH-dependent reductase (SsCR) was discovered by genome data mining. After SsCR was overexpressed in E. coli BL21, recombinant E. coli CCZU-A13 with high reductase activity and excellent stereoselectivity for the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-CHBE) was screened using one high-throughput colorimetric screening strategy. After the reaction optimization, a highly stereoselective bioreduction of COBE into (R)-CHBE (>99% ee) with the resting cells of E. coli CCZU-A13 was successfully demonstrated in n-butyl acetate-water (10:90, v/v) biphasic system. Biotransformation of 600mM COBE for 8h in the biphasic system, (R)-CHBE (>99% ee) could be obtained in the high yield of 100%. Moreover, the broad substrate specificity in the reduction of aliphatic and aromatic carbonyl compounds was also found. Significantly, E. coli CCZU-A13 shows high potential in the industrial production of (R)-CHBE (>99% ee) and its derivatives.
通过基因组数据挖掘发现了一种依赖 NADH 的还原酶（SsCR）。在大肠杆菌 BL21 中过表达 SsCR 后，使用一种高通量比色筛选策略筛选出具有高还原酶活性和对乙基 4-氯-3-氧代丁酸酯（COBE）还原为乙基（R）-4-氯-3-羟基丁酸酯（（R）-CHBE）具有优异对映选择性的重组大肠杆菌 CCZU-A13。在反应优化后，在正丁醇-水（10:90，v/v）两相体系中，用大肠杆菌 CCZU-A13 的静止细胞成功地将 COBE 高度立体选择性地生物还原为（R）-CHBE（>99%ee）。在两相体系中，600mM COBE 转化 8h，（R）-CHBE（>99%ee）的产率高达 100%。此外，还发现了该还原酶在还原脂肪族和芳香族羰基化合物时具有广泛的底物特异性。重要的是，大肠杆菌 CCZU-A13 在（R）-CHBE（>99%ee）及其衍生物的工业生产中具有很高的潜力。
