Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
Org Biomol Chem. 2011 Aug 7;9(15):5463-8. doi: 10.1039/c1ob05285c. Epub 2011 Jun 14.
A carbonyl reductase gene (yueD) from Bacillus sp. ECU0013 was heterologously overexpressed in Escherichia coli, and the encoded protein (BYueD) was purified to homogeneity and characterized. The NADPH-dependent reductase showed a broad substrate spectrum towards different aromatic ketones, and α- and β-ketoesters. Although the enantioselectivity was high to moderate for the reduction of α-ketoesters, all the tested β-ketoesters and aromatic ketones were reduced to the corresponding chiral alcohols in enantiomerically pure forms. Furthermore, the practical applicability of this enzyme was evaluated for the reduction of ethyl 4-chloro-3-oxobutanoate (1a). Using Escherichia coli cells coexpressing BYueD and glucose dehydrogenase, 215 g L(-1) (1.3 M) of 1a was stoichiometrically converted to ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-1b) in an aqueous-toluene biphasic system by using a substrate fed-batch strategy, resulting in an overall hydroxyl product yield of 91.7% with enantiomeric purity of 99.6% ee.
来自芽孢杆菌 ECU0013 的羰基还原酶基因 (yueD) 在大肠杆菌中被异源过表达，编码的蛋白 (BYueD) 被纯化至均一性并进行了表征。该 NADPH 依赖性还原酶对不同芳香酮和α-和β-酮酯具有广泛的底物谱。虽然对于α-酮酯的还原具有高到中等的对映选择性，但所有测试的β-酮酯和芳香酮都以对映体纯的形式还原为相应的手性醇。此外，还评估了该酶在还原 4-氯-3-氧代丁酸乙酯 (1a) 中的实际应用。使用共表达 BYueD 和葡萄糖脱氢酶的大肠杆菌细胞，通过使用底物分批进料策略，在水-甲苯两相体系中，将 215 g L(-1)（1.3 M）的 1a 定量转化为乙基 (R)-4-氯-3-羟基丁酸酯 ((R)-1b)，总羟基产物收率为 91.7%，对映体纯度为 99.6%ee。
