文献类型：期刊文献

英文题名：Significantly Improved Expression and Biochemical Properties of Recombinant Serratia marcescens Lipase as Robust Biocatalyst for Kinetic Resolution of Chiral Ester

作者：Wang, Yi[1];Zhao, Jian[1];Xu, Jian-He[1];Fan, Li-Qiang[1];Li, Su-Xia[1];Zhao, Li-Li[1];Mao, Xiao-Bo[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2010

卷号：162

期号：8

起止页码：2387

外文期刊名：APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000284295000024)】；

基金：This research was financially supported by the National High Technology Research and Development Program of China (No. 2007AA02Z225) and China National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204).

语种：英文

外文关键词：Serratia marcescens ECU1010; Lipase; Soluble expression; Purification; Characterization; Enantioselectivity; Biocatalytic resolution

摘要：A lipase gene from Serratia marcescens ECU1010 was cloned into expression vector pET28a, sequenced, and overexpressed as an N terminus His-tag fusion protein in Escherichia colt. Through the optimization of culture conditions in shake flask, the lipase activity was improved up to 1.09 x 10(5) U/1, which is a great improvement compared to our previous reports. It was purified to homogeneity by Ni-NTA affinity chromatography with an overall yield of 59.4% and a purification factor of 2.4-fold. This recombinant lipase displayed excellent stability below 30 C and within the pH range of 5.0-6.8, giving temperature and pH optima at 40 C and pH 9.0, respectively. The lipase activity was found to increase in the presence of metal ions such as Ca2+, Cu2+, and some nonionic surfactants such as PEG series. In addition, among p-nitrophenyl esters of fatty acids with varied chain length, the recombinant lipase showed the maximum activity on p-nitrophenyl laurate (C-12). Using racemic trans-3-(4'-methoxy-phenyl)-glycidyl methyl ester [(+/-)-MPGM] as substrate, which is a key chiral synthon for production of diltiazem, a 50% conversion yield was achieved after 4 h in toluene-water (100 mM KPB phosphate buffer, pH 7.5) biphasic system (5:5 ml) at 30 degrees C under shaking condition (160 rpm), affording (-)-MPGM in nearly 100% ee. The K-m and V-max values of the lipase for ()-MPGM were 222 mM and 1.24 mmol min(-1) mg(-1), respectively. The above-mentioned features make the highly enantioselective lipase from Serratia marcescens ECU1010 a robust biocatalyst for practical use in large-scale production of diltiazem intermediate.