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] State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, 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：EI(收录号：20104613387969)

语种：英文

外文关键词：Esters - Gene expression - Recombinant proteins - Escherichia coli - Fatty acids - Affinity chromatography - Lipases - Substrates - Cloning - Metal ions - Metals - Nonionic surfactants - Purification

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 coli. Through the optimization of culture conditions in shake flask, the lipase activity was improved up to 1.09×105 U/l, 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 (C12). 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 °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 mmolmin-1mg-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. ? 2010 Springer Science+Business Media, LLC.