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

Content Provider	Springer Nature Link
Author	Wang, Yi Zhao, Jian Xu, Jian He Fan, Li Qiang Li, Su Xia Zhao, Li Li Mao, Xiao Bo
Copyright Year	2010
Abstract	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 × 10$^{5}$ 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 Ca$^{2+}$, Cu$^{2+}$, 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 °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.
Starting Page	2387
Ending Page	2399
Page Count	13
File Format	PDF
ISSN	02732289
Journal	Applied Biochemistry and Biotechnology
Volume Number	162
Issue Number	8
e-ISSN	15590291
Language	English
Publisher	Humana Press Inc
Publisher Date	2010-06-24
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Serratia marcescens ECU1010 Lipase Soluble expression Purification Characterization Enantioselectivity Biocatalytic resolution Biochemistry Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Environmental Engineering Biochemistry Bioengineering Applied Microbiology and Biotechnology Biotechnology

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