1. Significantly Improved Expression and Biochemical Properties of Recombinant Serratia marcescens Lipase as Robust Biocatalyst for Kinetic Resolution of Chiral Ester
- Author
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Yi Wang, Jian Zhao, Suxia Li, Xiao-Bo Mao, Jian-He Xu, Li-Qiang Fan, and Li-Li Zhao
- Subjects
Triacylglycerol lipase ,Gene Expression ,Bioengineering ,Protein Engineering ,Applied Microbiology and Biotechnology ,Biochemistry ,Substrate Specificity ,Kinetic resolution ,Surface-Active Agents ,Affinity chromatography ,Enzyme Stability ,PEG ratio ,Cloning, Molecular ,Lipase ,Molecular Biology ,Serratia marcescens ,Soil Microbiology ,Chromatography ,biology ,Chemistry ,Temperature ,Substrate (chemistry) ,Esters ,Stereoisomerism ,General Medicine ,Hydrogen-Ion Concentration ,biology.organism_classification ,Recombinant Proteins ,Kinetics ,Solubility ,Metals ,Biocatalysis ,biology.protein ,Enantiomer ,Biotechnology - 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 x 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 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.
- Published
- 2010