Wang_2010_Appl.Biochem.Biotechnol_162_2387

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 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 degrees C and within the pH range of 5.0-6.8, giving temperature and pH optima at 40 degrees 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(1)(2)). 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.