Wang_2010_Appl.Biochem.Biotechnol_162_2387

Reference

Title : Significantly improved expression and biochemical properties of recombinant Serratia marcescens lipase as robust biocatalyst for kinetic resolution of chiral ester - Wang_2010_Appl.Biochem.Biotechnol_162_2387
Author(s) : Wang Y , Zhao J , Xu JH , Fan LQ , Li SX , Zhao LL , Mao XB
Ref : Appl Biochem Biotechnol , 162 :2387 , 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 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.

PubMedSearch : Wang_2010_Appl.Biochem.Biotechnol_162_2387
PubMedID: 20574813

Related information

Substrate Diltiazem

Citations formats

Wang Y, Zhao J, Xu JH, Fan LQ, Li SX, Zhao LL, Mao XB (2010)
Significantly improved expression and biochemical properties of recombinant Serratia marcescens lipase as robust biocatalyst for kinetic resolution of chiral ester
Appl Biochem Biotechnol 162 :2387

Wang Y, Zhao J, Xu JH, Fan LQ, Li SX, Zhao LL, Mao XB (2010)
Appl Biochem Biotechnol 162 :2387