Significantly improved expression and biochemical properties of recombinant Serratia marcescens lipase as robust biocatalyst for kinetic resolution of chiral ester.
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.
Wang
Yi
Y
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People's Republic of China.
Zhao
Jian
J
Xu
Jian-He
JH
Fan
Li-Qiang
LQ
Li
Su-Xia
SX
Zhao
Li-Li
LL
Mao
Xiao-Bo
XB
D055162:Biocatalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004952:Esters / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics; D012988:Soil Microbiology; D012995:Solubility; D013237:Stereoisomerism; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature
D004952:Esters; D008670:Metals; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D008049:Lipase