Cloning, expression and characterization of lipase from Pseudomonas alcaligenes

Abstract:

[Objective] The lipase gene of P. alcaligenes was cloned and expressed in Escherichia coli, the enzymatic properties were characterized. [Methods] The lipase gene was obtained by constructing the genomic library of P. alcaligenes and PCR. Then the gene overexpressed in E. coli BL21(DE3) with plasmid pET30a(+). The recombinant lipase was purified with HisTrapTM affinity chromatography and the enzymatic properties were determined. [Results] A 1 575 bp lipase gene was attained (GenBank assession number: JN674069). The molecular weight of lipase was 55 kD, the optimal substrate of the lipase was p-NPO, the optimal temperature and pH were 35 °C and pH 9.0. The lipase activity was increased to 156% after treated by 1 mmol/L Cu2+ for 30 min. Under the optimum conditions, the specific activity, Km and Vmax of the enzyme were 275 U/mg, 80 μmol/L and 290 mmol/(min·g protein), respectively. [Conclusion] The cloning and expressing of lipase provided the fundamental to the application in chiral resolution.