Improvement of Thermostability of Penicillium expansum Lipase by Site-directed Mutagenesis

Abstract

In order to improve the thermostability of Penicillium expansum lipase(PEL),the lipase gene was mutated by sitedirected mutagenesis.A recombinant plasmid pAO815-ep8-K115R which contains double mutant genes was constructed by overlap extension PCR using the cDNA of a random-mutant lipase ep8(a single site mutant) as the template and two special primers were used to generate another mutation site K115R.The recombinant vector was transformed into Pichia pastoris GS115 by electroporation and the recombinant mutant GS-pAO815-ep8-K115R can secret the double-mutant lipase PEL-ep8K115R-GS into the medium when it was induced by methanol.Thermostability analysis revealed that the residual activity of the double-mutant lipase PEL-ep8-K115R-GS after incubated at 40 ℃ for 30 min was 54% and 27% higher than that of the wild type lipase PEL-GS and the random-mutant lipase PEL-ep8-GS respectively.Tm of the double-mutant lipase PEL-ep8-K115RGS was 42.2 ℃,3.5 ℃ higher than that of the wild type lipase PEL-GS,and 2.0 ℃ higher than that of the random-mutant lipase PEL-ep8-GS.