Improvement of the Optimum Temperature of Penicillium expansum Lipase by Site-Directed Mutagenesis

Abstract

In order to improve the optimum temperature of lipases, the Penicillum expansum lipase (PEL) gene was mutated by site-directed mutagenesis using overlap extension PCR technique. The recombinant plasmid containing mutant E83V pPIC3.5K-lip-E83V was expressed in Pichia pastoris GS115. Comparison experiments of the mutant PEL-E83V-GS and the wild-type PEL-GS showed that the optimum temperature (45 °C) of the mutant was 5 °C higher than that of the wild type. The thermostability of the mutant was similar to that of the wild type. The enzymatic activity of the mutant was 188 U/ml at 37 °C, which was 80% that of the wild type in the same conditions. Hydrophobic interaction may be enhanced in the surface region by the hydrophilic amino acid Glu substituted with the hydrophobic amino acid Val, and may be responsible for the improvement of the optimum temperature.