Enhancement of the Enzymatic Activity of Escherichia coli Acetyl Esterase by a Double Mutation Obtained by Random Mutagenesis

摘要

A double mutant of Escherichia coli acetyl esterase (EcAE) with enhanced enzymatic activity was obtained by random mutagenesis using error-prone PCR and screening for enzymatic activity by observing halo formation on a tributyrin plate. The mutant contained Leu97Phe (L97F) and Leu209Phe (L209F) mutations. Single mutants L97F and L209F were also constructed and analyzed for kinetic parameters, as well as double mutant L97F/L209F. Kinetic analysis using p-nitro-phenyl butyrate as substrate indicated that the k_(cat) values of L97F and L97F/L209F were larger than that of the wild-type enzyme, by 8.3-fold and 12-fold respectively, whereas no significant change was observed in the k_(cat) value of L209F. The K_m values of L209F and L97F/ L209F were smaller than that of the wild-type enzyme, by 2.9-fold and 2.4-fold respectively, whereas no significant change was observed in the K_m value of L97F. These results indicate that a combination of an increase in k_(cat) values due to the L97F mutation and a decrease in K_m value due to the L209F mutation renders the k_(cat)/K_m value of the double mutant enzyme 29-fold higher than that of the wild-type enzyme.