Enhancement of the enzymatic activity of escherichia coli acetyl esterase by a double mutation obtained by random mutagenesis

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Abstract

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-nitrophenyl butyrate as substrate indicated that the kcat 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 kcat value of L209F. The Km 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 Km value of L97F. These results indicate that a combination of an increase in kcat values due to the L97F mutation and a decrease in Km value due to the L209F mutation renders the kcat/Km value of the double mutant enzyme 29-fold higher than that of the wild-type enzyme.

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Kobayashi, R., Hirano, N., Kanaya, S., & Haruki, M. (2012). Enhancement of the enzymatic activity of escherichia coli acetyl esterase by a double mutation obtained by random mutagenesis. Bioscience, Biotechnology and Biochemistry, 76(11), 2082–2088. https://doi.org/10.1271/bbb.120430

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