title: Enhancement of the enzymatic activity of Escherichia coli acetyl esterase by a double mutation obtained by random mutagenesis.
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 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.
authors.affiliation: Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, Koriyama, Fukushima 963-8642, Japan.
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Kobayashi
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Hirano
authors.forename: Shigenori
authors.initials: S
authors.lastname: Kanaya
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Haruki
mesh_terms: D000115:Acetylesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D013696:Temperature
chemical_list: D000115:Acetylesterase