Enhancement of the enzymatic activity of Escherichia coli acetyl esterase by random mutagenesis

Abstract

Random mutagenesis and screening for enzymatic activity was used to engineer Escherichia coli acetyl esterase to enhance its enzymatic activity. A gene encoding E. coli esterase was subjected to PCR random mutagenesis. The mutated esterase gene was expressed in E. coli cells, and the variants were screened for esterase activity by observing halo formation on a tributyrin plate. The screening of a variant with enhanced halo formation allowed us to isolate the Arg48Ser (R48S) mutation, which was predicted to be close to the substrate-binding site. Kinetics analysis using p-nitrophenyl butylate as the substrate demonstrated that the R48S mutant exhibits esterase activity significantly greater than that of the wild-type esterase with the kcat/Km value of the wild-type enzyme by 2.6-fold (3.0-fold in kcat). The replacement of Arg48 by Ala or Glu also increased the kcat value, whereas that by Lys did not. These results suggest that the replacement of Arg by neutral or acidic residues facilitates the release of the fatty acid product due to the loss of interaction between the positive charge of Arg and the negative charge of the fatty acid product, thereby enhancing the catalytic efficiency. Furthermore, kinetics analysis using tributyrin as the substrate demonstrated that the Km and kcat value of the R48S mutant are, respectively, 3.4-times smaller and 2.8-times larger than those of the wild-type enzyme, suggesting that the replacement of Arg48 by Ser could relieve the steric hindrance caused by the bulky alcohol moiety of the substrate, thereby facilitating substrate binding. Unexpectedly, the R48S mutation was found to enhance the thermostability of the enzyme as well. The temperature at which the enzyme activity is inactivated by 50% for a 10-min incubation of the R48S mutant was estimated to be 66 °C, which was 5 °C higher than that of the wild-type enzyme.