title: Increasing synthetic performance of penicillin G acylase from Bacillus megaterium by site-directed mutagenesis.
abstract: Site-directed mutagenesis based on predicted modeled structure of pencillin G acylase from Bacillus megaterium (BmPGA) was followed to increase its performance in the kinetically controlled synthesis of cephalexin with high reactant concentrations of 133 mM 7-amino-desaceto-xycephalosporanic acid (7-ADCA) and 267 mM D: -phenylglycine amide (D-PGA). We directed changes in amino acid residues to positions close to the active site that were expected to affect the catalytic performance of penicillin acylase: alpha Y144, alpha F145, and beta V24. Alpha F145 was mutated into tyrosine, alanine, and leucine. Alpha Y144 and beta V24 were mutated into arginine and phenylalanine, respectively. The S/H ratios of three mutants, BmPGAalpha144R, BmPGAbeta24F, and BmPGAbeta24F+alpha144R, were up to 1.3-3.0 times higher values. Compared to the wild-type BmPGA, BmPGAbeta24F+alpha144R showed superior potential of the synthetic performance, allowing the accumulation of up to twofold more cephalexin at significantly higher conversion rates.
authors.affiliation: Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China.
authors.forename: Jingang
authors.initials: J
authors.lastname: Wang
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
authors.forename: He
authors.initials: H
authors.lastname: Huang
authors.forename: Zhongyi
authors.initials: Z
authors.lastname: Yuan
authors.forename: Dafu
authors.initials: D
authors.lastname: Ding
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
authors.forename: Weihong
authors.initials: W
authors.lastname: Jiang
mesh_terms: D000596:Amino Acids / Q000235:genetics; D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D016297:Mutagenesis, Site-Directed; D010399:Penicillin Amidase / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*
chemical_list: D000596:Amino Acids; D010399:Penicillin Amidase