title: Expression and purification of penicillin G acylase enzymes from four different micro-organisms, and a comparative evaluation of their synthesis/hydrolysis ratios for cephalexin.
abstract: Several genes for the enzyme penicillin G acylase, as isolated from four different micro-organisms (Alcaligenes facaelis, Escherichia coli, Kluyvera cryocrescens or Providencia rettgeri) were modified at their carboxy-termini to include His-tag fusions, then were expressed from the plasmid pET-24a(+) in E. coli JM109(DE3) cells. All fusion proteins were next purified to homogeneity in a single step by agar-based Co-IDA chromatography, and were then evaluated as catalysts for the synthesis of cephalexin by a kinetically controlled strategy. We find here that the penicillin G acylase enzyme from K. cryocrescens shows a higher intrinsic synthesis/hydrolysis ratio, when compared to three other enzymes from A. facaelis or P. rettgeri, or E. coli.
authors.affiliation: Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, PR China.
authors.forename: Tianfan
authors.initials: T
authors.lastname: Cheng
authors.forename: Maolin
authors.initials: M
authors.lastname: Chen
authors.forename: Huabao
authors.initials: H
authors.lastname: Zheng
authors.forename: Jingang
authors.initials: J
authors.lastname: Wang
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
authors.forename: Weihong
authors.initials: W
authors.lastname: Jiang
mesh_terms: D042481:Alcaligenes faecalis / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002506:Cephalexin / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D044086:Kluyvera / Q000201:enzymology; D010399:Penicillin Amidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011532:Providencia / Q000201:enzymology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D010399:Penicillin Amidase; D002506:Cephalexin