title: Improved activity and pH stability of E. coli ATCC 11105 penicillin acylase by error-prone PCR.
abstract: Penicillin G acylase is the key enzyme used in the industrial production of β-lactam antibiotics. This enzyme hydrolyzes penicillin G and related β-lactam antibiotics releasing 6-aminopenicillanic acid, which is an intermediate in the production of semisynthetic penicillins. To improve the enzymatic activity of Escherichia coli penicillin acylase, sequential rounds of error-prone polymerase chain reaction were applied to the E. coli pac gene. After the second round of evolution, the best mutant M2234 with enhanced activity was selected and analyzed. DNA sequence analyses of M2234 revealed that one amino acid residue (K297I), located far from the center of the catalytic pocket, was changed. This mutant (M2234) has a specific activity 4.0 times higher than the parent enzyme and also displayed higher stability at pH 10.
authors.affiliation: Department of Molecular Biology and Genetics, Gebze Institute of Technology (GIT), 41400, Kocaeli, Turkey, hbalci@gyte.edu.tr.
authors.forename: Huseyin
authors.initials: H
authors.lastname: Balci
authors.forename: Merve Tuzlakoglu
authors.initials: MT
authors.lastname: Ozturk
authors.forename: Tjaard
authors.initials: T
authors.lastname: Pijning
authors.forename: Saliha Issever
authors.initials: SI
authors.lastname: Ozturk
authors.forename: Fusun
authors.initials: F
authors.lastname: Gumusel
mesh_terms: D019943:Amino Acid Substitution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D016296:Mutagenesis; D020125:Mutation, Missense; D010399:Penicillin Amidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering / Q000379:methods; D017422:Sequence Analysis, DNA
chemical_list: D029968:Escherichia coli Proteins; D010399:Penicillin Amidase