Improved activity and pH stability of E. coli ATCC 11105 penicillin acylase by error-prone PCR

• Published in: Applied microbiology and biotechnology 2014-05, Vol.98 (10), p.4467-4477
• Main Authors: Balci, Huseyin, Ozturk, Merve Tuzlakoglu, Pijning, Tjaard, Ozturk, Saliha Issever, Gumusel, Fusun
• Format: Article
• Language: English
• Subjects: amidase; Amino Acid Substitution; Amino acids; Antibiotics; benzylpenicillin; Beta lactamases; Biocatalysts; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Chemical properties; Cloning; DNA polymerase; E coli; Enzymatic activity; enzyme activity; Enzyme kinetics; Enzyme Stability; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; evolution; Gene expression; genes; Genetic engineering; High-throughput screening (Biochemical assaying); Hydrogen-Ion Concentration; Industrial production; Life Sciences; Microbial Genetics and Genomics; Microbiology; Mutagenesis; Mutation, Missense; nucleotide sequences; Penicillin; Penicillin Amidase - chemistry; Penicillin Amidase - genetics; Penicillin Amidase - metabolism; Peptides; Plasmids; Point Mutation; Polymerase chain reaction; Polymerase Chain Reaction - methods; Protein Engineering - methods; sequence analysis; Sequence Analysis, DNA
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-013-5476-7

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.