期刊
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
卷 98, 期 10, 页码 4467-4477出版社
SPRINGER
DOI: 10.1007/s00253-013-5476-7
关键词
Directed evolution; Error-prone PCR; E. coli penicillin acylase; 3D homology model; Catalytic activity
Penicillin G acylase is the key enzyme used in the industrial production of beta-lactam antibiotics. This enzyme hydrolyzes penicillin G and related beta-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.
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