title: Efficient enzymatic synthesis of ampicillin by mutant Alcaligenes faecalis penicillin G acylase.
abstract: Semi-synthetic β-lactam antibiotics (SSBAs) are one of the most important antibiotic families in the world market. Their enzymatic synthesis can be catalyzed by penicillin G acylases (PGAs). In this study, to improve enzymatic synthesis of ampicillin, site-saturating mutagenesis was performed on three conserved amino acid residues: βF24, αR146, and αF147 of thermo-stable penicillin G acylase from Alcaligenes faecalis (Af PGA). Four mutants βF24G, βF24A, βF24S, and βF24P were recovered by screening the mutant bank. Kinetic analysis of them showed up to 800-fold increased kcat/Km value for activated acyl donor D-phenylglycine methyl ester (D-PGME). When βF24G was used for ampicillin synthesis under kinetic control at industrially relevant conditions, 95% of nucleophile 6-aminopenicillanic acid (6-APA) was converted to ampicillin in aqueous medium at room temperature while 12% process time is needed to reach maximum product accumulation at 25% enzyme concentration compared with the wild-type Af PGA. Consequently, process productivity of enzymatic synthesis of ampicillin catalyzed by Af PGA was improved by more than 130 times, which indicated an enzyme viable for efficient SSBAs synthesis.
authors: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China.
authors: Senwen
authors: S
authors: Deng
authors: Enzyme and Fermentation Technology Laboratory, College of Light Industry Science and Engineering, Nanjing Forestry University, Nanjing 210037, PR China; Jiangsu Provincial Key Lab of Pulp and Paper Science and Technology, Nanjing Forestry University, Nanjing 210037, PR China. Electronic address: ezhsu@njfu.edu.cn.
authors: Erzheng
authors: E
authors: Su
authors: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China.
authors: Xiaoqiang
authors: X
authors: Ma
authors: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China.
authors: Shengli
authors: S
authors: Yang
authors: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China. Electronic address: dzhwei@ecust.edu.cn.
authors: Dongzhi
authors: D
authors: Wei
mesh_terms: D042481:Alcaligenes faecalis / Q000201:enzymology*; D000667:Ampicillin / Q000378:metabolism*; D010399:Penicillin Amidase / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*
chemical_list: D000667:Ampicillin; D010399:Penicillin Amidase
keywords: Ampicillin; Antibiotics; Enzyme catalysis; Penicillin G acylase; Protein engineering