Efficient enzymatic synthesis of ampicillin by mutant Alcaligenes faecalis penicillin G acylase

Senwen Deng; Erzheng Su; Xiaoqiang Ma; Shengli Yang; Dongzhi Wei

doi:10.1016/j.jbiotec.2015.01.004

Efficient enzymatic synthesis of ampicillin by mutant Alcaligenes faecalis penicillin G acylase

J Biotechnol. 2015 Apr 10:199:62-8. doi: 10.1016/j.jbiotec.2015.01.004. Epub 2015 Feb 11.

Authors

Senwen Deng¹, Erzheng Su², Xiaoqiang Ma¹, Shengli Yang¹, Dongzhi Wei³

Affiliations

¹ State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China.
² 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.
³ 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.

PMID: 25681630
DOI: 10.1016/j.jbiotec.2015.01.004

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.

Keywords: Ampicillin; Antibiotics; Enzyme catalysis; Penicillin G acylase; Protein engineering.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Alcaligenes faecalis / enzymology*
Ampicillin / metabolism*
Penicillin Amidase / genetics
Penicillin Amidase / metabolism*
Protein Engineering / methods*

Substances

Ampicillin
Penicillin Amidase