Two plate-based colorimetric assays for screening α-amino acid ester hydrolase with high synthesis/hydrolysis ratio

Lu Wang; Li-Juan Ye; Yue Pan; Yi Cao

doi:10.1016/j.enzmictec.2012.04.009

Two plate-based colorimetric assays for screening α-amino acid ester hydrolase with high synthesis/hydrolysis ratio

Enzyme Microb Technol. 2012 Jul 15;51(2):107-12. doi: 10.1016/j.enzmictec.2012.04.009. Epub 2012 May 3.

Authors

Lu Wang¹, Li-Juan Ye, Yue Pan, Yi Cao

Affiliation

¹ College of Life Sciences, Sichuan University, Chengdu 610064, China.

PMID: 22664195
DOI: 10.1016/j.enzmictec.2012.04.009

Abstract

α-Amino acid ester hydrolases (AEHs) are enzymes of interest to the semi-synthesis of β-lactam antibiotics with α-amino, such as cephalexin and cefaclor. An undesired side reaction, the hydrolysis of α-amino acid ester, had hindered applications in antibiotics synthesis. Although the enzymes' S/H ratio can be increased by protein engineering, such approaches require a suitable screening assay. Such a screening assay has not yet been described for AEHs. In this paper, we report a 96-well plate format screening procedure for AEHs based on two spectrophotometric assays. To reduce the hydrolysis reaction while maintaining synthesis activity, and to evaluate the effectiveness of the screening strategy, we introduced random mutations in part of the aeh gene from Xanthomonas rubrillineans by error-prone PCR. By a parallel plate-based screening strategy, three mutants with improved S/H ratio, R87L, T132N and N219I, were obtained.

Publication types

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

MeSH terms

Acinetobacter / enzymology
Acinetobacter / genetics
Anti-Bacterial Agents / biosynthesis
Anti-Bacterial Agents / chemistry
Bacterial Proteins / chemistry
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Base Sequence
Carboxylic Ester Hydrolases / chemistry
Carboxylic Ester Hydrolases / genetics
Carboxylic Ester Hydrolases / metabolism*
Catalytic Domain
Cefaclor / metabolism
Colorimetry / methods*
DNA, Bacterial / genetics
Enzyme Assays / methods*
Glycine / analogs & derivatives
Glycine / chemistry
Glycine / metabolism
Hydrolysis
Kinetics
Models, Molecular
Mutagenesis, Site-Directed
Protein Engineering
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Xanthomonas / enzymology
Xanthomonas / genetics
beta-Lactams / chemistry
beta-Lactams / metabolism

Substances

Anti-Bacterial Agents
Bacterial Proteins
DNA, Bacterial
Recombinant Proteins
beta-Lactams
2-phenylglycine
Cefaclor
Carboxylic Ester Hydrolases
alpha-amino acid esterase
Glycine