Two plate-based colorimetric assays for screening α-amino acid ester hydrolase with high synthesis/hydrolysis ratio.
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.
College of Life Sciences, Sichuan University, Chengdu 610064, China.
Lu
L
Wang
Li-Juan
LJ
Ye
Yue
Y
Pan
Yi
Y
Cao
D000150:Acinetobacter / Q000201:enzymology / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002433:Cefaclor / Q000378:metabolism; D003124:Colorimetry / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D057075:Enzyme Assays / Q000379:methods*; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014974:Xanthomonas / Q000201:enzymology / Q000235:genetics; D047090:beta-Lactams / Q000737:chemistry / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D047090:beta-Lactams; C008852:2-phenylglycine; D002433:Cefaclor; D002265:Carboxylic Ester Hydrolases; C058058:alpha-amino acid esterase; D005998:Glycine