Changing the specificity of α-amino acid ester hydrolase toward para-hydroxyl cephalosporins synthesis by site-directed saturation mutagenesis

• Published in: Biotechnology letters Vol. 34; no. 9; pp. 1719 - 1724
• Main Authors: Ye, Li-Juan, Wang, Lu, Pan, Yue, Cao, Yi
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer-Verlag 01.09.2012; Springer Netherlands; Springer
• Subjects: Antibiotics; Applied Microbiology; Biochemistry; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Carboxylic Ester Hydrolases - genetics; Carboxylic Ester Hydrolases - metabolism; Cephalosporin; cephalosporins; Cephalosporins - biosynthesis; Cloning, Molecular; Crystal structure; Escherichia coli; Escherichia coli - genetics; Esters; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes; Life Sciences; Microbiology; Models, Molecular; mutagenesis; Mutagenesis, Site-Directed - methods; mutants; Original Research Paper; Protein Conformation; Substrate Specificity; Synthesis; Xanthomonas; Xanthomonas - enzymology; Xanthomonas - genetics; Cefprozil; Model product screening; Substrate specificity; Site-directed saturation mutagenesis; α-Amino acid ester hydrolase; Enzyme; Saturation; Site directed mutagenesis; Esterases; Carboxylic ester hydrolases; Screening; Cephalosporin derivatives; α-Amino-acid esterase; Cefprozil Model product screening; Hydrolases; Models
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-012-0955-y

α-Amino acid ester hydrolases (AEHs) catalyze the synthesis of β-lactam antibiotics containing an α-amino group with decreased activity toward antibiotics with a p-hydroxyl group. The AEH gene from Xanthomonas rubrillineans was cloned and expressed in Escherichia coli. Based on the crystal structure of the AEH and cefprozil complex, 13 residues not directly involved in substrate recognition were mutated individually. The resulting ~1,300 mutants were screened for activity using cefprozil as a model product based on spectrophotometric assay in a 96-well format. Mutants with improved cefprozil synthetic activity revealed the particular importance of positions 87, 131 and 175 for specificity. The mutant V131S with the highest initial rates of synthesis toward three p-hydroxyl cephalosporins showed 23 %, 17 % and 64 % increase in maximum product accumulation of cefadroxil, cefprozil and cefatrizine, respectively.