alpha-Amino acid ester hydrolases (AEHs) catalyze the synthesis of beta-lactam antibiotics containing an alpha-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 similar to 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.