α-Amino ester hydrolases (αAEHs) are capable of the synthesis and hydrolysis of α-amino β-lactam antibiotics such as ampicllin and have only recently been cloned and over-expressed using E. coli [Barends:2003, Polderman-Tijmes:2002]. The enzymatic synthesis of ampicillin using α-amino ester hydrolases (αAEH) is an attractive alternative to using penicillin G acylase (PGA) for enzymatic synthesis of beta-lactam antibiotics. A 637 amino acids (~72 kDa) putative glutaryl 7-ACA acylase (gaa gene) from Xanthomonas campestris pv. campestris strain ATCC 33913 was identified in a genome study[daSilva:2002]. A homology search indicates that this protein is 93% identical to the known αAEH from Xanthomonas citri strain IFO 3835 and 59% identical to the Acetobacter turbidans strain ATCC 9325. Additionally, the catalytic triad (S174, D338, H370) and carboxylate cluster (D208, E340, D341) residues in the gaa protein are conserved, suggesting the putative glutaryl 7-ACA acylase from X. campestris pv. campestris strain ATCC 33913 belongs to the αAEH subclass of enzymes.

The gaa gene was cloned and expressed in E. coli. The X. campestris pv. campestris αAEH was characterized for activity, temperature, pH, and substrate specificity. The resulting kinetic parameters (kcat, KM) of ampicillin hydrolysis of this protein are comparable to other αAEH characterized from A. turbidans and X. citri proteins. Results on expression, optimal processing parameters and efforts to alter the substrate specificity of the X. campestris pv. campestris αAEH toward larger β-lactam resistant semi-synthetic antibiotics using site-directed mutagenesis around the active site will be discussed.