The development of enzymatic pathways to β-lactamase resistant antibiotics allows for cheaper, faster manufacturing of "second-line" antibiotics used to treat antibiotic resistant bacterial pathogens.  Enzymes have been successfully applied towards the synthesis of "first-line" semi-synthetic antibiotics such as ampicillin and amoxicillin, however, they have not been used in the production more complex β-lactamase resistant antibiotics, such as oxacillin. With increasing resistance by bacteria towards
 the currently used antibiotics, it is crucial to develop enzymes to
 improve manufacturing of β-lactamase resistant antibiotics.

            Penicillin G Acylase (PGA) and α-amino ester hydrolase (AEH) are promising template enzymes for protein engineering of novel biocatalysis toward the synthesis of β-lactamase resistant antibiotics. PGA is used commercially in both the production of 6-amino-penicillanic acid (6-APA) from Penicllin G and the semi-synthesis of antibiotics such as ampicllin and amoxicillin. An alternative enzyme to PGA is α-amino ester hydrolase (AEH).  AEH catalyzes the synthesis and hydrolysis of α-amino acylases, including ampicillin. AEH is less studied than PGA, having only been recently overexpressed in E.coli. in 2001.

            In this work, a putative glutaryl-7-ACA acylase precursor (gaa gene) from Xanthomonas campestris pv. campestris strain ATCC 33913 was cloned, over expressed and characterized in E.coli.  The gaa gene has 93% sequence identity on a nucleotide basis to the a-amino ester hydrolase from Xanthomonas citri strain IFO 3835, with all catalytic residues conserved. The gaa gene shows hydrolytic activity toward the substrate, ampicillin, using an HPLC assay.  Furthermore, a semi-rational design library was developed for the penicillin G. acylase from E.coli to alter the substrate specificity of penicillin G. acylase by modifying the substrate-binding pocket.  An NNK library for the binding pocket residues βF24, αR145, and αF146 was incorportated into the pAc vector. This library was screened for hydrolysis of oxacillin using a filter-lift Schiff base assay that detects the hydrolysis product 6-APA.  Efforts to further characterize the AEH from Xanthomonas campestris pv. campestris and engineer penicillin G. acylase toward improved properties will be reported.