(126e) Optimization of An α-Amino Ester Hydrolase for the Synthesis of β-Lactam Antibiotics

α-Amino ester hydrolases (αAEHs) are capable of the synthesis and hydrolysis of αamino β-lactam antibiotics such as ampicllin and have only recently been cloned and over-expressed using E. coli. αAEHs are serine hydrolases with a catalytic triad (S174, D307, H340) and carboxylate cluster (D208, E309, D310). The acidic carboxylate cluster has been linked to the enzyme's specificity toward α-amino substrates. Our lab has cloned and characterized an αAEH from the organism Xanthomonas campestris pv. campestris.

The enzymatic synthesis of ampicillin using αAEH is an attractive alternative to using more industrial common penicillin G acylase (PGA) for the enzymatic synthesis of β-lactam antibiotics. Additionally, the unique substrate specificity of αAEHs makes it an interesting candidate for the one-pot synthesis of ampicillin. PGA is competitively inhibited by phenylacetic acid(PAA), which is one of the byproducts of the hydrolysis of penicillin G to make 6-aminopenicillanic acid (6-APA). Due to the lack of an &alpha-amino group on PAA, αAEHs are not inhibited by PAA. Using a two-enzyme system with PGA and αAEH one can synthesize ampicillin directly from penicillin G, thus eliminating the 6-APA isolation step that is required when using PGA alone.

In this work, the stability and substrate profile of this enzyme will be reported. We will discuss results from rational protein engineering around the active site and site directed mutations of the carboxylate cluster to investigate the unique substrate specificity of this protein. Lastly, optimization of the ampicillin synthesis reaction and results from the one-pot synthesis reaction will be reported.