Ampicillin Synthesis Using a Two-Enzyme Cascade System with Both &alpha-Amino Ester Hydrolase and Penicillin G Acylase

The current enzymatic production of semi-synthetic penicillin antibiotics requires isolation and purification of the intermediate 6-aminopenicillanic acid (6-APA), which adds cost and complexity to the manufacturing process.  In this work we take advantage of the unique substrate specificity of α-amino ester hydrolases  (AEH, EC 3.1.1.43), which are specific for α-amino containing β-lactam antibiotics, to perform a purely aqueous one-pot production of ampicillin directly from penicillin G, catalyzed by both AEH and penicillin G acylase (PGA, EC 3.5.1.11).  The synthesis was performed in both a one-pot, one-step synthesis with 39% maximum conversion to ampicillin, and a one-pot, two-step process resulting in a maximum of 47% conversion to ampicillin.  This two-enzyme cascade reaction to semi-synthetic antibiotics is a promising alternative to the current two-step, two-pot enzymatic process with intermittent isolation of 6-APA.