(545b) Investigating the Behavior of Penicillin G Acylase From E. Coli in the Presence of Organic Co-Solvents

Beta-lactam antibiotics, such as ampicillin and amoxicillin, comprise 65% of the total antibiotics market with annual sales of $15 billon (Chandel:2008). Semi-synthetic antibiotics are synthesized (either chemically or enzymatically) from a beta-lactam nucleus and a side chain acyl donor. Chemical coupling of the beta-lactam moiety and the side chain acyl donor has dominated the industrial production of semi-synthetic beta-lactam antibiotics since their discovery in the early 1960s (Wegman: 2001). The chemical coupling process generates large amounts of waste and requires highly reactive reagents, large volumes of solvents and low temperatures. The enzymatic synthesis of these antibiotics would be environmentally benign as it minimizes waste, is carried out at ambient temperature and pH, and does not require toxic or hazardous reagents our solvents. The drawback of the enzymatic synthesis is its lower yield (30-40%) in comparison to the chemical synthesis (90%).

The lower yield in the enzymatic synthesis can be attributed to the fact that the enzyme that catalyzes the reaction, penicillin G acylase (PGA), has the ability to act as a hydrolase. As a result, PGA catalyzes the undesired primary hydrolysis of the side chain acyl donor and the secondary hydrolysis of the antibiotic in addition to the desired synthesis reaction. Like most enzymes, PGA shows significantly decreased stability and activity in all but a few organic co-solvents (Illanes: 2001). There are numerous advantages for enzymatic syntheses in organic solvents including increased solubilities of reactants and products, shifting reaction equilibria, and, most important for our purposes, suppressing hydrolysis reactions.

Our research examines the behavior of PGA in the presence of organic co-solvents, including those co-solvents that have nucleophilic capabilities. There have been numerous studies on the effects of different organic co-solvents on the synthesis of semi-synthetic beta-lactam antibiotics, but the majority of them focus on only a handful of solvents at only a few co-solvent concentrations. We will discuss the behavior of wild type PGA and thermostable PGA mutants in a wide variety of organic co-solvents, in terms of both log P values and denaturation capacities, over a wide range of co-solvent volume percentages.