(179b) Kinetic Modelling of Cephalexin Synthesis By ?-Amino Ester Hydrolase (AEH) from Xanthomonas Campestris Pv. Campestris: how Substrate Inhibition Affects Reactor Design

β-lactam antibiotics, especially those derived from penicillins and cephalosporins, are continually the most prescribed antibiotics in the world. Semi-synthetic β-lactam antibiotics can be produced enzymatically as an environmentally conscious and cost-effective alternative to traditional chemical synthesis [1]. The enzymatic reaction couples an activated acyl side chain with a β-lactam nucleus to produce cephalexin and amoxicillin, among other β-lactam antibiotics.

α-Amino ester hydrolases (AEHs) comprise a small class of enzymes capable of enantioselective production of semi-synthetic β-lactam antibiotics. Despite their rapid kinetics and high selectivity, AEH use is complicated by low stability and rapid deactivation [2]. Although AEH substrate specificity and reactivity have been thoroughly studied [3], [4], a complete mechanism for AEH catalyzed synthesis of β-lactam antibiotics has yet to be fully realized. While the general mechanism by Youshko and Svedas [5] is applicable under low reactant concentrations, we demonstrate that AEH suffers from substrate inhibition not previously shown.

We present a new kinetic model herein to more fully describe the AEH catalyzed synthesis of cephalexin as well as reactant and product hydrolysis pathways. More specifically, we demonstrate substrate inhibition as well as byproduct inhibition and the impact on cephalexin synthesis rate and selectivity. Based on this newly derived model, we predict the optimum reactor conditions for selective synthesis of cephalexin on a pilot plant scale using AEH and discuss the tradeoffs between fractional yield, conversion, and productivity under different conditions.

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[2] J.K. Blum, M.D. Ricketts, A.S. Bommarius, J. Biotechnol. 160 (2012) 214-221

[3] J.J. Polderman-Tijmes, P.A. Jekel, C.M. Jeronimus-Stratingh, A.P. Bruins, J.-M.Van Der Laan, T. Sonke, D.B. Janssen, J. Biol. Chem. 277 (2002) 28474–28482.

[4] J.K. Blum, A.S. Bommarius, J. Mol. Catal. B: Enzym. 67 (2010) 21-28

[5] M.I. Youshko, and V.K. Åšvedas, Biochemistry (Moscow) 65 (2000) 1367-1375.