Optimization of cephalexin synthesis with immobilized penicillin acylase in ethylene glycol medium at low temperatures

Abstract

Organic cosolvents, and among them, polyols, are suitable media to perform the enzymatic synthesis of β-lactam antibiotics with immobilized penicillin acylase, because they effectively reduce water activity, depressing hydrolytic reactions in favor of synthesis. Among polyols, ethylene glycol has proven to be particularly suited as reaction medium for their synthesis. Previous studies have shown that pH, temperature, and cosolvent concentration are the most relevant variables in the kinetically controlled synthesis of cephalexin from 7-amino-3-deacetoxy cephalosporanic acid and phenylglycine methyl ester, conversion yield increasing at low temperatures and high cosolvent concentrations. The objective of this work is the optimization of temperature, pH, and ethylene glycol concentration in the kinetically controlled synthesis of cephalexin with immobilized penicillin acylase at lower than ambient temperature in terms of substrate molar conversion yield. Phenylglycine was used as acyl donor and 7-amino-3-deacetoxy cephalosporanic acid was the limiting substrate at 30 mM. Optimization was performed using surface of response methodology, optimum conditions being 12 °C, pH 6.8, and 60% (v/v) ethylene glycol, at which cephalexin yield was close to stoichiometric with respect to the limiting nucleophile, which is unattainable in aqueous medium. Stability of the biocatalyst at optimum conditions for cephalexin synthesis was very high, with a projected half-life of 1500 h, making it a suitable catalyst for the large-scale production of cephalexin.

Introduction

Semi-synthetic cephalosporins are a family of antibiotics of considerable therapeutic and commercial relevance [1]. They are produced, mostly chemically, from leading molecules, like cephalosporin C [2], [3] and penicillin G [4] and can arise either from 7-amino-3-deacetoxy cephalosporanic (7ADCA), like cephalexin [5], cefadroxil [6], and cefaclor [7], or from 7-amino cephalosporanic (7ACA), like cephalotin [8], cefamandole [9], and cefazolin [10].

Biocatalysis can be considered a viable alternative to replace the chemical route in use for the production of semi-synthetic β-lactam antibiotics [3]. Higher specificity and lower environmental burden of biocatalytic processes are paving the way for such substitution [11], but better biocatalysts and higher yields than actually obtained are required [12]. Substantial advances have been made in penicillin acylase biocatalyst design by directed immobilization [13], [14], aggregation [15], [16] and derivatization [17] and fruits are still to come from protein engineering techniques, like site-directed mutagenesis and directed evolution [18], [19]. On the other hand, solvent engineering has been a major breakthrough in enzyme biocatalysis [20], [21]. The use or organic solvents as reaction medium for synthesis with penicillin acylase is appealing, because reduced water activity depresses water driven reactions in favor of synthesis [22], while increasing the proportion of reactive non-ionized species [23]. However, hydrophobic solvents are hardly compatible with penicillin acylase and the process conditions required for synthesis, so that in practice more hydrophilic organic cosolvents are to be preferred, even though they are reportedly deleterious for enzyme activity [24]. There are, however, notable exceptions like polyols, where penicillin acylase is as active and even more stable than in aqueous medium [25], which has been consistently proven in the case of ethylene glycol [26], [27], [28].

Synthesis of derived cephalosporins with penicillin acylase can be conducted under thermodynamic [29], [30], [31] or kinetic control [32], [33], [34].

The first strategy considers the displacement of equilibrium from hydrolysis to synthesis, this is, the direct condensation of the nucleophile and the acyl donor. Non-ionic forms of the substrates are required for the condensation reaction, so that pH will be an important variable in this strategy by altering the ionic equilibria of the nucleophile and the acyl donor substrates. Cosolvents may be helpful in this strategy by altering the pK of the carboxylic acids and by reducing the amount of water that pulls equilibrium in favor of hydrolysis of the condensation product. In this strategy, the yield is determined by the thermodynamic equilibrium of the reaction (which is largely independent of the biocatalyst used) and drastic conditions are usually required to displace it in favor of synthesis, so that very robust biocatalysts are required to perform adequately [44].

Synthesis under kinetic control requires an activated acyl donor, in the form of an ester [35] or an amide [36]. It is usually a better strategy when product yield is the main issue, since product concentration is not limited by the equilibrium of the reaction [37]. In the kinetically controlled synthesis of β-lactam antibiotics, the reaction of synthesis (synthetase activity) will occur simultaneously with the hydrolysis of both the activated acyl donor (esterase activity) and the antibiotic product (amidase activity) [32]. Yields will be then favored by reducing water activity, since the rates of the hydrolytic reactions will be reduced [22]. Because of the shape of its sorption isotherm, ethylene glycol is particularly effective in reducing water activity [38]. Previous studies of cephalexin synthesis with immobilized penicillin acylase, showed a strong impact of ethylene glycol on product yield, as a consequence of the increase in the ratio of synthesis to hydrolysis [39]. Temperature (in the range above ambient) and pH were relevant variables in the synthesis of cephalexin in ethylene glycol medium with another penicillin acylase biocatalyst, yields increasing at lower temperature [40].

This article presents the optimization of the kinetically controlled synthesis of cephalexin with immobilized penicillin acylase in ethylene glycol medium in the range of temperature below ambient. Temperature, pH, and ethylene glycol concentration were selected as the most relevant variables, and optimum conditions were determined using response surface methodology, having cephalexin molar yield as objective function. The hypothesis underlying is that yield should increase at low temperatures and high ethylene glycol concentrations at levels significantly higher than those obtained in aqueous medium and temperatures above ambient.