Enzyme and Microbial Technology 40 (2007) 195–203 Production of cephalexin in organic medium at high substrate concentrations with CLEA of penicillin acylase and PGA-450 A. Illanes a,∗ , L. Wilson a , C. Altamirano a , Z. Cabrera a , L. Alvarez a , C. Aguirre b a School of Biochemical Engineering, Pontificia Universidad Cat´ olica Valpara´ ıso, P.O. Box4059, Valpara´ ıso, Chile b Faculty of Sciences, Universidad Cat´ olica Ssma. Concepci ´ on, P.O. Box 297, Concepci´ on, Chile Received 30 January 2006; received in revised form 10 March 2006; accepted 31 March 2006 Abstract The kinetically controlled synthesis of cephalexin in ethylene glycol was previously optimized at moderate substrate concentrations obtaining yields close to stoichiometric. A study is now presented on the production of cephalexin at very high substrates concentrations, up to 750 mM acyl donor, with immobilized and cross-linked enzyme aggregates (CLEA) of penicillin acylase. Since conversion yield close to 100% was already obtained, attention was given to productivity under the hypothesis that increasing substrates concentration will produce a substantial increase in productivity without reducing yield. An increase of 29 times in volumetric productivity and 4.5 times in specific productivity was obtained with PGA-450 with respect to the results obtained at moderate substrates concentrations (below 100mM acyl donor). Volumetric productivity was lower for CLEA than for PGA-450, but specific productivity was almost the same for both. Sequential batch reactor operations were conducted to assess the biocatalyst operational stability and global productivity, considering one half-life as biocatalyst life cycle. Under such criterion, 40.1 and 135.5g of cephalexin/g of biocatalyst were obtained for PGA-450 and CLEA, respectively. Yields remained close to 100% during the whole cycle. These are very good values which can be improved by optimizing the biocatalyst replacement criterion. © 2006 Elsevier Inc. All rights reserved. Keywords: Penicillin acylase; CLEA; Cephalexin; Organic cosolvents 1. Introduction Penicillin acylase is one of the few examples of successful application of enzymes for the large-scale production of valuable pharmaceuticals [1,2]. Its current industrial application refers to the production of 6 amino penicillanic acid (6APA) from either penicillin G or V; however, the enzyme has the potential for performing several reactions of organic synthesis, including the synthesis of derived penicillins and cephalosporins from the corresponding -lactam nuclei and suitable acyl donors [3]. Syn- thesis can be performed under kinetic [4] or thermodynamic [5] control, but the former is preferred when high conversion yield is a process task [6,7]. In this strategy, the reaction of synthe- sis, that is the nucleophilic attack of the -lactam nucleus to the enzyme-acyl complex, competes with the reactions of hydrol- ysis of both the antibiotic product and the activated acyl donor [8]. The reduction of water activity in the reaction medium is ∗ Corresponding author. E-mail address: aillanes@ucv.cl (A. Illanes). beneficial, since it depresses the competing hydrolytic reactions in favor of synthesis [9,10]. Water activity can be conveniently depressed by using cosolvents, or high concentration of sub- strates, or both. Water miscible organic cosolvents, especially polyols, have proved to be suitable media for the synthesis of -lactam antibi- otics with immobilized penicillin acylase and in fact much higher yields can be obtained than in fully aqueous media [11,12]. However, initial rates are lower in organic cosolvents, so that productivity can be impaired. In addition, depending on the cosolvent, the enzyme can be less stable than in aqueous medium, so that robust biocatalysts are required. This problem has been thoroughly addressed and substantial improvements have been obtained recently for penicillin acylase, considering insolubilization with [13,14] and without inert matrices [15–17], directed immobilization and also genetic manipulations involv- ing site-directed mutagenesis and protein engineering [18,19]. Comprehensive reviews on the subject have been published recently [20,21]. Cross-linking enzyme aggregates (CLEAs), are particularly promising novel biocatalysts for organic syn- thesis: they are robust enough to withstand harsh conditions, 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.03.041