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 100 mM 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 135.5 g 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. (c) 2006 Elsevier Inc. All rights reserved.