Artículo de publicación ISIThe effect of co-solvents was studied on the kinetically controlled synthesis of cephalexin with immobilised penicillin acylase (PA). Yield correlated well with the ratio of initial rates of cephalexin synthesis to acyl donor hydrolysis and water activity. Yield was substantially higher in EG than in aqueous medium and was selected to study the effect of (apparent) pH on initial rates of synthesis and hydrolysis. The ratio of initial rates of product synthesis to hydrolysis (r2), adequate to assess the potential for synthesis in kinetically controlled systems under excess acyl donor, was maximal at pH 7.5 and correlated well with yield. The effect of ethylene glycol was very strong with an improvement of 230% in r2, whose value was higher than 1, hardly attainable in aqueous medium. This reflected in a considerable improvement (13%) in product yield, which highlights the potential of EG as a medium for the production of cephalexin.