The effect of temperature was studied for the kinetically controlled synthesis of cephalexin and ampicillin with penicillin acylase immobilised in glyoxyl agarose. Yield increased at low temperatures in the absence and presence of ethylene glycol, while the initial ratio of synthesis to hydrolysis decreased. Arrhenius equations were used to describe the temperature dependency of the hydrolysis and synthesis rates. The effect of ethylene glycol was stronger over the yield of synthesis of cephalexin than ampicillin. In the case of cephalexin, yield increased from 82.8% in aqueous buffer to 97.6% in 50% (v/v) ethylene glycol medium at 0 degrees C, while at 20 degrees C an increase from 68.8% to 78.7% was obtained. The presence of ethylene glycol produced a greater increase in the energies of activation of the hydrolysis reactions than of the synthesis reactions, which explains the higher conversion yields obtained in the presence of the cosolvent, both for cephalexin and ampicillin. Cephalexin synthesis was optimized using an experimental design based on surface of response methodology. (c) 2006 Elsevier Ltd. All rights reserved.