The synthesis of cephalexin with glyoxyl-agarose immobilised penicillin acylase (GAPA) in 50% (v/v) ethylene glycol at 90 mM acyl donor was optimised in terms of pH and temperature, using molar yield as objective function. Yield was substantially increased by the presence of the cosolvent and stability was also higher than in aqueous medium. pH and temperature optima were 7.0 and 28 degreesC and maximum yield was 96%, substantially higher than previously obtained with a commercial biocatalyst. The operational stability of the biocatalyst was determined as a half-life of 135 h at 28 degreesC. Synthesis of cephalexin at low temperatures is being studied to determine if the increase in biocatalyst stability will outweigh the longer times and higher enzyme loads required. Optimisation is still to be conducted at higher substrate concentrations. Reduction in acyl donor excess is advisable but it will reduce yield. (C) 2003 Elsevier Ltd. All rights reserved.