The effects of organic cosolvents on the synthesis of ampicillin from phenylglycine methyl ester (PGME) and 6-amino penicillanic acid (6-APA) using immobilized Bacillus megaterium penicillin G acylase have been examined. Several cosolvents were tested for their influence on the enzyme in terms of enzyme stability and hydrophobicity. Among the cosolvents tested, ethylene glycol was found to increase the yield of ampicillin by 39-50%. The effects of ethylene glycol on the pK(a) of PGME, the hydrolysis of ampicillin and PGME, and synthetase/amidase and esterase/amidase ratios were also studied. Experimental data indicated that ethylene glycol inhibited more the hydrolysis of the ampicillin than the hydrolysis of the PGME and the synthetase/amidase ratio varied from 0.2 to 0.88 when the concentration (v/v) of the cosolvent increased from 0 to 40%. The enhancement of the synthesis yield was mainly caused by the reduction in the hydrolysis of acyl donor (PGME) and product (ampicillin) in the water-cosolvent system. (C) 2003 Society of Chemical Industry.