The efficient enzymatic synthesis of antoxicillin using anhydrous tert-pentanol as a novel media has been demonstrated for the first time. p-OH-Phenylglycine methyl ester (HPGM) was selected as the activated acyl donor due to its good solubility in organic solvents. The screening results of 21 organic solvents showed that solvents with either strong polarity or poor substrate solubility were unfavorable. Remarkable catalytic activity of the immobilized penicillin acylase (IPA) from Escherichia coli was retained in tert-pentanol, and high yield could be obtained. Effects of various parameters such as acyl donor, water content or cosolvents of tert-pentanol, substrate concentration, temperature, etc., on the enzymatic synthesis of amoxicillin in tert-pentanol were investigated systematically. The best reaction medium, the optimal temperature, initial concentration of 6-APA and HPGM and concentration of enzyme were tert-pentanol, 15 degrees C, 100, 200 mM and 20 IU/mL, respectively. Under the optimal conditions, the yield of amoxicillin was as high as 88% after a reaction time of 20 h. (c) 2008 Elsevier Inc. All rights reserved.