A new penicillin acylase was isolated by cloning and functional screening of DNA isolated from a sand soil enrichment culture. Sequence analysis of this enzyme, PAS2, revealed homology to a group of prominent penicillin G acylases, including the intensively studied enzyme of Escherichia coli ATCC 11105. Accordingly, PAS2 was found to be an Ntn-hydrolase with an N-terminal serine as the catalytic nucleophile, located on its 61.9 kDa beta-subunit. The alpha-subunit was shown to have a molecular mass of 25.5 kDa. To evaluate the biocatalytic performance of the new enzyme, the complex kinetic parameters alpha, beta(0), and gamma were determined for the kinetically controlled synthesis of a number of important semi-synthetic penicillins and cephalosporins. While alpha is a measure for the relative affinity of the enzyme for the activated acyl donor (AD), beta(0) and gamma quantify the efficiency of acyl-transfer to the beta-lactam nucleophile. Compared to the penicillin acylase of E. coli, PAS2 showed superior potential for the synthesis of 6-aminopenicillanic acid (6-APA)-derived antibiotics, allowing the accumulation of up to 2.3-fold more target product at significantly higher conversion rates. In the synthesis of amoxicillin, for instance, 1.6-fold more antibiotic was formed using the new enzyme, making PAS2 an interesting candidate for biocatalytic application. (C) 2004 Elsevier Inc. All rights reserved.