A novel penicillin G acylase from the bacterial strain Achromobacter sp. CCM 4824 was characterized. The specific activity of purified enzyme was 27.6 U mg(-1) protein (6-nitro-3-phenylacetylamidobenzoic acid, NIPAB as the substrate). The enzyme consists of two dissimilar subunits alpha and beta with a molecular mass of 27.0 and 62.4 kDa, respectively. The isoelectric point was about pH 8.8. The N-terminal animo acid sequence of beta subunit (SNMWIVGRDHAKDARSILLN) and internal amino acid sequence of alpha subunit (YGYGYAVAQDRLFQMEMAR) exhibited a significant similarity with penicillin G acylases. The K-m values for penicillin G and NIPAB were 1.9 +/- 0.1 and 4.5 +/- 0.2 muM, respectively. The turnover rates k(cat) for penicillin G and NIPAB were 29 +/- 1 and 19 +/- 1 s(-1), respectively. The maximal hydrolytic activity of the enzyme was found at pH 7.5 and temperature of 60degreesC. In contrast to the published substrate specificities of penicillin G acylases, the enzyme exhibited an almost two-fold hydrolytic activity with ampicillin, amoxicillin and cephalexin compared to penicillin G. (C) 2003 Elsevier Science Inc. All rights reserved.