Cloning and biochemical characterization of Co2+-activated bromoperoxidase-esterase (perhydrolase) from Pseudomonas putida IF-3 strain

The gene encoding Co2+-activated bromoperoxidase (BPO)-esterase (EST), cata lyzing the organic acid-assisted bromination of some organic compounds with H2O2 and Br- and quite specific hydrolysis of (R)-acetylthioisobutyric aci d methyl ester, was cloned from the chromosomal DNA of the Pseudomonas puti da IF-3 strain. The bpo-est gene comprises 831 bp and encoded a protein of 30181 Da. The enzyme was expressed at a high level in Escherichia coli and purified to homogeneity by ammonium sulfate fractionation and two-step colu mn chromatographies. The recombinant enzyme required acetic acid, propionic acid, isobutyric acid or n-butyric acid in addition to H2O2 and Br- for th e brominating reaction and was activated by Co2+ ions. It catalyzed the bro mination of styrene and indene to give the corresponding racemic bromohydri n. Although the enzyme did not release free peracetic acid in the reaction mixture, chemical reaction with peracetic acid could well explain such enzy matic reactions via a peracetic acid intermediate. The results indicated th at the enzyme was a novel Co2+ -activated organic acid-dependent BPO (perhy drolase)-EST, belonging to the non-metal haloperoxidase-hydrolase family. ( C) 2001 Elsevier Science B.V. All rights reserved.