Amidase of Pseudomonas putida BR-1 having acyl transfer activity (ATA) was purified up to 2.9-fold with 23.2 % yield using ammonium sulfate fractionation, gel permeation and anion exchange chromatography. The SDS-PAGE analysis of the purified enzyme revealed that this enzyme is consisting of alpha- and beta-subunits with molecular masses of 55 and 48 kDa, respectively and the molecular mass of holoenzyme was estimated to be 128 kDa. Total 2.3-fold increase in ATA was observed after optimization of reaction conditions for purified enzyme (sodium phosphate buffer 0.125 M, pH 7.5, temperature 50 A degrees C). The purified amidase of P. putida BR-1 has K (m)A (226 mM) and V (max) (71 A mu mol/min/mg protein) with nicotinamide and K B-m (995 mM) and V (max) (806 A mu mol/min/mg protein) for hydroxylamine. This enzyme has very high potential for biotransformation of N-substituted aromatic amides and for the production of a variety of hydroxamic acids.