Bi-substrate Kinetic Analysis of Acyl Transfer Activity of Purified Amidase from Pseudomonas putida BR-1
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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 α- and β-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 °C). The purified amidase of P. putida BR-1 has K mA (226 mM) and V max (71 µmol/min/mg protein) with nicotinamide and K mB (995 mM) and V max (806 µmol/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.
KeywordsAcyl transfer activity Pseudomonas putida BR-1 Bi-substrate Purification Hydroxamic acid
The authors acknowledge the University Grants Commission, India for providing financial support in the form of Senior Research Fellowship to Mr. Ravi Kant Bhatia and Vijay Kumar, and Department of Biotechnology, for providing Senior Research Fellowship to Mr. Shashi Kant Bhatia.
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