Praveen Kumar Mehta

Purification and characterization of arylacetonitrile‐specific nitrilase of Alcaligenes sp. MTCC 10675

Arylacetonitrile‐hydrolyzing nitrilase (E.C. 3.5.5.5) of Alcaligenes sp. MTCC 10675 has been purified by up to 46‐fold to homogeneity and 32% yield using ammonium sulfate fractionation, Sephacryl S‐300 gel permeation, and anion exchange chromatography. The molecular weight of the native enzyme was estimated to be 520 ± 60 kDa. The subunit has a molecular weight of 60 ± 14 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE). The optimum pH and temperature of the purified enzyme were 6.5 and 50 °C, respectively. The purified arylacetonitrilase has a half‐life of 3 H 20 Min at its optimum temperature. The value for Vmax, Km, kcat, and ki of enzyme for mandelonitrile as a substrate was 50 ± 05 µmol/Min/mg, 13 ± 02 mM, 26 ± 03 Sec−, and 32.4 ± 03 mM, respectively. Alcaligenes sp. MTCC 10675 arylacetonitrilase amino acid sequence has variations from other reported arylacetonitrilase, namely, A11G, N21H, D149N, S170T, P171R, S179A, Q180N, and S191A, and it has a high thermal stability and catalytic rate as compared with the already purified arylacetonitrilase.

Bio-statistical enhancement of acyl transfer activity of amidase for biotransformation of N-substituted aromatic amides.

Acyl transfer activity (ATA) of amidase transfers an acyl group of different amides to hydroxylamine to form the corresponding hydroxamic acid. Bacterial isolate BR-1 was isolated from cyanogenic plant Cirsium vulgare rhizosphere and identified as Pseudomonas putida BR-1 by 16S rDNA sequencing. This organism exhibited high ATA for the biotransformation of N-substituted aromatic amide to the corresponding hydroxamic acid. Optimization of media, tryptone (0.6%), inducer, pH 8.5, and a growth temperature 25°C for 56 h, resulted in a 7-fold increase in ATA. Further, Response Surface Methodology (RSM) and multiple feeding approach (20 mM after 14 h) of inducer led to a 29% enhancement of ATA from this organism. The half life (t1/2) of this enzyme at 50°C and 60°C was 3 h and 1 h, respectively. The ATA of amidase of Pseudomonas putida BR-1 makes it a potential candidate for the production of a variety of N-substituted aromatic hydroxamic acid.