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

摘要

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 V_(max), K_m, k_(cat), and k_i 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.