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.
Department of Biotechnology, Himachal Pradesh University, Shimla, India.
S K
SK
Bhatia
Department of Biotechnology, Himachal Pradesh University, Shimla, India.
P K
PK
Mehta
Department of Biotechnology, Himachal Pradesh University, Shimla, India.
R K
RK
Bhatia
Department of Biotechnology, Himachal Pradesh University, Shimla, India.
T C
TC
Bhalla
D000421:Alcaligenes / Q000201:enzymology*; D055162:Biocatalysis; D004795:Enzyme Stability; D006836:Hydro-Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D013696:Temperature
D006836:Hydro-Lyases; C048831:nitrile hydratase
Alcaligenes sp. MTCC 10675; arylacetonitrile; chromatography; mandelonitrile