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Purification and characterization of arylacetonitrile‐specific nitrilase of Alcaligenes sp. MTCC 10675
Author(s) -
Bhatia S. K.,
Mehta P. K.,
Bhatia R. K.,
Bhalla T. C.
Publication year - 2014
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1192
Subject(s) - nitrilase , alcaligenes , chemistry , chromatography , polyacrylamide gel electrophoresis , alcaligenes faecalis , molecular mass , fractionation , gel permeation chromatography , enzyme , ion exchange , gel electrophoresis , biochemistry , bacteria , biology , organic chemistry , pseudomonas , ion , genetics , polymer
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.