Characterization of an inducible nitrilase from a thermophilic bacillus.
Nitrilase activity was induced in the thermophilic bacterium Bacillus pallidus strain Dac521 by growth on benzonitrile-supplemented minimal medium. The enzyme had a subunit relative molecular mass of 41 kDa but was purified as a complex with a putative GroEL protein (total M(r), 600 kDa). The enzyme catalyzed the hydrolysis of aliphatic, aromatic, and heterocyclic nitriles with widely varying kcat/KM values, primarily the result of differences in substrate affinity. Of the nitriles tested, 4-cyanopyridine was hydrolyzed at the fastest rate. Substitution of benzonitrile at the meta or para position either had no effect on catalytic rate or enhanced kcat, while orthosubstitution was strongly inhibitory, probably because of steric hindrance. The effect of catalytic inhibitors was consistent with the presence of active site thiol residues although activity was little affected by putative thiol reagents such as iodoacetate, iodoacetamide, and N-methylmaleimide. Enzymatic activity was constant between pH 6 and 9 with an optimum at pH 7.6. The optimal temperature for activity was 65 degrees C with rapid activity loss at higher temperatures. The purified nitrilase-GroEL complex had the following half-lives of activity: 8.4 h at 50 degrees C, 2.5 h at 60 degrees C, 13 min at 70 degrees C, and less than 3 min at 80 degrees C.
Almatawah
Q A
QA
Department of Biochemistry and Molecular Biology, University College London, UK.
Cramp
R
R
Cowan
D A
DA
D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology* / Q000502:physiology; D018834:Chaperonin 60 / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D006358:Hot Temperature; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018834:Chaperonin 60; D046911:Macromolecular Substances; D000619:Aminohydrolases; C021867:nitrilase