Characterisation of an Inducible Nitrilase from a Thermophilic Bacillus
Qadreyah ALMATAWAH*, Rebecca CRAMP, and Don COWAN
Department of Biochemistry and Molecular Biology, University College London, Gower St., London WC1E 6BT, U.K.
Nitrilase activity was induced in the thermophilic bacterium Bacillus pallidus strain Dac521 (1) by growth on benzonitrile-containing minimal medium. The enzyme was located in the periplasmic space and had a molecular mass of approx. 600 kDa with 52 kDa subunits as determined by gel filtration. Enzymatic activity was constant between pH 6 and 9 with an optimum at pH 7.6. The optimal temperature for activity was 65℃ and the enzyme was unstable at higher temperatures. The nitrilase had the following half-lives: 8 hr at 50℃, 2.5 hr at 60℃, less than 10 minutes at 70℃ and less than 5 minutes at 80℃. The partially purified nitrilase catalysed the hydrolysis of aliphatic, aromatic and heterocyclic nitriles. kcat/Km values varied widely with different substrates, primarily because of differences in Km values. Of the nitriles tested, 4-cyanopyridine was hydrolyzed at the fastest rate. Substitution of benzonitrile at the meta- or para- position did not inhibit attack on the nitrile; in some instances enzymatic rates were greater than on unsubstituted substrates. Ortho substitution, however, was incompatible with enzyme attack presumably due to steric hindrance.
Silver and mercuric ions inhibited activity while other divalent metal ions, metal ion chelating agents, carbonyl reagents and reductants had no effect on the enzyme activity. The effect of SH reagent was inconsistent: the enzyme was inhibited by 4- chloromercuricbenzoic acid, p-hydroxymercuribenzoic acid and phenylmercuric acetate but was not affected by other SH reagents such as iodoacetate, iodoacetamide and N-methylmaleimide. Ethylene glycol had some stabilising effect on the enzyme. The enzyme lost only 18% of it's activity in 50% (v/v) ethylene glycol and activity increased 22% at 20% (v/v) ethylene glycol concentration. Other organic solvents such as acetone, ethanol and methanol had destabilising effects at concentrations of more than 20% (v/v).
1. Cramp, R., Gilmour, M., and Cowan, D.A. (1997) Microbiol. 143, 2313-2320.
