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An inducible nitrilase from a thermophilic bacillus

Almatawah, Qadreyah A. M.; (1999) An inducible nitrilase from a thermophilic bacillus. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Bacillus pallidus strain Dac521 was previously isolated as a novel thermophilic bacterium that can producing separate aliphatic nitrile and aromatic nitrile degrading enzymes. The optimum culture conditions for aromatic nitrile (benzonitrile) degrading enzyme production was investigated. The aromatic nitrile degrading activity was found to be induced when benzonitrile was used as sole carbon and nitrogen source and the induction was concentration dependent. The highest specific activity (0.035 + 0.002 U/mg protein) was found when benzonitrile was used at concentration of 20 mM. The induction was repressed by readily utilisable carbon sources but not by weakly utilisable carbon source such as starch. However, induction was not repressed significantly by nitrogen sources and only partially repressed by complex nitrogen source such as casein and yeast extract. Studies on the effect of nitriles, acids and aromatic compounds as inducers show that benzonitrile and isovaleronitrile were the best inducers whereas other nitriles, acids and aromatic compounds acted as very poor inducers. Nitrilase activity, specifically localised in periplasmic space, was purified to near homogeneity by a two step chromatographic procedure, resulting in a 35 fold purification with 64% yield. The purified enzyme had a native relative molecular mass of approx. 600 kDa with 41 kDa subunits suggesting an ai4-ai6 quaternary structure. The N-terminal amino acid sequence of the nitrilase monomer was determined and compared to those of mesophiles. Homologies between 73% to 40% were observed between nitrilase subunit and the N-terminal sequences. Enzymatic activity was constant between pH 6 and 9 with an optimum pH at 7.6. The optimal temperature for activity was 65°C and the enzyme was unstable at higher temperatures. The partially purified nitrilase had the following half-lives: 8.4 hr at 50°C, 2.5 hr at 60°C, 13 minutes at 70°C and less than 3 minutes at 80°C. The nitrilase catalysed the hydrolysis of aliphatic, aromatic and heterocyclic nitriles with widely varied kcat/KM values, primarily due to differences in substrate affinity. Of the nitriles tested, 4-cyanopyridine was hydrolysed at the fastest rate. Substitution of benzonitrile at the meta- or para- position had no effect on catalytic rate or enhanced kcat, while ortho substitution was strongly inhibitory, probably due to steric hindrance. The effect of catalytic inhibitors was consistent with the presence of active sites thiol residues although activity was little affected by putative thiol reagents such as iodoacetate, iodoacetamide and N-methylmaleimide. Ethylene glycol had some stabilising effect on the enzyme. The enzyme activity was increased 22% at 20% (v/v) ethylene glycol concentration and only 18% of activity was lost at 50% (v/v) ethylene glycol concentration. Other organic solvents tested, such as acetone, ethanol and methanol, had destabilising effects at concentrations of more than 20% (v/v). B. pallidus Dac521 nitrilase catalysed the direct hydrolysis of 3-cyanopyridine to nicotinic acid without detectable formation of nicotinamide. The reaction conditions for nicotinic acid production were optimised using resting cells. Temperature and pH optima were 60°C and 8.0, respectively. Under optimised conditions, 100% of the 3-cyanopyridine substrate could be converted to nicotinic acid at a conversion rate of 76 jamol/min. Resting cells were effective in converting 3-cyanopyridine at concentrations of up to 0.3 M, but both 3-cyanopyridine and nicotinic acid inhibited the hydrolysis of 3-cyanopyridine at concentrations greater than 0.2 M. Immobilised bacteria in calcium alginate beads retained 98% of activity and were more stable than non-immobilised bacteria at 60°C. Calcium alginate immobilised bacteria used in a column bioreactor retained 100% of 3-cyanopyridinase activity for over 100 hr and 10 hr when continuously supplied with 0.1 M 3-cyanopyridine at 50°C and 60°C, respectively.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: An inducible nitrilase from a thermophilic bacillus
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Degrading enzymes
URI: https://discovery.ucl.ac.uk/id/eprint/10121683
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