Cramp, Rebecca Ann; (1998) Novel nitrile degrading enzymes in thermophilic bacteria. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Thermal sediment samples were screened for the production of nitrile degrading enzymes on nitrile containing minimal nutrient media plates. Twenty-eight isolates which grew on these plates were investigated for growth on aromatic and aliphatic nitriles and amides. These isolates grew in a variety of different nitriles and amides but the maximum optical densities reached were less than 0.2 (@ 600nm). Therefore, optimisation of growth and enzyme production was investigated and fourteen different minimal and complex media were tested. Repeat addition of nitrile to strains growing in a complex media resulted in high maximal optical densities and nitrile degrading specific activities for a high percentage of the strains. This media was used to investigate nitrile production in seven isolates. Expression of nitrile degrading enzyme activity ranged from intermittent to continuous and maximum specific activities ranged from 9*10^6 to 905*10^-6 units per milligram of cells (wet weight). A single strain that had high levels of nitrile degrading enzyme activity over the entire growth period was chosen for further study. This Bacillus pallidus Dac521 isolate grew in minimal media with acetonitrile, propionitrile, benzonitrile, acetamide, propionamide or benzamide as the sole carbon and nitrogen source and expressed a constitutive aliphatic nitrile hydratase and amidase in addition to an inducible aromatic nitrilase. The maximum rate of nitrile hydrolysis occurred at 55°C for whole cells and 50°C for cell-free extracts and both the nitrile hydratase and nitrilase were highly thermostable. Cell-free extracts containing the induced aromatic nitrilase (benzonitrilase) degraded benzonitrile, propionitrile, isovaleronitrile and acrylonitrile, were highly thermostable and exhibited nitrile degrading activity up to 70°C. This benzonitrilase was purified to apparent homogeneity in two chromatography steps with 117 fold purification and 1% recovery and was composed of identical subunits of 33kDa with a native mass of 165kDa. The specific activity of this purified benzonitrilase, was 7 μmole of ammonia per minute per milligram of protein based on benzonilrile hydrolysis at 50°C in pH 7.2 phosphate buffer. The constitutive nitrile hydratase was also purified to apparent homogeneity. This involved a three step chromatographic procedure, resulting in a 49 fold purification with 56% recovery. The nitrile hydratase was a 110kDa tetrameric enzyme composed of two α and two β subunits with relative molecular masses of 27,000 and 29,000, respectively. The specific activity of the enzyme was 68.4 μmole of ammonia per minute per milligram of protein based on acetonitrile hydrolysis at 50°C in pH 7.2 phosphate buffer. The N-terminal amino acid sequences of the nitrile hydratase subunits were determined and compared to those of mesophiles. Homologies between the β subunit, but not the alpha subunit, N-terminal sequences were observed. The native enzyme was stable over a broad pH range and exhibited ≥50% activity between pH 6.2 and 8.7. Two pIs of 4.7 and 5.5 were determined for the enzyme, suggesting the presence of isozymes. The nitrile hydratase hydrolysed a narrow range of aliphatic substrates including acetonitrile, propionitrile and acrylonitrile but did not hydrolyse any of the cyclic, hydroxy, di or aromatic nitriles tested. In addition, the nitrile hydratase activity was irreversibly inhibited by the aromatic nitrile, benzonitrile. Determination of the kinetic constants of acetonitrile, acrylonitrile and propionitrile hydrolysis indicated that acetonitrile was hydrolysed with greatest efficiency.

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