Pereira, Rui Alexandre Martins; (1998) A novel nitrile hydratase and amidase from a thermophilic bacillus isolate. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Screening of bacterial isolates from thermal soil and salt lake sediment at 60C and 70C yielded a nitrile-degrading organism isolate (RAPc8) growing at 60C and capable of utilising acetonitnle and propionitrile as the sole nitrogen source. The isolate was taxonomically assigned to the genus Bacillus using partial 16S ribosomal RNA sequence analysis. The most closely related strain was DSM 2349. Biomass and enzyme optimisation studies showed that Bacillus strain RAPc8 grew optimally at 65 C with the constitutive production of a thermostable intracellular nitrile hydratase. No "benzonitrilase" activity was detected under any of the conditions tested. Growth of Bacillus strain RAPc8 on complex media produced the highest biomass (A6oo of approximately 2.8 = 0.45g(dcw).L-1). Enzyme productivity was approximately 6000 Units L-1. The nitrile hydratase, an [alpha]2[beta]2 heteroletramer with a native molecular weight of 110kDa, was purified to apparent homogeneity. N-terminal sequence data showed no homology to know bacterial [alpha] subunit sequences but had a high level of identity with other bacterial N-terminal [beta] subunit sequences. The purified enzyme had a broad pH- activity range (50% activity limits were pH 5.1 and 8.7) and a half-life of 2.5 hours at 50 C in the absence of either substrates or know stabilisers. Nitrile hydratase substrate specificity was restricted to aliphatic nitriles, but an unusual preference for branched and cyclic nitriles was noted. Turnover rates tor propionitrile and acetonitrile under optimum reaction conditions were 436 sec-1 and 765 sec-1 respectively. Whole cell biotransformation studies indicated that Bacillus strain RAPc8 could be used for the efficient conversion of aliphatic nitriles to the corresponding carboxylic acids or amides. Urea or chloroacetone inhibited amidase activity with the accumulation of the amide intermediate. Bacillus Strain RAPc8 genomic phage libraries were constructed and probed for the nitrile hydratase gene with degenerate oligonucleotide primers designed from the N-terminal amino acid sequence and a DNA fragment amplified from genomic DNA using the same degenerate primers. Sequencing of positive clones did not conclusively identify the nitrile hydratase gene.

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