UCL Discovery
UCL home » Library Services » Electronic resources » UCL Discovery

Nitrile degrading enzymes from extreme environments

Cameron, Rory A.; (2003) Nitrile degrading enzymes from extreme environments. Doctoral thesis (Ph.D), UCL (University College London). Green open access

[thumbnail of Nitrile_degrading_enzymes_from.pdf]
Preview
Text
Nitrile_degrading_enzymes_from.pdf

Download (21MB) | Preview

Abstract

In a study prior to this work, screening experiments for thermophilic nitrile degrading organisms led to the identification of a moderately thermophilic Bacillus sp. (strain RAPc8). The organism was shown to constitutively express a nitrile hydratase (NHase), which was purified and extensively characterised. This work reports the cloning of a gene cluster containing the NHase genes of Bacillus sp. RAPc8 into Escherichia coli. Sequencing of a 5.9kb section of cloned DNA revealed the presence of eight complete open reading frames, encoding (in order), amidase (belonging to the nitrilase related aliphatic amidase family), nitrile hydratase β and α subunits (of the cobalt containing class), a putative 122 amino acid protein of unknown function, designated P14K, a homologue of the 2Fe-2S class of ferredoxins and three putative proteins with distinct homology to the cobalt uptake proteins cbiM, cbiN and cbiQ of the S. typhimurium LT2 cobalamin biosynthesis pathway. The NHase operon shares an exceptionally high sequence identity with that of Bacillus sp. BR449. The deduced NHase α subunit N-terminal sequence bore no similarity the empirically determined sequence of the previous study. However, in this study, N-terminal sequencing of the recombinant protein confirmed the sequence predicted by gene translation. The amidase and nitrile hydratase genes of Bacillus sp. RAPc8 were subcloned and actively expressed in E. coli. The recombinant NHase was partially purified and found to exhibit catalytic behaviour very similar to the native protein. Substrate specificity trials showed that the recombinant enzyme, as with the native enzyme, has broad aliphatic substrate specificity, but no detectable activity on aromatic nitriles. A rational design approach, based on the observation that the enzyme was irreversibly inhibited by benzonitrile, was applied in attempts to broaden the substrate specificity of the enzyme to include aromatic nitriles. A crude homology model of the enzyme structure was built using the crystal structure of the NHase of Rhodococcus sp. R312 as a template. Aromatic residues in the active site, identified from the model were replaced with non-polar or charged residues using site directed mutagenesis. Of the six mutants investigated, none demonstrated any catalytic activity on the aromatic nitriles tested. However, two mutants, βY67E and βW76G, did display an apparent decrease benzonitrile inhibition (rise in KI of -6.5mM and 29.25Mm respectively). A further two mutants (β36L and βY67A) also showed a marked increase in specific activity in crude cell extracts compared with that of the wild type enzyme. A PCR based method for the identification of NHase genes in environmental soil and sediment samples was developed and employed to verify the presence of such genes in samples taken from New Zealand hot pools. Six unique sequences corresponding to the partial sequence of the α subunit gene were identified. At the DNA level, these sequences showed significant homology with the Bacillus NHase sequences (between 83% and 93% sequence identity with the Bacillus sp. RAPc8 gene). DNA isolated from NHase positive sediment samples was used to create a substantial multi-genomic library for the purpose of screening for full-length NHase genes. However, attempts to identify functional nitrile hydratase genes through complementation screening on succinate-nitrile minimal media were unsuccessful.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Nitrile degrading enzymes from extreme environments
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Applied sciences; Nitrile hydratase
URI: https://discovery.ucl.ac.uk/id/eprint/10099907
Downloads since deposit
85Downloads
Download activity - last month
Download activity - last 12 months
Downloads by country - last 12 months

Archive Staff Only

View Item View Item