Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8

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dc.contributor.authorWilliamson, D S
dc.contributor.authorDent, K C
dc.contributor.authorWeber, B W
dc.contributor.authorVarsani, A
dc.contributor.authorFrederick, J
dc.contributor.authorThuku, R
dc.contributor.authorCameron, R A
dc.contributor.authorvan Heerden, J H
dc.contributor.authorCowan, D A
dc.contributor.authorSewell, B T
dc.date.accessioned2016-07-26T07:44:50Z
dc.date.available2016-07-26T07:44:50Z
dc.date.issued2010
dc.date.updated2016-07-15T15:53:12Z
dc.description.abstractGeobacillus pallidus RAPc8 (NRRL: B-59396) is a moderately thermophilic gram-positive bacterium, originally isolated from Australian lake sediment. The G. pallidus RAPc8 gene encoding an inducible nitrilase was located and cloned using degenerate primers coding for wellconserved nitrilase sequences, coupled with inverse PCR. The nitrilase open reading frame was cloned into an expression plasmid and the expressed recombinant enzyme purified and characterized. The protein had a monomer molecular weight of 35,790 Da, and the purified functional enzyme had an apparent molecular weight of ~600 kDa by size exclusion chromatography. Similar to several plant nitrilases and some bacterial nitrilases, the recombinant G. pallidus RAPc8 enzyme produced both acid and amide products from nitrile substrates. The ratios of acid to amide produced from the substrates we tested are significantly different to those reported for other enzymes, and this has implications for our understanding of the mechanism of the nitrilases which may assist with rational design of these enzymes. Electron microscopy and image classification showed complexes having crescent-like, “c-shaped”, circular and “figure-8” shapes. Protein models suggested that the various complexes were composed of 6, 8, 10 and 20 subunits, respectively.en_ZA
dc.identifierhttp://dx.doi.org/10.1007/s00253-010-2734-9
dc.identifier.apacitationWilliamson, D. S., Dent, K. C., Weber, B. W., Varsani, A., Frederick, J., Thuku, R., ... Sewell, B. T. (2010). Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8. <i>Applied Microbiology and Biotechnology</i>, http://hdl.handle.net/11427/20737en_ZA
dc.identifier.chicagocitationWilliamson, D S, K C Dent, B W Weber, A Varsani, J Frederick, R Thuku, R A Cameron, J H van Heerden, D A Cowan, and B T Sewell "Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8." <i>Applied Microbiology and Biotechnology</i> (2010) http://hdl.handle.net/11427/20737en_ZA
dc.identifier.citationWilliamson, D. S., Dent, K. C., Weber, B. W., Varsani, A., Frederick, J., Thuku, R. N., ... & Sewell, B. T. (2010). Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8. Applied microbiology and biotechnology, 88(1), 143-153.en_ZA
dc.identifier.issn0175-7598en_ZA
dc.identifier.ris TY - Journal Article AU - Williamson, D S AU - Dent, K C AU - Weber, B W AU - Varsani, A AU - Frederick, J AU - Thuku, R AU - Cameron, R A AU - van Heerden, J H AU - Cowan, D A AU - Sewell, B T AB - Geobacillus pallidus RAPc8 (NRRL: B-59396) is a moderately thermophilic gram-positive bacterium, originally isolated from Australian lake sediment. The G. pallidus RAPc8 gene encoding an inducible nitrilase was located and cloned using degenerate primers coding for wellconserved nitrilase sequences, coupled with inverse PCR. The nitrilase open reading frame was cloned into an expression plasmid and the expressed recombinant enzyme purified and characterized. The protein had a monomer molecular weight of 35,790 Da, and the purified functional enzyme had an apparent molecular weight of ~600 kDa by size exclusion chromatography. Similar to several plant nitrilases and some bacterial nitrilases, the recombinant G. pallidus RAPc8 enzyme produced both acid and amide products from nitrile substrates. The ratios of acid to amide produced from the substrates we tested are significantly different to those reported for other enzymes, and this has implications for our understanding of the mechanism of the nitrilases which may assist with rational design of these enzymes. Electron microscopy and image classification showed complexes having crescent-like, “c-shaped”, circular and “figure-8” shapes. Protein models suggested that the various complexes were composed of 6, 8, 10 and 20 subunits, respectively. DA - 2010 DB - OpenUCT DP - University of Cape Town J1 - Applied Microbiology and Biotechnology LK - https://open.uct.ac.za PB - University of Cape Town PY - 2010 SM - 0175-7598 T1 - Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8 TI - Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8 UR - http://hdl.handle.net/11427/20737 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/20737
dc.identifier.vancouvercitationWilliamson DS, Dent KC, Weber BW, Varsani A, Frederick J, Thuku R, et al. Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8. Applied Microbiology and Biotechnology. 2010; http://hdl.handle.net/11427/20737.en_ZA
dc.languageengen_ZA
dc.publisherSpringer Verlagen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.sourceApplied Microbiology and Biotechnologyen_ZA
dc.source.urihttp://www.springer.com/life+sciences/microbiology/journal/253
dc.subject.otherNitrilase
dc.subject.otherGeobacillus pallidus
dc.subject.otherTetrahedral intermediate
dc.titleStructural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8en_ZA
dc.typeJournal Articleen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceArticleen_ZA
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