Applied Microbiology and Biotechnology

, Volume 88, Issue 1, pp 143–153 | Cite as

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

  • Dael S. Williamson
  • Kyle C. Dent
  • Brandon W. Weber
  • Arvind Varsani
  • Joni Frederick
  • Robert N. Thuku
  • Rory A. Cameron
  • Johan H. van Heerden
  • Donald A. Cowan
  • B. Trevor Sewell
Biotechnologically Relevant Enzymes and Proteins

Abstract

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 well-conserved 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.

Keywords

Nitrilase Geobacillus pallidus Tetrahedral intermediate 

Supplementary material

253_2010_2734_MOESM1_ESM.ppt (444 kb)
Supplementary Figure 1A two-dimensional graphic representation of pairwise amino acid aequence identities of various nitrilases. Each block is colored representing the percentage sequence identity on a scale which ranges from dark blue (0%) to brown (100%). (PPT 443 kb)

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Dael S. Williamson
    • 1
    • 4
  • Kyle C. Dent
    • 1
    • 2
  • Brandon W. Weber
    • 1
  • Arvind Varsani
    • 1
    • 5
  • Joni Frederick
    • 2
    • 3
  • Robert N. Thuku
    • 1
    • 2
  • Rory A. Cameron
    • 4
  • Johan H. van Heerden
    • 2
  • Donald A. Cowan
    • 4
  • B. Trevor Sewell
    • 1
  1. 1.Electron Microscope UnitUniversity of Cape TownCape TownSouth Africa
  2. 2.Department of Molecular and Cell BiologyUniversity of Cape TownCape TownSouth Africa
  3. 3.Enzyme TechnologiesCSIR BiosciencesModderfonteinSouth Africa
  4. 4.Institute for Microbial Biotechnology and Metagenomics, Department of BiotechnologyUniversity of the Western CapeBellvilleSouth Africa
  5. 5.School of Biological SciencesUniversity of CanterburyChristchurchNew Zealand

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