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Applied Microbiology and Biotechnology

, Volume 75, Issue 4, pp 801–811 | Cite as

A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity

  • H. S. Makhongela
  • A. E. Glowacka
  • V. B. Agarkar
  • B. T. Sewell
  • B. Weber
  • R. A. Cameron
  • D. A. Cowan
  • S. G. Burton
Biotechnologically Relevant Enzymes and Proteins

Abstract

An amidase (EC 3.5.1.4) in branch 2 of the nitrilase superfamily, from the thermophilic strain Geobacillus pallidus RAPc8, was produced at high expression levels (20 U/mg) in small-scale fermentations of Escherichia coli. The enzyme was purified to 90% homogeneity with specific activity of 1,800 U/mg in just two steps, namely, heat-treatment and gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopic (EM) analysis of the homogenous enzyme showed the native enzyme to be a homohexamer of 38 kDa subunits. Analysis of the biochemical properties of the amidase showed that the optimal temperature and pH for activity were 50 and 7.0°C, respectively. The amidase exhibited high thermal stability at 50 and 60°C, with half-lives greater than 5 h at both temperatures. At 70 and 80°C, the half-life values were 43 and 10 min, respectively. The amidase catalyzed the hydrolysis of low molecular weight aliphatic amides, with d-selectivity towards lactamide. Inhibition studies showed activation/inhibition data consistent with the presence of a catalytically active thiol group. Acyl transfer reactions were demonstrated with acetamide, propionamide, isobutyramide, and acrylamide as substrates and hydroxylamine as the acyl acceptor; the highest reaction rate being with isobutyramide. Immobilization by entrapment in polyacrylamide gels, covalent binding on Eupergit C beads at 4°C and on Amberlite-XAD57 resulted in low protein binding and low activity, but immobilization on Eupergit C beads at 25°C with cross-linking resulted in high protein binding yield and high immobilized specific activity (80% of non-immobilized activity). Characterization of Eupergit C-immobilized preparations showed that the optimum reaction temperature was unchanged, the pH range was somewhat broadened, and stability was enhanced giving half-lives of 52 min at 70°C and 30 min at 80°C. The amidase has potential for application under high temperature conditions as a biocatalyst for d-selective amide hydrolysis producing enantiomerically pure carboxylic acids and for production of novel amides by acyl transfer.

Keywords

Amidase Enantioselectivity Substrate specificity Thermostable Characterization Immobilization 

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • H. S. Makhongela
    • 1
  • A. E. Glowacka
    • 1
  • V. B. Agarkar
    • 2
  • B. T. Sewell
    • 3
  • B. Weber
    • 3
  • R. A. Cameron
    • 2
  • D. A. Cowan
    • 2
  • S. G. Burton
    • 1
  1. 1.Bioprocess Engineering Research Unit, Department of Chemical EngineeringUniversity of Cape TownCape TownSouth Africa
  2. 2.Advanced Research Centre for Applied Microbiology, Department of BiotechnologyUniversity of the Western CapeCape TownSouth Africa
  3. 3.Electron Microscope UnitUniversity of Cape TownCape TownSouth Africa

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