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

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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.

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Authors and Affiliations

  1. Bioprocess Engineering Research Unit, Department of Chemical Engineering, University of Cape Town, Rondebosch, Cape Town, South Africa

    H. S. Makhongela, A. E. Glowacka & S. G. Burton

  2. Advanced Research Centre for Applied Microbiology, Department of Biotechnology, University of the Western Cape, Bellville, Cape Town, South Africa

    V. B. Agarkar, R. A. Cameron & D. A. Cowan

  3. Electron Microscope Unit, University of Cape Town, Rondebosch, Cape Town, South Africa

    B. T. Sewell & B. Weber

Authors
  1. H. S. Makhongela
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  2. A. E. Glowacka
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  3. V. B. Agarkar
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  4. B. T. Sewell
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  5. B. Weber
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  6. R. A. Cameron
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  7. D. A. Cowan
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  8. S. G. Burton
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Correspondence to S. G. Burton.

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Makhongela, H.S., Glowacka, A.E., Agarkar, V.B. et al. A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity. Appl Microbiol Biotechnol 75, 801–811 (2007). https://doi.org/10.1007/s00253-007-0883-2

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  • DOI: https://doi.org/10.1007/s00253-007-0883-2

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