A novel thermostable nitrilase superfamily amidase from <Emphasis Type="Italic">Geobacillus pallidus</Emphasis> showing acyl transfer activity |
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Authors: | H S Makhongela A E Glowacka V B Agarkar B T Sewell B Weber R A Cameron D A Cowan S G Burton |
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Institution: | (1) Bioprocess Engineering Research Unit, Department of Chemical Engineering, University of Cape Town, Rondebosch, Cape Town, South Africa;(2) Advanced Research Centre for Applied Microbiology, Department of Biotechnology, University of the Western Cape, Bellville, Cape Town, South Africa;(3) Electron Microscope Unit, University of Cape Town, Rondebosch, Cape Town, South Africa |
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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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Keywords: | Amidase Enantioselectivity Substrate specificity Thermostable Characterization Immobilization |
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