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

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dc.contributor.authorMakhongela, H S
dc.contributor.authorGlowacka, A E
dc.contributor.authorAgarkar, V B
dc.contributor.authorSewell, B T
dc.contributor.authorWeber, B
dc.contributor.authorCameron, R A
dc.contributor.authorCowan, D A
dc.contributor.authorBurton, S G
dc.date.accessioned2016-09-05T18:48:36Z
dc.date.available2016-09-05T18:48:36Z
dc.date.issued2007
dc.date.updated2016-09-05T13:05:45Z
dc.description.abstractAn 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.en_ZA
dc.identifierhttp://dx.doi.org/10.1007/s00253-007-0883-2
dc.identifier.apacitationMakhongela, H. S., Glowacka, A. E., Agarkar, V. B., Sewell, B. T., Weber, B., Cameron, R. A., ... Burton, S. G. (2007). A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity. <i>Applied Microbiology and Biotechnology</i>, http://hdl.handle.net/11427/21673en_ZA
dc.identifier.chicagocitationMakhongela, H S, A E Glowacka, V B Agarkar, B T Sewell, B Weber, R A Cameron, D A Cowan, and S G Burton "A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity." <i>Applied Microbiology and Biotechnology</i> (2007) http://hdl.handle.net/11427/21673en_ZA
dc.identifier.citationMakhongela, H. S., Glowacka, A. E., Agarkar, V. B., Sewell, B. T., Weber, B., Cameron, R. A., ... & Burton, S. G. (2007). A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity. Applied microbiology and biotechnology, 75(4), 801-811.en_ZA
dc.identifier.issn0175-7598en_ZA
dc.identifier.ris TY - Journal Article AU - Makhongela, H S AU - Glowacka, A E AU - Agarkar, V B AU - Sewell, B T AU - Weber, B AU - Cameron, R A AU - Cowan, D A AU - Burton, S G AB - 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. DA - 2007 DB - OpenUCT DP - University of Cape Town J1 - Applied Microbiology and Biotechnology LK - https://open.uct.ac.za PB - University of Cape Town PY - 2007 SM - 0175-7598 T1 - A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity TI - A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity UR - http://hdl.handle.net/11427/21673 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/21673
dc.identifier.vancouvercitationMakhongela HS, Glowacka AE, Agarkar VB, Sewell BT, Weber B, Cameron RA, et al. A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity. Applied Microbiology and Biotechnology. 2007; http://hdl.handle.net/11427/21673.en_ZA
dc.languageengen_ZA
dc.publisherSpringeren_ZA
dc.publisher.institutionUniversity of Cape Town
dc.sourceApplied Microbiology and Biotechnologyen_ZA
dc.source.urihttp://link.springer.com/journal/253
dc.subject.otherAmidase
dc.subject.otherEnantioselectivity
dc.subject.otherSubstrate specificity
dc.subject.otherThermostable
dc.subject.otherCharacterization
dc.titleA novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activityen_ZA
dc.typeJournal Articleen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceArticleen_ZA
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