Browsing by Author "Cowan, D A"

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    A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity
    (Springer, 2007) Makhongela, H S; Glowacka, A E; Agarkar, V B; Sewell, B T; Weber, B; Cameron, R A; Cowan, D A; Burton, S G
    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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    A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity.
    (Springer Verlag, 2007) Makhongela, H S; Glowacka, A E; Agarkar, V B; Sewell, B T; Weber, B; Cameron, R A; Cowan, D A; Burton, S G
    An amidase (EC 3.5.1.4) in branch 2 of the nitrilase superfamily, from the thermophilic strainGeobacillus 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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    The quaternary structure of the amidase from Geobacillus pallidus RAPc8 is revealed by its crystal packing
    (International Union of Crystallography, 2006) Agarkar, V B; Kimani, S W; Cowan, D A; Sayed, M F R; Sewell, B T
    The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase enzyme superfamily. It converts amides to the corresponding acids and ammonia and has application as an industrial catalyst. RAPc8 amidase has been cloned and functionally expressed in Escherichia coli and has been purified by heat treatment and a number of chromatographic steps. The enzyme was crystallized using the hanging-drop vapour-diffusion method. Crystals produced in the presence of 1.2 M sodium citrate, 400 mM NaCl, 100 mM sodium acetate pH 5.6 were selected for X-ray diffraction studies. A data set having acceptable statistics to 1.96 Å resolution was collected under cryoconditions using an in-house X-ray source. The space group was determined to be primitive cubic P4232, with unit-cell parameter a = 130.49 (±0.05) Å. The structure was solved by molecular replacement using the backbone of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB code 1j31 ) with all non-identical side chains substituted with alanine as a probe. There is one subunit per asymmetric unit. The subunits are packed as trimers of dimers with D3 point-group symmetry around the threefold axis in such a way that the dimer interface seen in the homologues is preserved.
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    Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8
    (Springer Verlag, 2010) Williamson, D S; Dent, K C; Weber, B W; Varsani, A; Frederick, J; Thuku, R; Cameron, R A; van Heerden, J H; Cowan, D A; Sewell, B T
    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 wellconserved 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.
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    Structure of an aliphatic amidase from Geobacillus pallidus RAPc8
    (International Union of Crystallography, 2007) Kimani, S W; Agarkar, V B; Cowan, D A; Sayed, M F R; Sewell, B T
    The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase superfamily and catalyzes the conversion of amides to the corresponding carboxylic acids and ammonia. It shows both amide-hydrolysis and acyl-transfer activities and also exhibits stereoselectivity for some enantiomeric substrates, thus making it a potentially important industrial catalyst. The crystal structure of G. pallidus RAPc8 amidase at a resolution of 1.9 Å was solved by molecular replacement from a crystal belonging to the primitive cubic space group P4232. G. pallidus RAPc8 amidase is homohexameric in solution and its monomers have the typical nitrilase-superfamily [alpha]-[beta]-[beta]-[alpha] fold. Association in the hexamer preserves the eight-layered [alpha]-[beta]-[beta]-[alpha]:[alpha]-[beta]-[beta]-[alpha] structure across an interface which is conserved in the known members of the superfamily. The extended carboxy-terminal tail contributes to this conserved interface by interlocking the monomers. Analysis of the small active site of the G. pallidus RAPc8 amidase suggests that access of a water molecule to the catalytic triad (Cys, Glu, Lys) side chains would be impeded by the formation of the acyl intermediate. It is proposed that another active-site residue, Glu142, the position of which is conserved in the homologues, acts as a general base to catalyse the hydrolysis of this intermediate. The small size of the substrate-binding pocket also explains the specificity of this enzyme for short aliphatic amides and its asymmetry explains its enantioselectivity.
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    Structure of an aliphatic amidase from Geobacillus pallidus RAPc8
    (International Union of Crystallography, 2007) Kimani, S W; Agarkar, V B; Cowan, D A; Sayed, M F.-R; Sewell, B T
    The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase superfamily and catalyzes the conversion of amides to the corresponding carboxylic acids and ammonia. It shows both amide-hydrolysis and acyl-transfer activities and also exhibits stereoselectivity for some enantiomeric substrates, thus making it a potentially important industrial catalyst. The crystal structure of G. pallidus RAPc8 amidase at a resolution of 1.9 Å was solved by molecular replacement from a crystal belonging to the primitive cubic space group P4232. G. pallidus RAPc8 amidase is homohexameric in solution and its monomers have the typical nitrilase-superfamily [alpha]-[beta]-[beta]-[alpha] fold. Association in the hexamer preserves the eight-layered [alpha]-[beta]-[beta]-[alpha]:[alpha]-[beta]-[beta]-[alpha] structure across an interface which is conserved in the known members of the superfamily. The extended carboxy-terminal tail contributes to this conserved interface by interlocking the monomers. Analysis of the small active site of the G. pallidus RAPc8 amidase suggests that access of a water molecule to the catalytic triad (Cys, Glu, Lys) side chains would be impeded by the formation of the acyl intermediate. It is proposed that another active-site residue, Glu142, the position of which is conserved in the homologues, acts as a general base to catalyse the hydrolysis of this intermediate. The small size of the substrate-binding pocket also explains the specificity of this enzyme for short aliphatic amides and its asymmetry explains its enantioselectivity.
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    Unique aliphatic amidase from a psychrotrophic and haloalkaliphilic Nesterenkonia isolate
    (American Society for Microbiology, 2011) Nel, A J M; Tuffin, I M; Sewell, B T; Cowan, D A
    Nesterenkonia strain AN1 was isolated from a screening program for nitrile- and amide-hydrolyzing microorganisms in Antarctic desert soil samples. Strain AN1 showed significant 16S rRNA sequence identity to known members of the genus. Like known Nesterenkonia species, strain AN1 was obligately alkaliphilic (optimum environmental pH, 9 to 10) and halotolerant (optimum environmental Na content, 0 to 15% [wt/vol]) but was also shown to be an obligate psychrophile with optimum growth at approximately 21°C. The partially sequenced genome of AN1 revealed an open reading frame (ORF) encoding a putative protein member of the nitrilase superfamily, referred to as NitN (264 amino acids). The protein crystallized readily as a dimer and the atomic structure of all but 10 amino acids of the protein was determined, confirming that the enzyme had an active site and a fold characteristic of the nitrilase superfamily. The protein was screened for activity against a variety of nitrile, carbamoyl, and amide substrates and was found to have only amidase activity. It had highest affinity for propionamide but demonstrated a low catalytic rate. NitN had maximal activity at 30°C and between pH 6.5 and 7.5, conditions which are outside the optimum growth range for the organism.