Browsing by Author "Burton, S G"

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    The effect of the particulate phase on coal biosolubilisation mediated by Trichoderma atroviride in a slurry bioreactor
    (Elsevier, 2008) Oborien, B O; Burton, S G; Cowan, D; Harrison S T L
    Low rank coal is currently under-utilised because of its low calorific value and high moisture and sulphur content. Its solubilisation by both bacterial and fungal cultures has been reported, the latter more commonly. Coal biosolubilisation processes have potential to convert low rank coal to either a clean, cost-effective energy source or complex aromatic compounds for biocatalytic conversion to value-added products. This can lead to an increased utilisation of low rank coal. In this study, the key variables of the slurry that affect biosolubilisation of low rank coal by Trichoderma atroviride in submerged culture were investigated. Results showed that the key operating variables that influence coal biosolubilisation in the slurry bioreactor are coal loading and particle size affecting available surface area. These factors affect the surface area available for coal biosolubilisation. The optimum coal loading occurred between 5 and 10% (w/v); an increase above this optimum led to inhibition of the fungal culture of T. atroviride (ES11) by fragmentation of the fungal mycelium. A decrease in particle size fraction led to an increase in the degree of coal solubilisation. Coal biosolubilisation was shown to increase 4-fold when particle size was decreased from 600–850 μm to 150–300 μm. A 28% biosolubilisation of coal of 150–300 μm, characterised by a surface specific area of 2.17 cm2 g−1 , was measured as coal weight loss over 14 days at solids loading at 5%. This can be compared with a 7.8% coal weight loss at 600–850 μm diameters (0.54 cm2 g−1 ). Soluble phenolic compounds are not a significant product of the coal biosolubilisation process. The change in pH observed in the presence of both coal and fungi was independent of coal loading and was not directly related to the extent of coal solubilisation. While soluble intermediates were observed as total organic, further metabolism resulted in complete oxidation of a significant fraction of the coal to CO2.
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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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    Open Access
    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.