3 results for Plant, Adrian R.

  • A cell-associated oligo-1,6-alpha-glucosidase from an extremely thermophilic anaerobic bacterium, Thermoanaerobium Tok6-B1.

    Plant, Adrian R.; Parratt, S.; Daniel, Roy M.; Morgan, Hugh W. (1988)

    Journal article
    University of Waikato

    Cell-associated oligo-1,6-alpha-glucosidase (EC 3.2.1.10) was isolated from Thermoanaerobium Tok6-B1 grown on starch-containing medium. Activity was purified 11.4-fold by salt precipitation, gel filtration, hydroxyapatite and anion-exchange chromatography. Molecular mass was determined as 30,000 by SDS/polyacrylamide-gel electrophoresis and 33,000 by analytical gel filtration. The probable order of specificity was p-nitrophenyl-alpha D-glucose greater than-isomaltose greater than-isomaltotriose greater than-panose greater than-nigerose and no activity was shown against malto-oligosaccharides, melezitose, melibiose, raffinose, cellobiose, sophorose, gentiobiose, lactose, pullulan, dextran or amylose. The optima for activity and stability were between pH 5.6 and 7.0 and the half-life at pH 6.5 was 1000 min at 70 degrees C and 20 min at 76 degrees C. Activity was stabilized by substrate, Mg2+, Mn2+ and Ca2+, but was destabilized by Zn2+ and EDTA. N-Ethylmaleimide, glucose and 1-O-methyl-alpha D-glucose were inhibitory but 1-O-methyl-beta D-glucose stimulated activity. The activation energy (Ea) was 109 kJ/mol.

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  • Active-site- and substrate-specificity of Thermoanaerobium Tok6-B1 pullulanase.

    Plant, Adrian R.; Clemens, Robyn M.; Morgan, Hugh W.; Daniel, Roy M. (1987)

    Journal article
    University of Waikato

    Thermoanaerobium Tok6-B1 pullulanase (EC 3.2.1.41) was active on alpha 1-6-glucosidic linkages of pullulan, amylopectin and glycogen and the alpha 1-4 linkages of amylose, amylopectin and glycogen but not of pullulan. Hydrolysis of short-chain-length malto-oligosaccharides (seven or fewer glucose residues) yielded maltose as product. Pullulan hydrolysis was pH-dependent and a plot of log(V/Km) versus pH implied a carboxy group with pKa 4.3 at the active site. Modification with 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide (EDAC) confirmed this view, and analysis of the order of reaction and inactivation kinetics suggested the presence of a single carboxy group at a catalytic centre of the active site. EDAC-mediated inhibition of pullulan alpha 1-6-bond hydrolysis was relieved by amylose or pullulan. Similarly both pullulan and amylose protected the activity directed at alpha 1-4 bonds of amylose from EDAC inhibition. When both amylose and pullulan were simultaneously present, the observed rate of product formation closely fitted a kinetic model in which both substrates were hydrolysed at the same active site.

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  • Purification and preliminary characterization of an extracellular pullulanase from Thermoanaerobium Tok6-B1

    Plant, Adrian R.; Clemens, Robyn M.; Daniel, Roy M.; Morgan, Hugh W. (1987)

    Journal article
    University of Waikato

    Extracellular pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41) was purified from cell free culture supernatants of Thermoanaerobium Tok6-B1 by ammonium sulphate precipitation, affinity precipitation, gel exclusion and ion exchange chromatography. A final purification factor of over 1600 was achieved. A molecular weight of 120 kD was determined by steric exclusion HPLC. Enzyme activity was specifically directed towards the 1–6 glucosidic linkages of pullulan resulting in 100% conversion to maltotriose and also possessed activity towards 1–4 linkages of starch, amylopectin and amylose producing maltooligosaccharides (DP2-DP4) as products. Maltotetraose was slowly hydrolysed to maltose. Values of K m (% w/v) were 7.3×10⁻³ for pullulan, 2.7×10⁻³ for amylopectin and 4.7×10⁻³ for Lintner's starch. Pullulanase activity was resistant to 6 M urea and was thermostable at temperatures up to 80°C (t 1/2 in the order of hours). Above 80°C thermal denaturation was significant (t 1/2=17 min at 85°C; 5 min at 90°C) but became less so in the presence of substrate (pullulan or starch). Thermostability was greatest at the pH activity optimum (pH 5.5) and was promoted by Ca²⁺ ions.

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