5 results for Petach, Helen H.

  • Review: The denaturation and degradation of stable enzymes at high temperatures

    Daniel, Roy M.; Dines, Mark; Petach, Helen H. (1996)

    Journal article
    University of Waikato

    Now that enzymes are available that are stable above 100 °C it is possible to investigate conformational stability at this temperature, and also the effect of high-temperature degradative reactions in functioning enzymes and the inter-relationship between degradation and denaturation. The conformational stability of proteins depends upon stabilizing forces arising from a large number of weak interactions, which are opposed by an almost equally large destabilizing force due mostly to conformational entropy. The difference between these, the net free energy of stabilization, is relatively small, equivalent to a few interactions. The enhanced stability of very stable proteins can be achieved by an additional stabilizing force which is again equivalent to only a few stabilizing interactions. There is currently no strong evidence that any particular interaction (e.g. hydrogen bonds, hydrophobic interactions) plays a more important role in proteins that are stable at 100 °C than in those stable at 50 °C, or that the structures of very stable proteins are systematically different from those of less stable proteins. The major degradative mechanisms are deamidation of asparagine and glutamine, and succinamide formation at aspartate and glutamate leading to peptide bond hydrolysis. In addition to being temperature-dependent, these reactions are strongly dependent upon the conformational freedom of the susceptible amino acid residues. Evidence is accumulating which suggests that even at 100 °C deamidation and succinamide formation proceed slowly or not at all in conformationally intact (native) enzymes. Whether this is the case at higher temperatures is not yet clear, so it is not known whether denaturation or degradation will set the upper limit of stability for enzymes.

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  • A general method for the synthesis of peptidyl substrate for proteolytic enzymes

    Whitmore, Andrew; Daniel, Roy M.; Petach, Helen H. (1995)

    Journal article
    University of Waikato

    The synthesis of an heterogeneous peptide substrate for the assay of proteases was carried out by cleaving a protein using a protease to generate small peptides which were then coupled to a chromophore, p-nitroaniline. The chromophoric peptide product could be used to assay for the protease which produced the original peptides.

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  • Properties and stabilization of an extracellular α-glucosidase from the extremely thermophilic archaebacteria Thermococcus strain AN 1: enzyme activity at 130°C

    Piller, Karin; Daniel, Roy M.; Petach, Helen H. (1996)

    Journal article
    University of Waikato

    An extracellular α-glucosidase from the thermophilic archaebacterium Thermococcus strain AN1 was purified 875-fold in five steps (Hiload Q-Sepharose, phenyl Sepharose, HPHT-hydroxyapatite, gel filtration and Mono Q chromatography) with a yield of 4%. It is a monomer with a molecular mass of about 60 kDa and a pI around 5. At 98°C, the purified enzyme in buffer has a half-life around 35 min, which is increased to around 215 min in presence of l% (w/v) dithiothreitol and 1% (w/v) BSA. Dithiothreitol (1%, w/v) and BSA (0.4%, w/v) also substantially increase the enzyme activity. The Km at 75°C is 0.41 mM with pNP-α- -glucopyranoside as substrate. The substrate preference of the enzyme is: pNP-α-D-glucoside > nigerose > panose > palatinose > isomaltose > maltose and turanose. No activity was found against starch, pullulan, amylose, maltotriose, maltotetraose, isomaltotriose, cellobiose and β-gentiobiose. A variety of techniques including immobilization (e.g., on epoxy and glass beads), chemical modification (cross- and cocross-linking) and the use of additives (including polyhydroxylic molecules, BSA, salts, etc.) were applied to enhance stability at temperatures above 100°C. The half-life could be increased from about 4 min at 110°C to 30–60 min at 130°C in presence of 90% (w/v) sorbitol, 1% (w/v) dithiothreitol and l% (w/v) BSA, and by cocross-linking with BSA in the presence of 90% (w/v) sorbitol. The stabilized enzyme showed good activity at 130°C.

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  • The effect of low temperatures on enzyme activity

    More, N.; Daniel, Roy M.; Petach, Helen H. (1995)

    Journal article
    University of Waikato

    The stability of two enzymes from extreme thermophiles (glutamate dehydrogenase from Thermococcales strain AN1 and beta-glucosidase from Caldocellum saccharolyticum expressed in Escherichia coli) has been exploited to allow measurement of activity over a 175 degrees C temperature range, from +90 degrees C to -85 degrees C for the glutamate dehydrogenase and from +90 degrees C to -70 degrees C for the beta-glucosidase. The Arrhenius plots of these enzymes, and those for two mesophilic enzymes (glutamate dehydrogenase from bovine liver and beta-galactosidase from Escherichia coli), exhibit no downward deflection corresponding to the glass transition, found by biophysical measurements of several non-enzymic mesophilic proteins at about -65 degrees C and reflecting a sharp decrease in protein flexibility as the overall motion of groups of atoms ceases.

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  • Arginase from kiwifruit: properties and seasonal variation

    Hale, C.A.; Clark, Christopher J.; Petach, Helen H.; Daniel, Roy M. (1997)

    Journal article
    University of Waikato

    The in vitro activity of arginase (EC 3.5.3.1) was investigated in youngest-mature leaves and roots (1-3 mm diameter) of kiwifruit vines (Actinidia deliciosa var. deliciosa) during an annual growth cycle, and enzyme from root material partially purified. No seasonal trend in the specific activity of arginase was observed in roots. Measurements in leaves, however, rose gradually during early growth and plateaued c. 17 weeks after budbreak. Changes in arginase activity were not correlated with changes in the concentration of arginine (substrate) or glutamine (likely end-product of arginine catabolism) in either tissue during the growth cycle. Purification was by (NH4)2SO4 precipitation and DEAE-cellulose chromatography. The kinetic properties of the enzyme, purified 60-fold over that in crude extracts, indicated a pH optimum of 8.8, and a Km (L-arginine) of 7.85 mM. Partially-purified enzyme was deactivated by dialysis against EDTA, and reactivated in the presence of Mn²⁺, Co²⁺, and Ni²⁺.

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