131 results for Daniel, Roy M., Journal article

  • Isolation from Rhizobium of factors which transfer electrons to soybean nitrogenase

    Phillips, D.A.; Daniel, Roy M.; Appleby, C.A.; Evans, H.J. (1973-01)

    Journal article
    University of Waikato

    Rhizobium japonicum grown anaerobically with nitrate produced several factors capable of transferring electrons from spinach chloroplasts lacking photosystem II to soybean root nodule nitrogenase. The same organism grown aerobically produced no factors with detectable activity. It is concluded that the bacterium is potentially capable of producing these proteins in the low oxygen conditions of the leguminous root nodule.

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  • Isolation of anaerobic, extremely thermophilic, sulphur metabolising archaebacteria from New Zealand hot springs

    Patel, B.K.C.; Jasperse-Herst, P.M.; Morgan, Hugh W.; Daniel, Roy M. (1986)

    Journal article
    University of Waikato

    Enrichments of New Zealand geo-thermal samples, initiated in anaerobic sulphur-containing media and incubated at temperatures above 85°C, yielded rod and coccal shaped organisms which possessed archaebacterial characteristics. Pure cultures were isolated and characterised. Five of the seven isolates, which were rod-shaped organisms and did not have an obligate requirement for sulphur respiration, were similar to Ther-moproteus sp. but had more neutral pH optima for growth. Three of these five Thermoproteus sp. were obligate heterotrophs, which has not previously been reported. The two coccal isolates had an obligate requirement for sulphur as an electron acceptor and were similar to Desulfurococcus sp. but again with more neutral pH optima for growth.

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  • The industrial potential of enzymes from extremely thermophilic bacteria

    Daniel, Roy M.; Cowan, Don A.; Morgan, Hugh W. (1981)

    Journal article
    University of Waikato

    The thermal regions of the central North Island of New Zealand are some of the most extensive in the world. In addition, they are readily accessible and contain a diversity of ecological habitats, including a large number at 100°C. These areas are regarded as an important tourist attraction, and as a source of geothermal power, It is now clear that they also contain an important and unique genetic resource.

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  • Review: Fundamental and biotechnological applications of neutron scattering measurements for macromolecular dynamics

    Tehei, Moeava; Daniel, Roy M.; Zaccai, Giuseppe (2006)

    Journal article
    University of Waikato

    To explore macromolecular dynamics on the picosecond timescale, we used neutron spectroscopy. First, molecular dynamics were analyzed for the hyperthermophile malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, the lactate dehydrogenase from Oryctolagus cunniculus muscle. Hyperthermophiles have elaborate molecular mechanisms of adaptation to extremely high temperature. Using a novel elastic neutron scattering approach that provides independent measurements of the global flexibility and of the structural resilience (rigidity), we have demonstrated that macromolecular dynamics represents one of these molecular mechanisms of thermoadaptation. The flexibilities were found to be similar for both enzymes at their optimal activity temperature and the resilience is higher for the hyperthermophilic protein. Secondly, macromolecular motions were examined in a native and immobilized dihydrofolate reductase (DHFR) from Escherichia coli. The immobilized mesophilic enzyme has increased stability and decreased activity, so that its properties are changed to resemble those of the thermophilic enzyme. Are these changes reflected in dynamical behavior? For this study, we performed quasielastic neutron scattering measurements to probe the protein motions. The residence time is 7.95 ps for the native DHFR and 20.36 ps for the immobilized DHFR. The average height of the potential barrier to local motions is therefore increased in the immobilized DHFR, with a difference in activation energy equal to 0.54 kcal/mol, which is, using the theoretical rate equation, of the same order than expected from calculation.

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  • Review: Detection and impact of protease and lipase activities in milk and milk powders

    Chen, L.; Daniel, Roy M.; Coolbear, Tim (2003)

    Journal article
    University of Waikato

    Freshly drawn milk contains indigenous enzymes, including proteases and lipases. During handling and processing, milk acquires contaminating bacteria that produce further proteases and lipases, adding to the enzyme loading in the milk. Even when several heat treatment steps are used to prepare milk products, these will not be sufficient to inactivate all of the enzymes. Adverse effects of heat on the product limit the extent of heat treatments that can be used. The activities of proteinases and lipases that survive the heat treatments may cause changes in functionality and flavour of milk products, including milk powders, during storage. This review focuses on the characteristics of proteases (specifically proteinases) and lipases in milk and milk powders, detection methods for such enzymes and the effects of these enzymes on milk and milk powders during storage.

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  • Review: Biomolecular stability and life at high temperatures

    Daniel, Roy M.; Cowan, Don A. (2000)

    Journal article
    University of Waikato

    It is not clear what the upper temperature limit forlife is, or what specific factors will set this limit, but it is generally assumed that the limit will be dictated by molecular instability. In this review, we examine the thermal stability of two key groups of biological molecules: the intracellular small molecules/metabolites and the major classes of macromolecules. Certain small molecules/metabolites are unstable in vitro at the growth temperatures of the hyperthermophiles in which they are found. This instability appears to be dealt with in vivo by a range of mechanisms including rapid turnover, metabolic channelling and local stabilisation. Evidence to date suggests that proteins have the potential to be stable at substantially higher temperatures than those known to support life, but evidence concerning degradative reactions above 100p°C is slight. DNA duplex stability is apparently achieved at high temperature by elevated salt concentrations, polyamines, cationic proteins, and supercoiling rather than manipulation of C-G ratios. RNA stability seems dependent upon covalent modification, although secondary structure is probably also critical. The diether-linked lipids, which make up the monolayer membrane of most organisms growing above 85p°C are chemically very stable and seem potentially capable of maintaining membrane integrity at much higher temperatures. However, the in vivo implications of the in vitro instability of biomolecules are difficult to assess, and in vivo data are rare.

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  • 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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  • The upper limits of enzyme thermal stability

    Daniel, Roy M. (1996)

    Journal article
    University of Waikato

    Some enzymes, such as those from extreme thermophiles, have significant half-lives above 100°C. The differences in structure and function between these very stable and less stable enzymes are relatively small and are comparable with those differences found among enzymes of similar stability. Recent evidence suggests that protein degradative reactions at high temperatures (>80°C) occur only slowly in conformationally intact proteins, so that conformational stability may still dictate the upper temperature limit for enzyme activity. The interrelationship of both conformational stability and enzymatic activity with protein flexibility suggests that in naturally occurring enzymes, we cannot expect to find stability at temperatures far above those which are optimum for the growth of the organism. Genetic and enzyme engineering studies are promising in terms of enhancing conformational stability, but are likely to require case-by-case knowledge of the enzyme concerned, and stability enhancements achieved so far are relatively small. Furthermore, engineered increases in stability may well be accompanied by a decrease in specific activity. Nevertheless, it should be feasible to engineer enzymes to be substantially more stable than any of those found so far in nature where useful half-lives above 120°C are already available.

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  • Rapid purification of two thermophilic proteinases using dye-ligand chromatography

    Cowan, Don A.; Daniel, Roy M. (1996)

    Journal article
    University of Waikato

    Dye-ligand chromatography has been used successfully for the purification of extracellular thermostable proteinases from thermophilic Bacillus and Thermus cultures. Single step purification factors of up to 115-fold (for Thermus protease) and 2195-fold (for Bacillus protease) were obtained. Elution studies suggested that the mode of binding involved the enzyme active sites. The method was readily scaleable to 600 l volume.

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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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  • Description of Caldicellulosiruptor saccharolyticus gen. nov., sp. nov: An obligately anaerobic, extremely thermophilic, cellulolytic bacterium

    Rainey, F.A.; Donnison, A.M.; Janssen, P.H.; Saul, David; Rodrigo, A.; Bergquist, Peter L.; Daniel, Roy M.; Stackebrandt, E.; Morgan, Hugh W. (1994)

    Journal article
    University of Waikato

    A new obligately anaerobic, extremely thermophilic, cellulolytic bacterium is described. The strain designated Tp8T 6331 is differentiated from thermophilic cellulolytic clostridia on the basis of physiological characteristics and phylogenetic position within the Bacillus/Clostridium subphylum of the Gram-positive bacteria. Strain Tp8T 6331 is assigned to a new genus Caldicellulosiruptor, as Caldicellulosiruptor saccharolyticus gen., nov., sp. nov.

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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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  • A pepstatin-insensitive aspartic proteinase from a thermophilic Bacillus sp.

    Toogood, H.S.; Prescott, Mark; Daniel, Roy M. (1995)

    Journal article
    University of Waikato

    Bacillus sp. strain Wp22.A1 produced a cell-associated aspartic proteinase which was purified to homogeneity using phenyl-Sepharose (hydrophobic and affinity chromatography) and Mono Q. The proteinase has a molecular mass of 45 kDa by SDS/PAGE and a pI of 3.8. It is insensitive to pepstatin, but is sensitive to the other aspartic proteinase-specific inhibitors diazoacetyl-DL-norleucine methyl ester (DAN) and 1,2-epoxy-3-(p-nitrophenoxy)propane. Inactivation by DAN was only partial, suggesting that it had non-specifically modified an aspartate residue at a site other than the active site. The enzyme was not inhibited by any of the serine or cysteine proteinase inhibitors tested. Maximum proteolytic activity was observed at pH 3.5. The proteinase had a higher activity with haemoglobin, but was more specific (Vmax./Km) for cytochrome c. Substrate inhibition was observed with both these substrates. The cleavage of oxidized insulin B chain tended to occur at sites where the P1 amino acid was bulky and non-polar, and the P1' amino acid was bulky and polar, such as its primary cleavage site of Val2-Asn3. The proteinase was stable in the pH range 2.5-5.5. Thermostability was increased in the presence of Ca2+, although to a lesser extent at higher temperatures. The thermostabilities at 60, 70, 80 and 90 degrees C were 45 h, 102, 21 and 3 min respectively in the presence of Ca2+.

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  • Characterisation of a thermostable pepstatin-insensitive acid proteinase from a Bacillus sp.

    Prescott, Mark; Peek, Keith; Daniel, Roy M. (1995)

    Journal article
    University of Waikato

    An acid proteinase, Wai 21a, produced by a thermophilic Bacillus species (strain Wai 21a) has been purified to homogeneity by cation-exchange chromatography, phenyl-Sepharose chromatography and anion-exchange chromatography. A pI of 3.8 was determined by isoelectric focussing. The protein contained some associated carbohydrate (20 mol hexose equiv/mol proteinase). Optimal proteolytic activity was observed at pH 3.0 (at 60°C). The Leu¹⁵-Tyr¹⁶ bond was the major site of hydrolysis for the oxidized B chain of insulin. Enzyme activity was not affected by inhibitors of the cysteine, metallo or serine class of proteinases. The aspartate proteinase inhibitor, pepstatin, did not inhibit enzyme activity. Inhibition of enzyme activity by 1,2-epoxy-3-(p-nitrophenoxy)-propane indicated the presence of at least one carboxyl group essential to the catalytic mechanism of the enzyme. Proteinase activity was inhibited by diazoacetyl- -norleucine methyl ester in a slow and non-specific manner atypical of pepstatin-sensitive aspartate proteinases. Wai 21a proteinase may be classified as member of the pepstatin-insensitive group of aspartate proteinases. The thermal stability at pH 3.0 and 60°C increased 2.1-fold (t1/2, 4.5–9.7 hr) in the presence of 5 mM Ca⁺⁺. An increase in both pH (3.0–4.5) and Ca⁺⁺ concentration (0–30 mM) resulted in a 15-fold increase (t1/2, 15–230 min) in thermal stability at 75°C. The amino acid composition of Wai 21a proteinase was found to be similar to other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the thermostable enzyme, kumamolysin.

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  • Did primitive microorganisms use nonhem iron proteins in place of NAD/P?

    Daniel, Roy M.; Danson, Michael J. (1995)

    Journal article
    University of Waikato

    Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are of universal occurrence in living organisms and play a central role in coupling oxidative with reductive reactions. However, the evidence that the origin and early evolution of life occurred at high temperatures (>95°C) is now strong, and at these temperatures some modern metabolites, including both the reduced and oxidized forms of these coenzymes, are unstable. We believe there is good evidence that indicates that in the most primitive organisms nonhem iron proteins carried out many or all of the functions of NAD/P(H). This has important implications for the way in which investigations of archaebacterial metabolism are conducted.

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  • Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A

    Wilson, Shelley-Ann; Daniel, Roy M.; Peek, Keith (1994)

    Journal article
    University of Waikato

    A proteinase isolated from Thermus RT41a was immobilized to controlled pore glass beads and was used in the free and immobilized forms for peptide synthesis. The observed maximum yield was the same in both cases. a number of dipeptides were produced from amino acid esters and amides. The best acyl components, from those tested, were found to be Ac-Phe-OEt and Bz-Ala-OMe. Tur-NH2, Trp-NH2, Leu-pNA, and Val-pNA were all reactive nucleophiles.The kinetically controlled synthesis of Bz-ala-Tyr-NH2 was optimized by studying the effect of pH, temperature, solvent concentration, ionic strength, and nucleophile and acyl donor concentration, ionic strength, and nucleophile and acyl donor concentration on the maximum yield. The initial conditions used were 25 mM Bz–ala-OMe, 25 mM Tyr-NH2, 70°C, pH 8.0, and 10% v/v dimethylformamide (DMF). The optimum conditions were 90% v/v DMF using 80 mM bz-Ala-OMe and 615 mM Tyr-NH2 at 40°C and pH 10. These conditions increased the maximum conversion from 0.75% to 26% (of the original ester concentration). In a number of other cosolvents, the best peptide yields were observed with acetonitrile and ethyl acetate. In 90% acetonitrile similar yields were observed to those in 90% DMF under optimized conditions except that the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100mM, respectively. The effect of the blocking group on the nucleophile was also investigated; -βNA and -pNA as blocking groups improved the yields markedly. The blocking and leaving groups of the acyldonor had no effect on the dipeptide yield.

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  • Citrate synthases from the Archaea: Development of a bio-specific, affinity chromatography purification procedure

    James, Keith D.; Russell, Rupert J.M.; Parker, Lynne; Daniel, Roy M.; Hough, David W.; Danson, Michael J. (1994)

    Journal article
    University of Waikato

    Citrate synthases from both thermophilic and halophilic Archaea have been purified to homogeneity using affinity chromatography on Matrex Gel Red A and elution with a combination of substrate (oxaloacetate) and product (coenzyme A). In a number of cases, purification from cell-extract to protein suitable for N-terminal sequencing can be achieved by this single-step procedure. The method is particularly useful in the rapid purification of a thermophilic archaeal citrate synthase from a cloned gene expressed in a mesophilic host.

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  • A hydrothermally precipitated catalytic iron sulphide membrane as a first step toward life

    Russell, Michael J.; Daniel, Roy M.; Hall, Allan J.; Sherringham, John A. (1994)

    Journal article
    University of Waikato

    We propose that life emerged from growing aggregates of iron sulphide bubbles containing alkaline and highly reduced hydrothermal solution. These bubbles were inflated hydrostatically at sulphidic submarine hot springs sited some distance from oceanic spreading centers four billion years ago. The membrane enclosing the bubbles was precipitated in response to contact between the spring waters and the mildly oxidized, acidic and iron-bearing Hadean ocean water. As the gelatinous sulphide bubbles aged and were inflated beyond their strength they budded, producing contiguous daughter bubbles by the precipitation of new membrane. [Fe2S2]+/0 or [Fe4S4]2+/+ clusters, possibly bonded by hydrothermal thiolate ligands as proferredoxins, could have catalyzed oxidation of thiolates to disulphides, thereby modifying membrane properties. We envisage the earliest iron sulphide bubbles (pro botryoids) first growing by hydrostatic inflation with hydrothermal fluid, but evolving to grow mainly by osmosis (the protocellular stage), driven by (1) catabolism of hydrothermal abiogenic organics trapped on the inner walls of the membrane, catalyzed by the iron sulphide clusters; and (2) cleavage of hydrophobic compounds dissolved in the membrane to hydrophilic moieties which were translocated, by the proton motive force inherent in the acidic Hadean ocean, to the alkaline interior of the protocell. The organics were generated first within the hydrothermal convective system feeding the hot springs operating in the oceanic crust and later in the pyritizing mound developing on the sea floor, as a consequence of the reduction of CO, CO2, and formaldehyde by Fe2+- and S2–-bearing minerals. We imagine the physicochemical interactions in and on the membrane to have been sufficiently complex to have engendered auto- and cross-catalytic replication. The membrane may have been constructed in such a way that a successful parent could have informed the daughters of membrane characteristics functional for the then-current level of evolution.

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  • Future research

    Holm, Nils G.; Cairns-Smith, Graham; Daniel, Roy M.; Ferris, James P.; Hennet, Remy J.-C.; Shock, Everett L.; Simoneit, Bernd R.T.; Yanagawa, Hiroshi (1992)

    Journal article
    University of Waikato

    What field studies need to be carried out in order to test the hydrotherrnal model for the origin of life? This question was already raised in 1989 by the workshop in Prague on 'Chemical Evolution and Neo-Abiogenesis in Marine Hydrothermal Systems' (Holm, 1990). The full range and diversity of hydrothermal environments can only be revealed if more exploration work is carried out on a world-wide basis. Much progress has been made in understanding the controls on the hydrothermal fluids of individual areas, but extension to a global context remains difficult (cf. Von Datum, 1990). In Chapter 2 off-axis hydrothermal systems are discussed, pointing out that only minor surveys have been directed towards this type of hydrothermal activity, even though the amount of water which circulates through off-axis systems is estimated to exceed that of on-axis systems by a factor of between 20 and 25.

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