131 results for Daniel, Roy M., Journal article

  • 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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  • Cellulolytic and hemicellulolytic enzymes functional above 100℃

    Ruttersmith, L.D.; Daniel, Roy M.; Simpson, H.D. (1992)

    Journal article
    University of Waikato

    Cellulases and hemicellulases have a variety of potential industrial applications, including the production of fermentable sugars from biomass and enzyme-assisted pulp bleaching. There are several advantages, in industrial terms, to be gained from employing thermostable enzymes in processes operating at elevated temperatures. For example, the lignin component of hemicellulose, which often blocks enzyme access, softens and melts over the temperature range 90-180°C.

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  • A correlation between protein thermostability and resistance to proteolysis.

    Daniel, Roy M.; Cowan, Don A.; Morgan, Hugh W.; Curran, M.P. (1982)

    Journal article
    University of Waikato

    The loss of activity due to proteolysis of purified L-asparaginase and beta-galactosidase from different sources correlates with the thermal instability of the enzymes. A similar correlation is found when populations of soluble proteins from micro-organisms grown at different temperatures are compared for proteolytic susceptibility and thermal stability. It is proposed that there is a general correlation between the thermostability of proteins and their resistance to proteolysis.

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  • Thermus filiformis sp. nov., a Filamentous Caldoactive Bacterium

    Hudson, J. Andrew; Morgan, Hugh W.; Daniel, Roy M. (1987)

    Journal article
    University of Waikato

    In a preliminary investigation the isolation of a caldoactive filamentous microorganism from a New Zealand hot spring was reported. This organism is described here as a new species belonging to the genus Thermus, namely, Thermus filiformis, based on ultrastructural, phenotypic, and anomalous Gram type characteristics. The cell wall of T. filiformis resembles that of Thermus aquaticus apart from the presence of an extra layer. The Thermus species tested, including T. filiformis, are negative for the aminopeptidase test, which is unusual for a gram-negative genus. T. filiformis is nonproteolytic, unlike most other Thermus strains, and also differs radically from other strains in morphology when it is observed by using phase-contrast microscopy. The single strain of the species has been deposited with the American Type Culture Collection as strain ATCC 43280T (T = type strain).

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  • An extremely thermostable xylanase from the thermophilic eubacterium Thermotoga.

    Simpson, H.D.; Haufler, U.R.; Daniel, Roy M. (1991)

    Journal article
    University of Waikato

    Endo-1,4-beta-xylanase (EC 3.2.1.8) was isolated from the culture supernatant of Thermotoga sp. strain FjSS3-B.1, an extremely thermophilic anaerobic eubacterium which grows optimally at 80 degrees C. Activity was purified 165-fold by anion-exchange and hydroxyapatite chromatography. The enzyme has an Mr of 31,000 as determined by SDS/PAGE and 35,000 by analytical gel filtration. The optima for activity and stability for purified xylanase were between pH 5.0 and 5.5. At pH 5.5, which is the optimum pH for thermostability, t1/2 (95 degrees C) is 90 min. The thermostability was improved by immobilization of the xylanase on to porous glass beads; t1/2 (105 degrees C) is 10 min. Several additives, such as sorbitol and xylan, were also found to increase the thermostability. At 130 degrees C, the half-life of immobilized xylanase in the presence of 90% sorbitol was 1.3 min. At 130 degrees C in molten sorbitol half of the enzyme denatured rapidly, but the remainder appeared to have a half-life of about 60 min.

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  • Modern life at high temperatures

    Daniel, Roy M. (1992)

    Journal article
    University of Waikato

    A variety of micro-organisms are now known which grow optimally above 65°C, and are defined as extreme thermophiles. As might be expected they are found in both natural and artificial hot environments. Until comparatively recently the upper optimum temperature for the growth of any living organism was about 85°C. Then in 1982 Stetter described an organism, isolated from the hot sea floor of a submarine solfatara field, which grew optimally at 105°C. Since then several other organisms have been found with optimum growth temperatures at 100"C or above and a few are capable of growth at 110*C (e.g. Huber et al., 1987; Fiala and Stetter, 1986; Zillig et al., 1987; Stetter et al., 1990).

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  • Cellulolytic properties of an extremely thermophilic anaerobe

    Hudson, J. Andrew; Morgan, Hugh W.; Daniel, Roy M. (1990)

    Journal article
    University of Waikato

    An extremely thermophilic anaerobe was isolated from a New Zealand hot spring by incubating bacterial mat strands in a medium containing xylan. The Gramreaction-negative organism that was subsequently purified had a temperature optimum of 70° C and a pH optimum of 7.0. The isolate, designated strain H173, grew on a restricted range of carbon sources. In batch culture H173 could degrade Avicel completely when supplied at 5 or 10 g l⁻¹. There was an initial growth phase, during which a cellulase complex was produced and carbohydrates fermented to form acetic and lactic acids, followed by a phase where cells were not metabolising but the cellulase complex actively converted cellulose to glucose. When co-cultured with strain Rt8.B1, an ethanologenic extreme thermophile, glucose was fermented to ethanol and acetate, and no reducing sugars accumulated in the medium. In pH controlled batch culture H173 produced an increased amount of lactate and acetate but there was again a phase when reducing sugars accumulated in the medium, and these were converted to ethanol by co-culture with Rt8.B1.

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  • Interactions of calcium and other metal ions with caldolysin, the thermostable proteinase from Thermus aquaticus strain T351.

    Khoo, T.C.; Cowan, Don A.; Daniel, Roy M.; Morgan, Hugh W. (1984)

    Journal article
    University of Waikato

    Caldolysin, the extracellular proteinase from the extreme thermophile Thermus aquaticus strain T351, is stabilized by Ca2+. A variety of metal ions were able to substitute for Ca2+. Most were unable to confer as much stability as Ca2+, with the exception of the lanthanide ions, which increased the half-life at 95 degrees C from 1 h to more than 4 h. Results from a variety of separation methods indicated that caldolysin binds 6 Ca2+ ions/molecule of enzyme. The presence of non-linear Ca2+ titration plots, and the removal of 4 Ca2+ ions/molecule by treatment with a cationic ion-exchange gel suggested that caldolysin possesses at least two different types of Ca2+-binding sites, with different affinities. Average binding constants of the two types of binding sites were 2.8 X 10(4)M-1 (for the low-affinity sites) and 7.5 X 10(5) M-1 (for the high-affinity sites). The total Ca2+-binding free energy for caldolysin was shown to be greater than for either thermolysin or Bacillus subtilis neutral proteinase. It appears that the higher thermostability of caldolysin is due to the presence of 6 Ca2+ ions rather than 4 Ca2+ ions/molecule.

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  • Purification and characterization of a pepstatin-insensitive, thermostable, extracellular acid proteinase from a bacterium

    Prescott, Mark; Peek, Keith; Prendergast, Elizabeth; Daniel, Roy M. (1992)

    Journal article
    University of Waikato

    Aspartate proteinases (also referred to as acid or carboxyl) represent one of the four major classes of proteinase. Work on members of this group has centered largely around those enzymes isolated from mammalian, fungal, or plant sources. Some of these have proven to be of great economic importance, for example, the renneting enzyme, chymosin.

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  • Anaerobic growth, nitrate reduction and denitrification in 46 rhizobium strains

    Daniel, Roy M.; Limmer, A.W.; Steele, K.W.; Smith, I.M. (1982)

    Journal article
    University of Waikato

    A total of 46 rhizobial strains were assessed for anaerobic growth in the presence of nitrate, and, using the criteria of nitrate utilization and nitrous oxide and nitrogen production, for their ability to denitrify. Nitrite production was also measured. Half of the strains were denitrifiers: these included all five strains of R. meliloti tested which produced N2 from nitrate and most of the slow-growing rhizobia, but none of the 14 strains of R. trifolii.

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  • Enumeration of thermophilic heterotrophs in Geothermally heated Soils from Mount Erebus, Ross Island, Antarctica

    Hudson, J. Andrew; Daniel, Roy M. (1988)

    Journal article
    University of Waikato

    Soil samples with temperatures up to 64°C were collected from Mount Erebus, an active volcano located on Ross Island, Antarctica. Acridine orange direct counts and most probable number counts of soil samples stored at 4°C for 2 months showed a wide variation in the number of thermophilic microorganisms in different soils. Organisms similar to Clostridium thermohydrosulfuricum, Bacillus schlegelii, and Bacillus acidocaldarius, as well as neutrophilic Bacillus strains, were isolated.

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  • Cellulases from extremely thermophilic bacteria

    Sharrock, Keith R.; Sissons, Christopher H.; Daniel, Roy M.; Morgan, Hugh W. (1983)

    Journal article
    University of Waikato

    Cellulose is the most abundant biopolymer on earth, and is the major component of urban waste. Thus cellulose must be seen as a very significant renewable source of chemical foodstocks when fossil fuels become restricted.

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  • Denitrification by rhizobia: A possible factor contributing to nitrogen losses from soils

    Daniel, Roy M.; Steele, K.W.; Limmer, A.W. (1980)

    Journal article
    University of Waikato

    The intensive pastoral farming system on which New Zealand animal production is based is almost completely dependent upon the rhizobium-legurne symbiosis for the fixed nitrogen required for pasture production. The average annual fixation has been measured as 184 kg nitrogen/ha in developed lowland pastures Hoglund et cii., 1979 and about 13 kg nitrogen/ha in poorly developed bill country pastures (Grant and Lambert, 1979). From these figures it can be estimated that rhizobia in New Zealand pastures fix in excess of one million tonnes of nitrogen an nually. The current annual application of fertilizer nitrogen to pastures is about 12 500 tonnes (O'Connor, 1979).

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  • Distribution of reverse gyrase in representative species of eubacteria and archaebacteria

    Collin, R.G.; Morgan, Hugh W.; Musgrave, D.R.; Daniel, Roy M. (1988)

    Journal article
    University of Waikato

    Reverse gyrase is a topoisomerase which positively supercoils closed circular plasmid DNA. Reverse gyrase activity is restricted to the thermoacidophilic group of archaebacteria. Thermophilic methanogens and eubacteria and all mesophilic organisms screened had no reverse gyrase activity. The result supports the deep phylogenetic divergence in archaebacterial evolution.

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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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  • Proteases from extreme thermophiles

    Coolbear, Tim; Daniel, Roy M.; Cowan, Don A.; Morgan, Hugh W. (1988)

    Journal article
    University of Waikato

    Extremely thermophilic bacteria are those that grow optimally at 65 ℃ or higher. Comparative data are presented on extracellular proteases from two extremely thermophilic eubacteria and one extremely thermophilic archaebacterium. The eubacteria were a Bacillus isolate (protease unnamed) and a Thermus isolate (protease named caldolysin) with optimum growth temperatures of 65 ℃ and 75 ℃, respectively. The archaebacterium was a Desulfurococcus isolate (protease named archaelysin) with an optimum growth temperature of 88 ℃.

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  • Nitrate dependent anaerobic acetylene-reduction and nitrogen-fixation by soybean bacteroids

    Rigaud, J.; Bergersen, F.J.; Turner, G.L.; Daniel, Roy M. (1973)

    Journal article
    University of Waikato

    Bacteroids isolated from nodules produced by one strain of Rhizobium japonicum (CC705) had strong nitrate-reducing activity and reduced C₂H₂ to C₂H₄ and N₂ to NH₃ anaerobically with nitrate. Bacteroids of another strain (CB1809) were much less active nitrate reducers and reduced little C₂H₂ anaerobically. Nitrite, which accumulated in the medium in anaerobic assays, was an inhibitor of C₂H₂reduction in both aerobic and anaerobic conditions. Succinate, at about 25 mM, stimulated both nitrate reduction and C₂H₂ reduction under aerobic conditions. Glucose stimulated C₂H₂ reduction up to 120 mM but nitrate reduction was inhibited in the presence of glucose. In terms of electrons transferred, the aerobic pathway appeared to be about 2.5 times more efficient than the anaerobic pathway in supporting nitrogenase activity of CC705 bacteroids.

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  • The alleged absence of ubiquinone from elasmobranchs

    Daniel, Roy M.; Redfearn, E.R. (1966)

    Journal article
    University of Waikato

    It has recently been suggested (Diplock & Haslewood, 1964) that certain lower vertebrates such as elasmobranchs and crocodylians are lacking in ubiquinone. Also, in a study of the lesser-spotted dogfish (Scyliorhinus caniculus) (Class Elasmobranchii, Order Selachii) Diplock & Haslewood (1965) were unable to demonstrate the biosynthesisof ubiquinone. In view of the fact that in all other vertebrate species so far examined the presence of ubiquinone is well authenticated (Crane, 1965), including in such a closely related animal as the shark (Nazir & Magar, 1964), these results seemed somewhat surprising. However, in view of these findings it seemed that a study of the respiratory enzyme systems of these organisms might be worth while. As a preliminary to this work an investigation on the alleged absence of ubiquinone from elasmobranch tissues was carried out.

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  • Characterization of a chelator-resistant proteinase from Thermus strain Rt4A2

    Freeman, S.A.; Peek, Keith; Prescott, Mark; Daniel, Roy M. (1993)

    Journal article
    University of Waikato

    The Thermus isolate Rt4A2 was found to produce an extracellular chelator-resistant proteinase. The proteinase was purified to homogeneity by (NH4)2SO4 precipitation, cation-exchange chromatography, gel-filtration chromatography, and weak anion-exchange chromatography. The Rt4A2 proteinase was found to have properties typical of an alkaline serine proteinase. It had a pH optimum of 9.0 and was specifically inhibited by phenylmethanesulphonyl fluoride. Its isoelectric point was greater than 10.25. Its molecular-mass was 31.6 kDa as determined by SDS/PAGE. N-terminal sequencing has shown it to have high sequence similarity with other serine proteinases from Thermus species. The proteinase hydrolysed a number of substrates including fibrin, casein, haemoglobin, collagen, albumin and the synthetic chromogenic peptide substrate Suc-Ala-Ala-Pro-Phe-NH-Np. The specific activity of the purified proteinase using azocasein as substrate was 313 units/mg. Substrate inhibition was observed above an azocasein concentration of 0.05% (w/v). Esterase activity was directed mainly towards those substrates containing the aliphatic or aromatic residues of alanine, glycine, tryptophan, tyrosine and phenylalanine. Thermostability half-lives of greater than 7 days at 70 degrees C, 43 h at 80 degrees C and 90 min at 90 degrees C were found in the presence of 5 mM CaCl2. At 90 degrees C increasing the CaCl2 concentration 100-fold (0.5 mM to 50 mM) caused a 4.3-fold increase in the half-life of the enzyme from 30 to 130 min. Half-lives of 19.4 min at 100 degrees C and 4.4 min at 105 degrees C were found in the presence of 50 mM CaCl2. The metal chelators EGTA and EDTA reduced the stability at higher temperatures but had no effect on the activity of the proteinase. Activity was not stimulated by common metal activators such as Ca2+, Mg2+ and Zn2+.

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  • Purification of a Thermostable DNA Polymerase from a Thermotoga Species

    Simpson, H.D.; Coolbear, Tim; Daniel, Roy M. (1990)

    Journal article
    University of Waikato

    Most of the initial studies on the fundamental cellular process of biosynthesis and repair of deoxyribonucleic acid (DNA) have been carried out on the bacterium Escherichia coli.¹When a single-stranded DNA template is provided with a primer, the 5’ - 3’ polymerase copies the template in the presence of deoxyribonucleotide triphosphates and a divalent cation, for example, Mg²⁺.

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