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R M Daniel

Publications and source records attributed to R M Daniel.

At least 73 records · Page 4Linked to original sources
PubMed1987

Purification and properties of a stable beta-glucosidase from an extremely thermophilic anaerobic bacterium.

A beta-glucosidase (EC 3.2.1.21) was purified to homogeneity from cell-free extracts of an extremely thermophilic anaerobic bacterium. The enzyme has an Mr of 43,000 as determined by molecular-exclusion chromatography, has a pI of 4.55 and shows optimum activity at pH 6.2. The enzyme is active against a wide range of aryl beta-glycosides and beta-linked disaccharides, with beta-galactosidase activity only slightly less than beta-glucosidase activity, and significant beta-xylosidase activity. Lineweaver-Burk plots for p-nitrophenyl beta-glucoside, o-nitrophenyl beta-glucoside and cellobiose substrates are biphasic concave-downwards. Inhibition of the beta-glucosidase by substrates and glucose is negligible. Thermal inactivation follows first-order kinetics, with t1/2 (65 degrees C) 45 h, t1/2 (75 degrees C) 47 min and t1/2 (85 degrees C) 1.4 min and a deactivation energy of 380 kJ/mol at pH 6.2. At pH 7.0, which is the optimum pH for thermostability, t1/2 (75 degrees C) is 130 min. At 75 degrees C, at pH 6.2, the thermostability is enhanced about 8-fold by 10% (w/v) glycerol, about 6-fold by 0.2 M-cellobiose and about 3-fold by 5 mM-dithiothreitol and 5 mM-2-mercaptoethanol.

Bacteria, Anaerobic↗
PubMed1987

Some observations on the inhibition and activation of a thermophilic protease.

The activity of Caldolysin, the extracellular protease from the extreme thermophile Thermus aquaticus strain T351, was reduced in the presence of high protein concentrations. The absence of this effect after enzyme immobilization, or when using chromogenic substrates, suggests that a steric mechanism is involved. The apparent activation of caldolysin under certain conditions was shown to be related to both temperature and the ionic strength of the aqueous environment. The effects on activity, substrate affinity and thermostability of chemical modification with various reagents are also discussed.

Endopeptidases↗
PubMed1987

The specific activities of mesophilic and thermophilic proteinases.

1. Most enzymes from extreme thermophiles do not possess higher specific activities than similar enzymes from mesophiles (measured at their respective growth temperatures). 2. However, using protein substrates, the specific activities of thermophilic proteinases are considerably higher than those of most microbial and eukaryotic proteinases. 3. This property could be attributed to purely kinetic influences on the enzyme, to some specific "design" feature of the proteinase, or to the effects of temperature on the substrate. 4. Comparisons of the rates of hydrolysis of large and small substrates by both mesophilic and thermophilic proteinases suggest that temperature-induced changes in substrate susceptibility are a major factor.

Bacillus↗
PubMed1987

Isolation of cellulolytic anaerobic extreme thermophiles from new zealand thermal sites.

Avicel enrichment cultures from 47 thermal-pool sites in the New Zealand Rotorua-Taupo region were screened for growth and carboxymethyl cellulase activity at 75 degrees C. Eight anaerobic cellulolytic cultures were obtained. The effect of temperature on carboxymethyl cellulase activity was measured, and bacteria were isolated from the five best cultures. Bacteria from two sources designated TP8 and TP10 grew at 75 degrees C, accumulated reducing sugar in the growth medium and gave free cellulases with avicelase activity. Bacteria from sources designated Tok4, Tok8, and Wai21 grew at 75 degrees C, accumulated no free sugars in the medium, and gave free carboxymethyl cellulases with virtually no avicelase activity. All were obligate anaerobic nonsporeforming rods which stained gram negative, grew on pentoses as well as hexoses, and gave ethanol and acetate as major fermentation end products. The isolated strain which produced the most active and stable cellulases (trivially designated TP8.T) had lower rates of free endocellulase accumulation at 75 degrees C than did Clostridium thermocellum at 60 degrees C, but its cellulase activity against avicel and filter paper in culture supernatants was comparable. Tested at 85 degrees C, TP8.T carboxymethyl cellulases included components which were very stable, whereas C. thermocellum carboxymethyl cellulases were all rapidly inactivated. The TP8.T avicelase activity was relatively unaffected by Triton X-100, EDTA, and dithiothreitol. Evidence was obtained for the existence of unisolated, cellulolytic extreme thermophiles producing cellulases which were more stable and active than those from TP8.T.

Journal Article↗
PubMed1986

Comparison of Cellulolytic Activities in Clostridium thermocellum and Three Thermophilic, Cellulolytic Anaerobes.

Avicelase, carboxymethyl cellulase (CMCase), and beta-glucosidase activities have been compared between Clostridium thermocellum and three extremely thermophilic, cellulolytic anaerobes, isolates TP8, TP11, and KT8. The three isolates were all small, gram-negative staining, oval-ended rods which occurred singly and, at exponential phase, in long chains. They were nonflagellated and no spores were visible. The KT8 and TP11 isolates caused clumping of the cellulose during growth. In all four organisms the CMCase activity paralleled cell growth; however, in C. thermocellum and TP8 the avicelase activity did not increase until early stationary phase. Total CMCase activity in C. thermocellum was significantly higher than in the three isolates; however, avicelase activities were much more comparable among the four organisms. C. thermocellum produced higher levels of ethanol, and all four organisms produced similar concentrations of acetate. The amounts of free and bound CMCase and avicelase activities were investigated. In C. thermocellum and TP8 most of the CMCase and avicelase activities were bound to the cellulose in the medium. In contrast, most of the CMCase activity in TP11 and KT8 was free in the culture supernatant; a significant percentage of avicelase activity was also free. The TP8 isolate was also grown on a defined medium with urea as sole nitrogen source and cellulose serving as the carbon source. Under these conditions the pattern of enzyme production was the same as that in the enriched medium, although the level of that production was considerably reduced.

Journal Article↗
PubMed1985

A specific L-asparaginase from Thermus aquaticus.

The L-asparaginase from an extreme thermophile, Thermus aquaticus strain T351, was highly substrate- and stereospecific, with no activity against glutamine or D-asparagine. It had a high Km of 8.6 mM. In these aspects it closely resembled the corresponding enzymes from thermophilic bacteria. The enzyme had a molecular weight of 80,000, an isoelectric point of 4.6, and a pH optimum of 9.5. It showed some substrate inhibition above 20 mM asparagine and was also inhibited by L-aspartic acid, D- and L-lysine (Ki of 5.2 and 1.25 mM, respectively), and D- and L-serine. The half-life of the enzyme at 85 degrees C was 40 min. The Arrhenius plot showed a change in slope at 55 degrees C.

Asparaginase↗
PubMed1984

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

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.

Apoenzymes↗
PubMed1983

A soluble NADH dehydrogenase (NADH: ferricyanide oxidoreductase) from Thermus aquaticus strain T351.

A soluble NADH dehydrogenase (NADH:ferricyanide oxidoreductase) has been obtained by simple disruption of cells of Thermus aquaticus strain T351, and purified. The enzyme is of low molecular mass, 50 000 Da, and displays many of the properties of the membrane-bound enzyme, including inhibition by both NADH and ferricyanide, and the same Km for ferricyanide. The enzyme contains 0.05 mol of FMN, 0.16 mol of labile sulphur and 2.2 mol of iron per mol of protein. The enzyme is inhibited by NAD and cupferron competitively with ferricyanide, and by ATP (but not ADP) competitively with NADH. The enzyme is particularly thermostable, having a half-life at 95 degrees C of 35 min. The effect of temperature on the molar absorption coefficient and the stability of NADH was determined.

Enzyme Activation↗
PubMed1983

Effect of rhizobial strain and host plant on nitrogen isotopic fractionation in legumes.

Lotus pedunculatus L., Medicago sativa L., Macroptilium atropurpureum, Glycine max, and Trifolium repens L. were grown in a N-free medium and inoculated with one of ten Rhizobium strains. Dry matter, N content, and delta(15)N values were determined for various plant parts.Nodules, with the exception of those from lucerne, were enriched in (15)N relative to atmospheric N. Considerable variation was found in delta(15)N values of plant herbage (-4.5 to +0.8). The extent of isotopic discrimination was dependent on both the host plant and the infecting rhizobial strain. This further complicates, but does not invalidate, the use of small variations in the natural abundance of (15)N to estimate the contribution of symbiotically fixed N(2) to the N in legume herbage. Some other implications of the observed differences are also discussed.

Journal Article↗
PubMed1982

A correlation between protein thermostability and resistance to proteolysis.

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.

Asparaginase↗
PubMed1982

Purification and some properties of an extracellular protease (caldolysin) from an extreme thermophile.

An extracellular metal-chelator-sensitive lytic protease (assigned the trivial name caldolysin) was isolated from a Thermus-like organism, Thermus T-351. Caldolysin was purified by affinity chromatography on Cbz-D-phenylalanine-TETA-Sepharose 4B and by gel filtration. It contained 13% carbohydrate, a single zinc atom, had a molecular weight of approx. 21,000, a pH optimum of 8 (azocasein substrate), and an isoelectric point of about 8.5. It was capable of hydrolysing many soluble and insoluble protein substrates, including collagen and elastin. No esterase activity was detected, and small peptides (less than four amino acids) and low molecular weight chromogenic substrates were not hydrolysed. A specificity for small aliphatic amino acids on either side of the splitting point was indicated. Caldolysin lysed heat-killed Gram-negative bacterial cells, but had little effect on Gram-positive organisms. Caldolysin exhibited a very high degree of thermostability (t 1/2(80 degrees C) approximately 30 h, t 1/2(90 degrees C) = 1 h). The stability (but not activity) was shown to be dependent on the presence of Ca2+ (t 1/2(75 degrees C, 10 mM calcium) greater than 193 h; t 1/2(75 degrees C, no calcium) = 4.8 min). None of the other metal ions tested (Co, Zn, Sr, Mg, Ba and Cu) was as effective as calcium in conferring thermostability of EDTA-treated caldolysin. Caldolysin was stable at room temperature in moderately acid and alkaline (pH 5 to 11) buffers for periods of greater than 90 days. Little loss of enzyme activity was detected after the incubation of caldolysin at 18 degrees C in the presence of 8 M urea, 6 M guanidine hydrochloride or 1% sodium dodecyl sulphate for 24 h. At 75 degrees C, the activity half-life of caldolysin in these denaturing agents was reduced to approx. 1 h, 1 h and more than 5 h, respectively.

Carbohydrates↗
PubMed1982

The purification and some properties of a stereospecific D-asparaginase from an extremely thermophilic bacterium, Thermus aquaticus.

A specific D-asparaginase was isolated and crystallized from Thermus aquaticus strain T351. It is present in larger amounts than the L-asparaginase. The enzyme has a molecular weight of 60 000, an isoelectric point of 4.8 and a Km of 2 mM. It has 6 disulphide bonds/molecule, and a histidine residue at the active site. It is inhibited by keto acids and by high salt concentrations.

Asparaginase↗
PubMed1982

A modification for increasing the sensitivity of the casein-agar plate assay: a simple semiquantitative assay for thermophilic and mesophilic proteases.

A casein-agar plate assay was used for the quantitative determination of both mesophilic and thermophilic proteases. Because many proteases are thermostable, assay at higher temperatures is possible. The sensitivity of the plate assay increased with temperature, the optimum assay temperature depending on the thermostability of the enzyme (e.g. Thermus protease, 75 degrees C; thermolysin, 65 degrees C; trypsin, 65 degrees C; alpha-chymotrypsin, 45 degrees C). A positive correlation was observed between incubation temperature and the density of the para-casein precipitate, increasing the accuracy of diameter measurement. Using this modified method, thermostable proteases could be assayed at levels well below the limits of detection of other methods (e.g. 40 pg of thermolysin and 300 pg of trypsin detectable at 65 degrees C, a 16-fold increase in the sensitivity for trypsin compared with a conventional plate assay (Fossum, K. (1970) Acta Pathol. Microbiol. Scand. Sect. B 78, 350-361)). The sensitivity of the plate assay could be further increased by the inclusion of some detergents and chaotropic agents in the gel.

Agar↗
PubMed1981

A method for the determination of nitrate reductase.

A procedure for the assay of nitrate reductase based on Szekely's diaminodiphenylsulphone method of nitrate determination (Szekely, E. (1967) Talanta 14, 941-950) is described. The method is simple and sensitive, allowing determination of 1 microgram of nitrate in a volume of 1 ml or less. It is particularly suited to the determination of nitrate reductase.

Cystamine↗
PubMed1976

Nitrate reductase from anaerobically grown Rhizobium japonicum.

The activity of nitrate reductase in Rhizobium japonicum is controlled by oxygen tension, and not by nitrate. The enzyme from R. japonicum grown anaerobically in the presence of nitrate resembles that from bacteroids in having a molecular weight of about 69000 daltons; the enzyme from aerobically grown cells ahs a molecular weight of about 170000 daltons. Both types of enzyme have similar Km values, but differ in their sensitivity to KCN.

Anaerobiosis↗
PubMed1975

Immunological evidence for the capability of free-living Rhizobium japonicum to synthesize a portion of a nitrogenase component.

Immunodiffusion tests conducted under aerobic conditions demonstrated that cross-reactive material to antiserum prepared against the Mo-Fe protein component of nitrogenase from soybean nodule bacteroids was detectable in extracts of free-living Rhizobium japonicum cells cultured in a standard medium under: aerobic conditions; aerobic conditions with nitrate; aerobic conditions with ammonia; anaerobic conditions with nitrate; and anaerobic conditions with nitrate and ammonia. The most intense precipitin bands resulted from cross-reaction of the antiserum with extracts of cells cultured anaerobically with nitrate or anaerobically with ammonia and nitrate. Immunodiffusion experiments with crude bacteroid extract and purified Mo-Fe protein revealed a greater number of precipitin bands in tests conducted under aerobic conditions than those conducted under anaerobic conditions. These results indicate that some of the cross-reactive material observed under aerobic conditions resulted from breakdown of the Mo-Fe protein. Bacteroid extracts of nodules from plants supplied with ammonia exhibited only a trace of nitrogenase activity. The addition of an excess of the Fe protein component of nitrogenase, however, resulted in 270-fold enhancement of activity indication the presence of active Mo-Fe protein in these extracts. Our experiments together with results published elsewhere provide evidence that the genetic information for synthesis of a part of the Mo-Fe component of nitrogenase is carried by Rhizobium.

Aerobiosis↗