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R M Daniel

Publications and source records attributed to R M Daniel.

At least 37 records · Page 2Linked to original sources
PubMed1996

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

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 1 volume.

Bacillus↗
PubMed1996

Properties and stabilization of an extracellular alpha-glucosidase from the extremely thermophilic archaebacteria Thermococcus strain AN1: enzyme activity at 130 degrees C.

An extracellular alpha-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 degrees C, the purified enzyme in buffer has a half-life around 35 min, which is increased to around 215 min in presence of 1% (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 degrees C is 0.41 mM with pNP-alpha-D-glucopyranoside as substrate. The substrate preference of the enzyme is: pNP-alpha-D-glucoside > nigerose > panose > palatinose > isomaltose > maltose and turanose. No activity was found against starch, pullulan, amylose, maltotriose, maltotetraose, isomaltotriose, cellobiose and beta-gentiobiose. A variety of techniques including immobolization (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 degrees C. The half-life could be increased from about 4 min at 100 degrees C to 30-60 min at 130 degrees C in presence of 90% (w/v) sorbitol, 1% (w/v) dithiothreitol and 1% (w/v) BSA, and by cross-linking with BSA in the presence of 90% (w/v) sorbitol. The stabilized enzyme showed good activity at 130 degrees C.

Archaea↗
PubMed1996

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

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 scalable to 600 1 volume.

Bacillus↗
PubMed1995

Steady state kinetics of the glutamate dehydrogenase from an archaebacterial extreme thermophile, isolate AN1.

A steady state kinetic study was carried out with the glutamate dehydrogenase from the thermophilic, archaebacterial isolate AN1. Initial velocity studies of the oxidative deamination reaction showed the mechanism is sequential and indicated that the order of substrate addition is random, while inhibition studies with products and substrate analogues suggested a strong preference for NADP+ to bind first. Initial velocity studies of the reductive amination reaction showed that the mechanism is sequential and indicated that the order of substrate addition is random, while product inhibition studies and the effect of substrate saturation on the initial velocity suggested that the preferred order of substrate addition is NADPH, 2-ketoglutarate, ammonia.

Ammonia↗
PubMed1995

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

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+.

Amino Acid Sequence↗
PubMed1995

The effect of low temperatures on enzyme activity.

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.

Animals↗
PubMed1995

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

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 degrees 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-DL-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 degrees 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 degrees 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 other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the thermostable enzyme, kumamolysin.

Amino Acid Sequence↗
PubMed1994

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

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.

Bacteria, Anaerobic↗
PubMed1993

Glutamate dehydrogenase from the extremely thermophilic archaebacterial isolate AN1.

Glutamate dehydrogenase (L-glutamate:NADP+ oxidoreductase, deaminating and transaminating, EC 1.4.1.4) was purified to homogeneity from the extremely thermophilic archaebacterial isolate AN1 (a member of the Thermococcales). The enzyme comprised a large proportion of the soluble cell protein (11%) and was purified in high yield. The molecular mass of the native enzyme was 204 kDa, while the subunit molecular mass was 47 kDa, indicating a tetrameric structure. The enzyme is specific for NADP(H) rather than NAD(H) by a factor of greater than 1000, as judged by Vmax/Km. Glutamate synthase activity was about 50% of the glutamate dehydrogenase activity. Activity was markedly enhanced by calcium, magnesium and manganese ions. The enzyme was highly thermostable with t1/2 values of 12.5 h and 47 min at 90 degrees C and 103 degrees C, respectively.

Archaea↗
PubMed1993

Thermostable beta-glucosidase and beta-xylosidase from Thermotoga sp. strain FjSS3-B.1.

A beta-D-glucosidase and a beta-D-xylosidase were purified to homogeneity from the thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1. Both enzymes were largely cell-associated and were probably associated with the 'toga' structures of this organism. Using SDS-PAGE they were found to have M(r) values of 75,000 and 92,000, respectively. The beta-glucosidase was active against cellobiose, sophorose and gentiobiose with Km values of 59 mM, 2.7 mM and 6 mM, respectively. The beta-xylosidase had a Km of 2 mM for xylobiose, showed strong activity against p-nitrophenyl alpha-L-arabinofuranoside and p-nitrophenyl alpha-L-arabinopyranoside, but was subject to strong substrate inhibition by p-nitrophenyl beta-D-xylopyranoside. Both enzymes were extremely thermostable, with half-lives of several hours at 98 degrees C. The thermostabilities of both enzymes were increased further by the addition of either trehalose or betaine.

Betaine↗
PubMed1993

Purification and properties of a beta-1,4-xylanase from a cellulolytic extreme thermophile expressed in Escherichia coli.

1. An endoxylanase (EC 3.2.1.8) was purified from an Escherichia coli strain carrying a xylanase gene from the extreme thermophile "Caldocellum saccharolyticum" strain Tp8T6.3.3.1. It was found to have an M(r) of 42,000 and an isoelectric point of approx. 5.0. 2. The enzyme showed optimum activity at pH 5.0-7.7 and had an activation energy of 44 kJ mol-1. It was stable at room temperature at pH 4.5-11.5 in the presence of 0.5 mg ml-1 bovine serum albumin. The half-life of the enzyme at 75 degrees C was 20 min at pH 6.0 in the presence of 0.5 mg ml-1 bovine serum albumin. 3. The xylanase had highest activity on oat spelts xylan, releasing xylobiose and some xylotriose. The Km for oat spelts xylan was 0.021% (w/v) at pH 6.0. 4. The enzyme had high activity on sugar cane bagasse hemicelluloses A and B, lower activity on larchwood xylan and also hydrolysed carboxymethylcellulose, 4-methylumbelliferyl beta-D-cellobioside and p-nitrophenyl beta-D-cellobioside, but could not hydrolyse xylobiose. 5. It showed transferase activity on p-nitrophenyl beta-D-xylopyranoside. Xylose did not inhibit the enzyme.

Bacteria↗
PubMed1993

The use of phenyl-Sepharose for the affinity purification of proteinases.

Phenyl-Sepharose is most often used as an adsorbent for hydrophobic interaction chromatography (HIC). We report on its effective use for the affinity purification of some extracellular thermostable proteinases from bacterial sources. Proteinases belonging to the serine, aspartate and metallo mechanistic classes were effectively retained by the media. Purification factors in the range of 2.9-60 and enzyme activity yields in excess of 88% were obtained. In some cases homogeneous enzyme was obtained from culture supernatants in a single step. A number of other proteinases from mammalian sources were also retained. The specificity of the enzyme/support interaction was studied. Proteinases complexed with peptide inhibitors (pepstatin and chymostatin) showed reduced binding to phenyl Sepharose indicating interaction with the active site cleft whereas modification with low molecular weight active site directed inactivators such as PMSF and DAN did not, indicating that binding may not be dependent on the catalytic site. Pepsinogen and the pro-enzyme form of the serine proteinase from the thermophilic Bacillus sp. strain Ak.1 were not retained by the media and could be resolved in an efficient manner from their active counterparts.

Bacteria↗
PubMed1993

Some characteristics of a proteinase from a thermophilic Bacillus sp. expressed in Escherichia coli: comparison with the native enzyme and its processing in E. coli and in vitro.

Proteinase Ak.1 was produced during the stationary phase of Bacillus sp. Ak.1 cultures. It is a serine proteinase with a pI of 4.0, and the molecular mass was estimated to be 36.9 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 60 and 70 degrees C, with half-lives of 13 h and 19 min at 80 and 90 degrees C, respectively. Maximum proteolytic activity was observed at pH 7.5 with azocasein as a substrate, and the enzyme also cleaved the endoproteinase substrate Suc-Ala-Ala-Pro-Phe-NH-Np (succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanalide). Major cleavage sites of the insulin B chain were identified as Leu-15-Tyr-16, Gln-4-His-5, and Glu-13-Ala-14. The proteinase gene was cloned in Escherichia coli, and expression of the active enzyme was detected in the extracellular medium at 75 degrees C. The enzyme is expressed in E. coli as an inactive proproteinase at 37 degrees C and is converted to the mature enzyme by heating the cell-free media to 60 degrees C or above. The proproteinase was purified to homogeneity and had a pI of 4.3 and a molecular mass of 45 kDa. The NH2-terminal sequence was Ala-Ser-Asn-Asp-Gly-Val-Glu-, showing the exact signal peptide cleavage point. Heating the proenzyme resulted in the production of active proteinase with an NH2-terminal sequence identical to that of the native enzyme. The characteristics of the cloned proteinase were identical to those of the native enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

The effect of metal ions on the activity and thermostability of the extracellular proteinase from a thermophilic Bacillus, strain EA.1.

The proteinase from the extremely thermophilic Bacillus strain EA.1 exhibits maximum stability at a pH of approx. 6.5. In the presence of calcium ions the half-life at 95 degrees C of the enzyme at this pH was 17 min, and loss of activity followed first-order decay kinetics. The role of metal ions in the activity and stability of the enzyme was studied using the holoenzyme, the metal-depleted apoenzyme, and a zinc-enriched apoenzyme preparation. Zinc and calcium ions were the preferred bivalent cations for the active site and stabilization site(s) respectively. Stabilization by metal ions was not in itself a highly stringent process, but ions other than calcium which stabilized the enzyme generally had a concomitant inhibitory effect on activity. Inhibition and stabilization of the enzyme by cations were concentration-dependent effects and certain ions activated the apoenzyme but not the holoenzyme. Manganese(II) ions conferred some stability and also activated the enzyme, but in the latter case were not as effective as zinc ions. The results are discussed with reference to the ionic radii, co-ordination number and preferred ligand donors of the ions. Mercury(II) ions severely compromised enzyme activity and stability, and the effects of thiol-reactive agents suggest that thiol groups also have a role in enzyme integrity.

Apoproteins↗
PubMed1992

Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A.

Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25-10.5) which can be exploited in purification by using cation-exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0-10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0-10.0 and maximum activity, in a 10-min assay, was observed at 90 degrees C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70 degrees C and the half-lives at 80 degrees C and 90 degrees C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60 degrees C and the half-life at 70 degrees C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents. Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1' amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1' position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1' sites.

Amino Acid Sequence↗