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At least 613 records · Page 34Linked to original sources
PubMed1989

One-step affinity purification of Bacillus neutral proteases using bacitracin-silica.

A purification procedure for neutral proteases from bacilli is described, in which bacitracin-silica was used as affinity medium. This enabled a one-step purification of the proteases directly from culture supernatant. Since neutral proteases are extremely sensitive towards autodigestion, conditions were chosen such, that autodigestion was largely prevented. Isopropanol appeared to be useful in both eluting the enzymes from the affinity medium, and inhibiting enzymatic activity during this step. The bacitracin-silica medium allowed high flow rates: with columns prepared for use in an FPLC system flow rates up to one column volume per minute were feasible, and still gave satisfactory results. The neutral proteases purified by this method were found to be homogeneous both by SDS-PAGE and analytical gel filtration.

Bacillus subtilis↗
PubMed1990

Expression of the copy DNA for human A4 and B4 L-lactate dehydrogenases in Escherichia coli.

The human LDH-A and LDH-B cDNAs, containing the coding regions for the L-lactate dehydrogenase A4 (M) and B4 (H) polypeptides respectively have been cloned into Escherichia coli to place the cDNAs under the control of hybrid E. coli/Bacillus stearothermophilus transcriptional and translational signals. Human A4- and B4-isoenzymes are produced in E. coli cells harbouring the expression plasmids pHLDHA22 and pHLDHB10 at levels of 6.5 and 1.5% of the soluble protein of the cell, respectively. The tac promoter of these vectors was not induced by isopropyl beta-D-thiogalactopyranoside. The A4 and B4 human isoenzymes synthesized in E. coli were purified to homogeneity and show the same properties as isoenzymes isolated from human tissue. The amino acid sequences of 12 N-terminal residues of the human isoenzymes synthesized in E. coli were determined to be identical to those deduced from the DNA sequence of the cloned cDNAs except that the N-terminal methionine was absent from both. However, in contrast to LDH made in human cells, acetylation of the N-terminal alanine does not take place in E. coli cells.

Amino Acid Sequence↗
PubMed1993

Purification and characterisation of Bst LVI restriction endonuclease, a thermostable isoschizomer of ClaI from Bacillus stearothermophilus LV.

This work describes the purification and biochemical characterization of BstLVI restriction endonuclease, a thermostable isoschizomer of ClaI, from Bacillus stearothermophilus LV. The enzyme was purified by successive DEAE-cellulose, Affi-Gel Blue and Heparin-Sepharose CL-6B column chromatography. A molecular weight of 37,000 was determined for Bst LVI by gel filtration. As expected from thermophilic proteins, the enzyme showed a high stability towards heat and also to other known protein-denaturing agents.

DNA↗
PubMed1994

Gene structure and amino acid sequences of alcohol dehydrogenases of Bacillus stearothermophilus.

Partial amino acid sequences of the two alcohol dehydrogenases of Bacillus stearothermophilus and the oligonucleotide sequence of a cloned fragment containing the gene for ADH 2334 were determined and compared with the known, derived ADH 1503 amino acid sequence. The two proteins are identical at 244 of 349 positions. ADH 2334 is encoded in a transcription unit containing an aldehyde dehydrogenase.

Alcohol Dehydrogenase↗
PubMed1996

Construction of single amino acid substitution mutants of cloned Bacillus stearothermophilus DNA polymerase I which lack 5'-->3' exonuclease activity.

Two individual amino acid substitutions were engineered at a selected site in the 5' --> 3' exonuclease domain of the cloned Bacillus stearothermophilus DNA polymerase I gene. These mutations resulted in the expression of enzymes lacking the 5' --> 3' exonuclease activity while maintaining normal polymerizing activity. The mutated and non-mutated enzymes were each constitutively expressed in an Escherichia coli host without the use of an exogenous or inducible promoter, and the mutated enzymes were demonstrated to be equivalent to the subtilisin large fragment of the native holoenzyme in sequencing reactions.

Amino Acids↗
PubMed1985

Changes in the state of subunit association of lactate dehydrogenase from Bacillus stearothermophilus.

Time-resolved measurements of the fluorescence anisotropy of an extrinsic dye-group attached to lactate dehydrogenase from B. stearothermophilus revealed that the rotational correlation time of the enzyme at low concentrations is 55 ns, while at high enzyme concentrations or in the presence of fructose 1,6-bisphosphate (Fru-1,6-P2) the correlation time increases to 95 ns. These correlation times are consistent with a change in Mr from 85 000 +/- 12 000 (dimer) to 150 000 +/- 22 000 (tetramer) and show that the tetrameric state can be induced either by raising the protein concentration or by the addition of the ligand. We have confirmed this change in molecular weight by gel-filtration experiments. In the ligand-induced tetramer, two Fru-1,6-P2 molecules are bound.

Binding Sites↗
PubMed1987

The engineering of a more thermally stable lactate dehydrogenase by reduction of the area of a water-accessible hydrophobic surface.

A site-directed mutant of Bacillus stearothermophilus lactate dehydrogenase (lactate:NAD+ oxidoreductase, EC 1.1.1.27) has been engineered in which the conserved hydrophobic residue isoleucine-250 has been replaced by the more hydrophilic residue asparagine. This isoleucine forms a large part of a water-accessible, hydrophobic surface in the active site of the apo-enzyme which is covered by the B-face of the nicotinamide ring when coenzymes are bound. Reduction in the area of this hydrophobic surface results in the mutant tetramer being more thermally stable than the wild-type enzyme.

Binding Sites↗
PubMed1987

The use of site-directed mutagenesis and time-resolved fluorescence spectroscopy to assign the fluorescence contributions of individual tryptophan residues in Bacillus stearothermophilus lactate dehydrogenase.

Site-directed mutagenesis has been used to generate two mutant Bacillus stearothermophilus lactate dehydrogenases: in one, Trp-150 has been replaced with a tyrosine residue and, in the other, both Trp-150 and -80 are replaced with tyrosines. Both enzymes are fully catalytically active and their affinities for substrates and coenzymes, and thermal stabilities are very similar to those of the native enzyme. Time-resolved fluorescence measurements using a synchrotron source have shown that all three tryptophans in the native enzyme fluoresce. By comparing the mutant and native enzymes it was possible, for the first time, to assign, unambiguously, lifetimes to the individual tryptophans: Trp-203 (7.4 ns), Trp-80 (2.35 ns) and Trp-150 (less than 0.3 ns). Trp-203 is responsible for 75-80% of the steady-state fluorescence emission, Trp-80 for 20%, and Trp-150 for less than 2%.

Animals↗
PubMed1987

A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure.

We have engineered a variant of the lactate dehydrogenase enzyme from Bacillus stearothermophilus in which arginine-173 at the proposed regulatory site has been replaced by glutamine. Like the wild-type enzyme, this mutant undergoes a reversible, protein-concentration-dependent subunit assembly, from dimer to tetramer. However, the mutant tetramer is much more stable (by a factor of 400) than the wild type and is destabilized rather than stabilized by binding the allosteric regulator, fructose 1,6-biphosphate (Fru-1,6-P2). The mutation has not significantly changed the catalytic properties of the dimer (Kd NADH, Km pyruvate, Ki oxamate and kcat), but has weakened the binding of Fru-1,6-P2 to both the dimeric and tetrameric forms of the enzyme and has almost abolished any stimulatory effect. We conclude that the Arg-173 residue in the wild-type enzyme is directly involved in the binding of Fru-1,6-P2, is important for allosteric communication with the active site, and, in part, regulates the state of quaternary structure through a charge-repulsion mechanism.

Allosteric Regulation↗
PubMed1987

A strong carboxylate-arginine interaction is important in substrate orientation and recognition in lactate dehydrogenase.

Using site-directed mutagenesis, Arginine-171 at the substrate-binding site of Bacillus stearothermophilus, lactate dehydrogenase has been replaced by lysine. In the closely homologous eukaryotic lactate dehydrogenase, this residue binds the carboxylate group of the substrate by forming a planar bifurcated bond. The mutation diminishes the binding energy of pyruvate, alpha-ketobutyrate and alpha-ketovalerate (measured by kcat/Km) by the same amount (about 6 kcal/mol). For each additional methylene group on the substrate, there is a loss of about 1.5 kcal/mol of binding energy in both mutant and wild-type enzymes. From these parallel trends in the two forms of enzyme, we infer that the mode of productive substrate binding is identical in each, the only difference being the loss of a strong carboxylate-guanidinium interaction in the mutant. In contrast to this simple pattern in kcat/Km, the Km alone increases with substrate-size in the wild-type enzyme, but decreases in the mutant. These results can be most simply explained by the occurrence of relatively tight unproductive enzyme-substrate complexes in the mutant enzyme as the substrate alkyl chain is extended. This does not occur in the wild-type enzyme, because the strong orienting effect of Arg-171 maximizes the frequency of substrates binding in the correct alignment.

Arginine↗
PubMed1987

Structural and spectroscopic comparison of manganese-containing superoxide dismutases.

Predicted secondary structures and optical properties of four manganese-containing superoxide dismutases isolated from Saccharomyces cerevisiae, Bacillus stearothermophilus, Escherichia coli and human liver are compared. The structural predictions are further compared with the known crystal structure of the manganese-containing superoxide dismutase from Thermus thermophilus HB8. The secondary structures of the four dismutases are predicted by the methods of Chou and Fasman (Adv. Enzymol. 47 (1978) 45-148), Garnier et al. (J. Mol. Biol. 120 (1978) 97-120) and Lim (J. Mol. Biol. 88 (1974) 873-894). The three models show satisfactory agreement and predict that the enzymes have a mixed alpha-helix and beta-sheet structure, and that they have homologous structures. The former conclusion is also reached from an analysis of the hydrophobic character of the amino-acid sequences of the four proteins according to Kyte and Doolittle (J. Mol. Biol. 157 (1982) 105-132). The calculation of the secondary structure based on the 185-260 nm circular dichroism spectrum of manganese-containing superoxide dismutase from S. cerevisiae reveals that the enzyme consists of 61% alpha-helix, 13% beta-sheet, 11% turn and 8% random coil conformations, which is in good accordance with the prediction based on the amino-acid sequences. Comparison of the 400-700 nm circular dichroism spectra of manganese-containing superoxide dismutase from S. cerevisiae, E. coli and T. thermophilus demonstrates that manganese atoms have homologous coordination in the three enzymes. This investigation based on primary structures and spectral properties indicates that the four dismutases have the same overall structure. Since the structural predictions are in good agreement with the structure found for the manganese-containing superoxide dismutase from T. thermophilus HB8, it can be concluded that this structure is representative for the four enzymes and probably for manganese-containing superoxide dismutases in general.

Amino Acid Sequence↗
PubMed1989

Isolation and characterisation of the glycerol dehydrogenase from Bacillus stearothermophilus.

A protocol for the rapid purification of the glycerol dehydrogenase (glycerol: NAD+ 2-oxidoreductase, EC 1.1.1.6) from the thermophile Bacillus stearothermophilus has been developed using a combination of chromatographic techniques including affinity chromatography on a Sepharose-immobilised triazine dye (Procion red, HE3B, ICI). Substrate specificity has been examined and Km values determined. The protein has been shown to have an oligomeric Mr of approx. 180,000 and consists of four identical subunits of Mr 42,000. Exposure to chelating agents (e.g., EDTA) leads to total loss of activity; the EDTA-inactivated enzyme can be reactivated by Zn2+ and requires 1 mol equivalent of zinc per subunit for full catalytic activity. Other divalent cations such as Cd2+ and Co2+ will reactivate the apo-enzyme but yields an enzyme of lower specific activity. The enzyme binds 1 equivalent of NADH per subunit and during catalysis transfers the 4-pro-R hydride from the nicotinamide ring of the reduced-coenzyme to the substrate. Glycerol increases the dissociation constant for the interaction between NADH and Zn-metallo-glycerol dehydrogenase (ZnGDH) but has no effect on the equilibrium between NADH and metal-depleted enzyme.

Amino Acids↗
PubMed1990

Electron transferase activity of diaphorase (NADH: acceptor oxidoreductase) from Bacillus stearothermophilus.

Electrochemical kinetic measurements were carried out for electron-transfer between NADH and the oxidized forms of mediators (ferrocenylmethanol (FMA), ferrocenyl-1-ethanol (FEA), N,N,N',N'-tetramethylphenylenediamine (TMPD), Co(Phen)2+(3) and Fe(CN)4-(6)) catalyzed by diaphorase (NADH: acceptor oxidoreductase, EC 1.6.99.-) purified from Bacillus stearothermophilus. Cyclic voltammograms for the mediators with excess NADH in the presence of diaphorase gave steady-state currents. The quantitative analysis of the dependence of the current on the mediator concentration yielded a Michaelis constant (Km) and molecular activity (ko), which are difficult to determine by the conventional spectrophotometric method. Small Km and large ko values were observed for the oxidized forms of FMA, FEA and TMPD compared to those for Co(Phen)3+(3) and Fe(CN)3-(6). It is suggested that the reaction pocket of the present diaphorase is hydrophobic. The present electrochemical procedure for the determination of the kinetic parameters is applicable widely to similar enzyme reactions.

Amino Acids↗
PubMed1990

Studies on the interactions of glycerol dehydrogenase from Bacillus stearothermophilus with Zn2+ ions and NADH.

The interactions of the essential divalent cation, Zn2+, with the binary complex formed between glycerol dehydrogenase (glycerol:NAD+ 2-oxidoreductase, EC 1.1.1.6) and its coenzyme NADH have been examined by fluorescence spectroscopy. Both the metallo and non-metallo form of the enzyme bind the coenzyme NADH. The addition of Zn2+ ions to a solution of the binary complex formed between metal-depleted enzyme and NADH results in a rapid increase in fluorescence emission at 430 nm. This has been used to determine the on rate for Zn2+ to the enzyme/binary complex. A dissociation constant of 3.02 +/- 0.25.10(-9) M for the equilibrium between Zn2+ ions and the enzyme has been determined.

Geobacillus stearothermophilus↗
PubMed1990

Amino acid sequences of the ribosomal proteins HL30 and HmaL5 from the archaebacterium Halobacterium marismortui.

The complete amino acid sequences of the ribosomal proteins HL30 and HmaL5 from the archaebacterium Halobacterium marismortui were determined. Protein HL30 was found to be acetylated at its N-terminal amino acid and shows homology to the eukaryotic ribosomal proteins YL34 from yeast and RL31 from rat. Protein HmaL5 was homologous to the protein L5 from Escherichia coli and Bacillus stearothermophilus as well as to YL16 from yeast. HmaL5 shows more similarities to its eukaryotic counterpart than to eubacterial ones.

Amino Acid Sequence↗
PubMed1991

Bacterial catalase-peroxidases are gene duplicated members of the plant peroxidase superfamily.

Bacterial catalase-peroxidases are enzymes containing 0.5-1.0 heme per subunit. The identical subunits are generally 80 kDa in size, and the sequenced subunits of E. coli, S. typhimurium and B. stearothermophilus contain 726-731 amino acid residues per subunit. The heme-containing peroxidases of plants, fungi and yeast are monomeric, homologous and 290-350 residues in size. Analyses of the amino acid sequences indicate that the double length of the bacterial peroxidases can be ascribed to gene duplication. Each half is homologous to eukaryotic, monomeric peroxidase and can be modelled into the high-resolution crystal structure of yeast cytochrome c peroxidase. The comparisons and modelling have predicted: (1) the C-terminal half does not bind heme, and bacterial peroxidases have one heme per subunit; (2) the ten dominating helices observed in the yeast enzyme are highly conserved and connected by surface loops which are often longer in the bacterial peroxidases; and (3) yeast cytochrome c peroxidase has evolved more slowly than other known peroxidases. The study has revealed ten invariant residues and a number of highly conserved residues present in peroxidases of the plant peroxidase superfamily and provides a basis for rationally engineered peroxidases.

Amino Acid Sequence↗
PubMed1991

Characterization of Bacillus stearothermophilus cyclodextrin glucanotransferase in ascorbic acid 2-O-alpha-glucoside formation.

In this study, we characterized cyclodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus in L-ascorbic acid-2-O-alpha-D-glucoside (AA-2G) formation and compared its enzymological properties with those of rat intestinal and rice seed alpha-glucosidases which had the ability to form AA-2G. CGTase formed AA-2G efficiently using alpha-cyclodextrin (alpha-CD) as a substrate and ascorbic acid (AA) as an acceptor. Several AA-2-oligoglucosides were also formed in this reaction mixture, and they could be converted to AA-2G by the additional treatment of glucoamylase. The optimum temperature for AA-2G formation was 70 degrees C and its optimum pH was around 5.0. CGTase also utilized beta- and gamma-CDs, maltooligosaccharides, dextrin, amylose, glycogen and starch as substrates, but not any disaccharides except maltose. CGTase showed the same acceptor specificity as two alpha-glucosidases, whereas its hydrolyzing activity towards AA-2G was very low compared with those of alpha-glucosidases. Cleavage profiles of AA-2-oligoglucosides by CGTase present a possible mechanism for AA-2G formation that CGTase transfers a glucose-hexamer to an acceptor at the first step and then a glucose is stepwisely removed from the non-reducing end of the product through glucoamylase-like action of this enzyme. These results indicate that CGTase is able to synthesize AA-2G more efficiently than rat and rice alpha-glucosidases and utilization of this enzyme makes the mass production of AA-2G possible.

Animals↗
PubMed1991

The effect of kirromycin on the metal ion coordination in complexes of elongation factor Tu from Bacillus stearothermophilus as inferred from the 17O-55Mn superhyperfine interaction.

The binding of the antibiotic kirromycin to elongation factor Tu (EF-Tu) leads to a severe line broadening of the Q-band manganese EPR lines of EF-Tu.Mn, EF-Tu.Mn.GDP, as well as EF-Tu.Mn.GDP.Pi indicating that the coordination sphere of the metal ion is changed by this interaction. The number of coordinated water molecules was determined from the 17O-55Mn superhyperfine coupling observable in H2(17)O enriched water; it is pH-dependent in the EF-Tu.Mn.GDP complex, at pH 6.8 probably four water molecules are coordinated with the protein bound manganese, at pH 8.4 one of the water molecules is replaced by a functional group from the protein. Independent of the pH, probably four and five water molecules are bound to the metal ion in the EF-Tu.Mn and in the EF-Tu.Mn.GDP.Pi complex, respectively. Kirromycin does not influence the number of water molecules bound to EF-Tu.Mn.GDP and EF-Tu.Mn.GDP.Pi, but leads to an increase of the number of water molecules coordinated to the metal ion in EF-Tu.Mn. The 17O-55Mn superhyperfine coupling constant in Mn(H2O)6 was determined from the EPR-spectra as 0.24 mT.

Anti-Bacterial Agents↗