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PubMedDate not supplied

Phylogenetic analysis of the putative phosphorylation domain in the pyruvate kinase of Bacillus stearothermophilus.

All sequenced phosphoenolpyruvate synthases (PPS), pyruvate:phosphate dikinases (PPDK) and enzymes I (EI) of the phosphoenolpyruvate:sugar phosphotransferase system comprise the PEP family. Linked to the C terminus of the sequenced pyruvate kinase from Bacillus stearothermophilus (PKBst) is a domain that is homologous to the putative phosphorylation domains of PEP family enzymes. We report sequence and phylogenetic analyses that lead to the following conclusions: (1) the phosphorylation domain of PKBst was derived from a PPS, late in the evolutionary process, after the divergence of PPSs from PPDKs and EIs; (2) this domain is probably functional in phosphoryl transfer; (3) the C-terminal phosphorylation domain in PKBst probably defines a compact domain in all PEP family proteins that is linked to other domains in these proteins via flexible linkers.

Amino Acid Sequence↗
PubMed1994

Solvent isotope effects and the nature of electrophilic catalysis in the action of the lactate dehydrogenase of Bacillus stearothermophilus.

Deuterium oxide at atom fractions of deuterium from 0.0 to 0.97 has an effect of less than 20% on the kinetic term kcat/KmB (believed to reflect the transition state for the hydride-transfer step) for the reduction of pyruvic acid by NADH at 55 degrees C, with catalysis by the tetrameric form of the lactate dehydrogenase of Bacillus stearothermophilus. This observation suggests that the hydride-transfer event is not assisted by protonic bridging to the carbonyl group being reduced. The results are consistent with protonic bridging only if an opposing isotope effect is present, for example from a generalized conformation or solvation change. The results are consistent with other forms of electrophilic catalysis.

Deuterium Oxide↗
PubMed1996

Properties of diacetyl (acetoin) reductase from Bacillus stearothermophilus.

The cells of Bacillus stearothermophilus contain an NADH-dependent diacetyl (acetoin) reductase. The enzyme was easily purified to homogeneity, partially characterised, and found to be composed of two subunits with the same molecular weight. In the presence of NADH, it catalyses the stereospecific reduction of diacetyl first to (3S)-acetoin and then to (2S,3S)-butanediol; in the presence of NAD+, it catalyses the oxidation of (2S,3S)- and meso-butanediol, respectively, to (3S)-acetoin and to (3R)-acetoin, but is unable to oxidise these compounds to diacetyl. The enzyme is also able to catalyse redox reactions involving some endo-bicyclic octen- and heptenols and the related ketones, and its use is suggested also for the recycling of NAD+ and NADH in enzymatic redox reactions useful in organic syntheses.

Alcohol Oxidoreductases↗
PubMed1996

Biotechnology and biomimetic with crystalline bacterial cell surface layers (S-layers).

Crystalline bacterial cell surface layers (S-layers) are the outermost cell envelope component of many eubacteria and archaeobacteria. S-layers are composed of a single protein or glycoprotein species and exhibit oblique, square or hexagonal lattice symmetry. Pores passing through these monomolecular arrays show identical size and morphology, and functional groups are aligned in well-defined positions and orientations. Due to these unique features, S-layers have broad application potential in biotechnology including functioning as biomimetic membranes. Presently, S-layers are used (i) for the production of isoporous ultrafiltration membranes with very well defined molecular sieving and adsorption properties, (ii) as matrices for the controlled immobilization of biologically active macromolecules (e.g., enzymes, antibodies, ligands) as required for biosensors, affinity membranes and affinity microparticles as well as for solid phase assays, (iii) as stabilizing structures for Langmuir-Blodgett films and liposomes and (iv) as carriers and adjuvants for weakly immunogenic antigens and haptens.

Adjuvants, Immunologic↗
PubMed1996

Purification and characterization of the alcohol dehydrogenase from a novel strain of Bacillus stearothermophilus growing at 70 degrees C.

The biocatalysts isolated from thermophilic microorganisms are the object of ever-growing scientific interest for (i) the comprehension of the molecular basis of their thermal tolerance, and (ii) their use in different bio-industrial fields. Here we report the purification and characterization of an alcohol dehydrogenase (designated ADH-hT) from the novel strain LLD-R of Bacillus stearothermophilus which grows at 70 degrees C. ADH-hT was obtained in pure form by anion exchange chromatography and two affinity chromatographies, with a final yield of about 30%. ADH-hT was found to be a tetramer of 37 kDa-subunits, and to have a pI of 4.9. ADH-hT displayed a broad substrate specificity; its activity was highest for aldehydes, and decreased progressively for alcohols and ketones. ADH-hT was endowed with catalytic activity and resistance in the presence of several denaturing agents (organic solvents, detergents, chaotropic agents). ADH-hT shared with ADH 1503 (the alcohol dehydrogenase from B. stearothermophilus strain NCA 1503 which grows at 55 degrees C) the optimal temperature of 65 degrees C, but it was more resistant than ADH 1503 towards heating. In conclusion, due to its stability and broad substrate specificity ADH-hT could be utilized in bio-industrial processes. Furthermore, we believe that ADH-hT could represent a good model system for studying the mechanism(s) which proteins exploit to gain heat resistance.

Alcohol Dehydrogenase↗
PubMed2005

Examination of MgATP binding in a tryptophan-shift mutant of phosphofructokinase from Bacillus stearothermophilus.

A tryptophan-shift variant of Bacillus stearothermophilus phosphofructokinase (BsPFK), W179F/F76W, was constructed to evaluate the binding and allosteric characteristics associated with MgATP. W179F/F76W BsPFK has a specific activity of 77+/-1 U/mg at pH 7 and 25 degrees C, which is a 35% decrease compared to the wild-type enzyme. The K(m) for MgATP increases from 43+/-3 microM for wild-type BsPFK to 160+/-7 microM in the variant. Binding and allosteric interaction between Fru-6-P and PEP for the variant are similar to those of the wild-type enzyme. W179F/F76W BsPFK has distinct fluorescence properties relative to wild-type or other tryptophan-shifted mutants of BsPFK. The binding of MgATP produces an 80% decrease in the fluorescence intensity while MgADP causes a 70% decrease. Capitalizing on these fluorescence changes, dissociation constants of 30+/-1 microM and 0.53+/-0.02 mM were measured for MgATP and MgADP, respectively. In addition, PEP was shown to enhance MgATP binding by 2.6-fold.

Adenosine Diphosphate↗
PubMed2004

Antibodies specific to modified glyceraldehyde-3-phosphate dehydrogenase induce inactivation of the native enzyme and change its conformation.

The antibodies specific to an inactive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus prepared by the treatment of the tetrameric holoenzyme with glutaraldehyde were obtained. They were purified from the pool of polyclonal rabbit antibodies to GAPDH with the use of immobilized GAPDH cross-linked by glutaraldehyde as an affinity sorbent. Such antibodies were capable of interacting with the native enzyme, inducing its time-dependent inactivation; the effect was different with the apo- and holoenzyme forms. Differential scanning calorimetry of the purified [GAPDH].[antibody] complex revealed a large shift of the temperature corresponding to the maximal heat capacity of the holoenzyme towards the lower temperature. Again, the effect appeared to be different with the apoenzyme. Together, the results are consistent with the hypothesis that a specific antibody is able to exercise a certain strain on the target protein, altering its conformation toward the structure of the species which served to select the antibody. The possibility of preparing selective enzyme inhibitors based on the antibodies specific to inactive enzyme conformations is considered.

Animals↗
PubMed2005

Opposite roles of domains 2+3 of Escherichia coli EF-Tu and Bacillus stearothermophilus EF-Tu in the regulation of EF-Tu GTPase activity.

The effect of noncatalytic domains 2+3 on the intrinsic activity and thermostability of the EF-Tu GTPase center was evaluated in experiments with isolated domains 1 and six chimeric variants of mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) EF-Tus. The isolated catalytic domains 1 of both EF-Tus displayed similar GTPase activities at their optimal temperatures. However, noncatalytic domains 2+3 of the EF-Tus influenced the GTPase activity of domains 1 differently, depending on the domain origin. Ecdomains 2+3 suppressed the GTPase activity of the Ecdomain 1, whereas those of BstEF-Tu stimulated the Bstdomain 1 GTPase. Domain 1 and domains 2+3 of both EF-Tus positively cooperated to heat-stabilize their GTPase centers to attain optimal activity at a temperature close to the optimal growth temperature of either organism. This can be explained by a stabilization effect of domains 2+3 on alpha-helical regions of the G-domain as revealed by CD spectroscopy.

Escherichia coli↗
PubMed2006

Post-transcriptional regulation of the xynA expression by a novel mRNA binding protein, XaiF.

XaiF, a novel 32kDa protein encoded by the ORF located in the immediate downstream of the xynA gene of Bacillus stearothermophilus No. 236, was identified to be the xylanase-specific trans-activator. In this study, the positive effect of XaiF was confirmed to be xylanase-specific, and the results from Northern blot and in vitro transcription assays showed that the XaiF increased the xynA transcripts at post-transcriptional step. Moreover, analysis of the mRNA decay rate led to the assertion that the XaiF functions to stabilize the xynA mRNA. Intriguingly, in vitro RNA-protein binding assay and analysis using gst-xynA 3'-UTR chimeric gene constructs demonstrated that the XaiF stabilizes xynA mRNA by direct binding onto the 3'-UTR of the mRNA.

Bacterial Proteins↗
PubMed2005

Effect of digestion temperature and pH on treatment efficiency and evolution of volatile fatty acids during thermophilic aerobic digestion of model high strength agricultural waste.

Thermophilic aerobic digestion (TAD) of a model agricultural waste, potato peel slurry, at soluble chemical oxygen demand (COD) load equivalent to approximately 8.0 gl(-1), was carried out under batch conditions at 0.5 vvm aeration rate. Digestions were carried out at temperatures of 45, 50, 55, 60 and 65 degrees C (or left unregulated) without pH control to study the effect of digestion temperatures on TAD. The effects of digestion pH on the process were studied at pH 6.0, 7.0, 8.0, 9.0 and 9.5 (and in unregulated control) all at 55 degrees C. Except for digestion at 65 degrees C, which was inoculated extraneously using culture of Bacillus strearothermophilus all reactions were carried out using the populations indigenous to the waste. During digestion at different temperatures, the removal of soluble COD increased with temperature to reach a peak at 60 degrees C before declining slightly, removal of soluble solid (SS) followed similar pattern and reached peak at 65 degrees C being the highest temperature studied, while the degradation of TSS and TS (TSS + TS) decreased with an increase in temperature. Digestion at pH 7.0 was more efficient than at other pH values. Acetate was the predominant volatile fatty acid (VFA) in all the reactions and accounted for up to 90% of the total. Digestion at 60 degrees C led to the greatest accumulation of acetate, and this coincided with the period of highest oxygen uptake, and rapid consumption of soluble carbohydrate. Iso-valerate was also produced at all pH values. Digestion at 55 degrees C and also at pH 7.0 led to rapid and efficient processes with least accumulation of VFA and should be of interest in full-scale processes whenever it is practicable to regulate the digestion pH and temperature. The result of digestion at unregulated pH indicates that gradual adaptation may be used to achieve efficient treatment at elevated pH values. This would be of interest in full-scale processes where it is not practicable to tightly regulate digestion pH, and where the waste is produced at a pH value much higher than neutral.

Aerobiosis↗
PubMed2004

Synthesis of polyhydroxylated piperidines and evaluation as glycosidase inhibitors.

A series of 16 new chiral nonracemic polyhydroxylated piperidines was synthesized utilizing several chiral beta-amino-alcohols. They act as a nitrogen source, chirality inducer and iminium stabilizer, in the desymmetrization of meso-trihydroxylated glutaraldehyde. The biological activity of these compounds towards several glycosidases (alpha-D-glucosidase, alpha-D-mannosidase, alpha-L-fucosidase) has been evaluated.

Bacterial Proteins↗
PubMed2005

Aglycon specificity profiling of alpha-glucosidases using synthetic probes.

We designed and synthesized hydrogen bond based probes 1-8 with the exception of known glycosidase inhibition mechanisms, and aglycon specificity of 11 different sources of alpha-glucosidases were investigated using their probes. Probe 4 (2,6-anhydro-1-deoxy-1-[(1-oxopentyl-5-hydroxy)amino]-D-glycero-D-ido-heptitol) showed a potent inhibition of S. cerevisiae alpha-glucosidase among all alpha-glucosidases. Probe 4 was found to be a competitive inhibitor for S. cerevisiae alpha-glucosidase with Ki 0.13 mM.

1-Deoxynojirimycin↗
PubMed2004

A novel alpha-glucosidase inhibitor from pine bark.

Inhibitors of carbohydrate-hydrolysing enzymes play an important role for the treatment of diabetes. To our knowledge, a number of inhibitors such as, 1-deoxynojirimycin, acarbose and voglibose have been identified as a result of screening of secondary metabolites up to now. In this note, we report the inhibitory effect on carbohydrate hydrolysis of ethanol extracts from more than 1400 species of plants with the aim of identifying a potential antihyperglycemic drug. Pinus densiflora bark extracts showed the highest inhibition activity against several carbohydrate-hydrolysing enzymes.

Animals↗