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At least 631 records · Page 35Linked to original sources
PubMed1992

Formycins A and B and some analogues: selective inhibitors of bacterial (Escherichia coli) purine nucleoside phosphorylase.

Formycin B (FB), a moderate inhibitor (Ki approximately 100 microM) of mammalian purine nucleoside phosphorylase (PNP), and formycin A (FA), which is totally inactive vs. the mammalian enzyme, are both effective inhibitors of the bacterial (Escherichia coli) enzyme (Ki approximately 5 microM). Examination of a series of N-methyl analogues of FA and FB led to the finding that N(6)-methyl-FA, virtually inactive vs. the mammalian enzyme, is the most potent inhibitor of E. coli purine nucleoside phosphorylase (Ki approximately 0.3 uM) at neutral pH. Inhibition is competitive not only with respect to Ino, but also relative to 7-methyl-Guo and 7-methyl-Ado, as substrates. Both oxoformycins A and B are relatively poor inhibitors. For the most potent inhibitor, N(6)-methyl-FA, it was shown that the enzyme preferentially binds the neutral, and not the cationic, form. In accordance with this the neutral, but not the cationic form, of the structurally related N(1)-methyl-Ado was found to be an excellent substrate. Reported data on tautomerism of formycins were profited from, and extended, to infer which tautomeric species and ionic forms are the active inhibitors. A commercially available (Sigma) bacterial PNP, of unknown origin, was shown to differ from the E. coli enzyme by its inability to phosphorylase Ado; this enzyme was also resistant to FA and FB. These findings have been extended to provide a detailed comparison of the substrate/inhibitor properties of PNP from various microorganisms.

Bacillus cereus↗
PubMed1993

The identification of a lysine residue reactive to pyridoxal-5-phosphate in the glycerol dehydrogenase from the thermophile Bacillus stearothermophilus.

The glycerol dehydrogenase (GDH) from Bacillus stearothermophilus is inactivated by incubation with pyridoxal-5-phosphate (PALP). The complex formed between the two can be trapped by reduction with sodium borohydride to yield a protein with an absorbance band at 325 nm and a fluorescence emission band at 430 nm, typical of trapped pyridoxal-5-phosphate moieties. Total loss of catalytic activity of the enzyme is associated with the modification of approximately one equivalent of the reagent; the incorporation of the reagent and the loss of activity can be prevented by the additional presence of the oxidised or reduced coenzyme. Peptides derived from the labelled protein have been sequenced and have identified Lys-97 as the reactive residue. Site-directed mutagenesis had been used to replace Lys-97 by a His residue. This mutated enzyme has no catalytic activity and fluorescence spectroscopy studies suggest that it is unable to bind NADH.

Amino Acid Sequence↗
PubMed1995

Sucrose reduces the efficiency of protein denaturation by a chaotropic agent.

Sugars and polyols are used to stabilize proteins. The degree of stabilization conferred on a model protein by sucrose was calculated in terms of the free energy of folding. Phosphoglycerate kinase (PGK) was denatured by guanidine hydrochloride (GuHCl) in different sucrose concentrations. The linear extrapolation method [1,2] was used to calculate the free energy of folding in the absence of denaturant. Although sucrose increased the concentration of GuHCl required to unfold the protein, the free energy of folding in water was unchanged. In order to probe the nature of the stabilizing effect of sucrose, an FT-Raman spectroscopic study of denaturant-polyol systems was undertaken. Investigations of interactions between GuHCl, urea or formamide and polyhydric compounds, revealed no evidence for hydrogen bonding or dipole-dipole associations. Polyhydric compounds caused minor changes in denaturant spectra although the converse was not observed. The structure of deuterated water changed on addition of denaturants. For non-ionic denaturants, addition of polyhydric solutes countered this change in water structure. Thus polyhydric compounds oppose the effect of denaturants on water structure. The observed increase in GuHCl concentration required to unfold PGK in the presence of sucrose may be attributed to this property of sucrose.

Formamides↗
PubMed1996

Conformational analysis of pentapeptide sequences matching a proposed recognition motif for lysosomal degradation.

A selective pathway for the degradation of specific long-lived cytosolic proteins is activated in response to starvation in vivo or to serum withdrawal from cultured cells. It involves recognition of a targeting motif by a member of the hsp70 family. A 5-residue targeting motif has been proposed on the basis of sequence comparisons. We investigate whether there is any structural basis for this motif being the true recognition signal. We examine the conformations of four motif peptides in proteins that are either known to be serum regulated or are from related vertebrate species, and two equivalent peptides in bacterial proteins that closely resemble other regulated proteins. Our studies show that all the motif sequences are located near the ends of surface helices with one or more of the residues buried in the structure, yet it is known that members of the hsp70 family tend to interact with extended peptide chains. Furthermore, recognition by these proteins generally requires a specific ordering of key residues, yet the motif implies a largely order-independent sequence characterized by residue type only. We conclude that the proposed motif is unlikely to be the true targeting signal for lysosomal degradation unless additional factors apply.

Amino Acid Sequence↗
PubMed1996

Structural consequences of neopullulanase mutations.

Bacillus stearothermophilus neopullulanase (NPL) structure was modeled based on Aspergillus oryzae alpha-amylase (TAA) to understand the structure-function relationships of this pullulan hydrolyzing enzyme. The NPL structure seems to consist of a central (alpha/beta)8 barrel to which the other domains are attached. The immediate surroundings of the NPL catalytic site were found to have very similar structure to TAA. The more distant sites are different due to the stereochemical requirements of accommodating in the substrate alpha-1,6-linkages at every third position instead of alpha-1,4-linkages. The substrate binding cleft is wider than in alpha-amylases. The NPL structure, function, substrate binding and the consequences of mutations were discussed based on the modeled structure.

Amino Acid Sequence↗
PubMed1990

Thermal analysis of bacteria by differential scanning calorimetry: relationship of protein denaturation in situ to maximum growth temperature.

Differential scanning calorimetry (DSC) was used to analyze thermal transitions in two strains of the thermophile Bacillus stearothermophilus (ATCC 12016 and WAT), the mesophile Bacillus megaterium and the psychrotroph Bacillus psychrophilus. The observed transitions, representing lipid melting and DNA and protein unfolding, are compared to the maximum growth temperature (Tmax) in each species as a means of identifying critical, thermolabile targets responsible for heat-induced inhibition of growth. A low temperature, lipid transition was detected in B. stearothermophilus and B. megaterium which varied slightly with Tmax but whose high temperature end is always 22-33 degrees C below Tmax. The transition temperature (Tm) of the main melting of DNA varies from 88 to 92 degrees C, 23-32 degrees C above Tmax. The main part of the profile representing irreversible transitions is resolvable into at least three distinct peaks and is identified primarily with protein denaturation. The onset temperature for denaturation (Tl), i.e., minimum temperature of detectable denaturation, is somewhat dependent on growth temperature (Tg). Tmax for B. stearothermophilus ATCC and WAT is 69 and 56 degrees C, respectively. For cells grown between 4 and 20 degrees C below Tmax, Tl is 2-4 degrees C lower than Tmax, demonstrating that some denaturation can be tolerated before complete inhibition of growth and suggesting that inhibition of growth is due to the denaturation of a critical protein with a Tm a few degrees above Tl or to the accumulation of denatured protein to a critical level. A similar pattern holds for B. megaterium and B. psychrophilus, except that Tmax is 48 and 32.5 degrees C (Tl = 45-46 degrees C and 30 degrees C), respectively. Thus, there is an excellent correlation between the onset of protein denaturation and maximum growth temperature for these three species of the same genus. This study also demonstrates the applicability of DSC for resolving transitions in intact cells on the basis of thermostability of cellular constituents and for obtaining an overall view of macromolecular stability.

Bacillus↗
PubMed1994

Engineering surface loops of proteins--a preferred strategy for obtaining new enzyme function.

A prerequisite for the rational redesign of enzymes is that altering amino acids in an attempt to obtain new biological function does not unexpectedly alter the globular, natural framework of the native protein on which the design is being executed. The results of combinatorial-mutagenesis strategies suggest that random variation of amino acid sequence is most easily tolerated at the solvent-exposed surfaces of a protein. This review analyses effective redesigns of Bacillus stearothermophilus lactate dehydrogenase (bsLDH), in which all residue variations are at solvent-exposed surfaces. The majority of these variations were located within surface loops, which interconnect stable secondary structures traversing the globular core of the protein.

Geobacillus stearothermophilus↗
PubMed1988

The evaluation of the recovery capacity of media for heat-treated Bacillus stearothermophilus spore strips.

Six media were assessed for their capacity to recover spores of Bacillus stearothermophilus from three commercially available types of spore strip heated for up to 5 min at 121 degrees C in steam. All media recovered similar numbers of spores from unheated strips; however, there was considerable difference in survivor counts when the media were used to recover heated spores. Media containing starch consistently recovered the highest number of spores. The greatest difference in recovery capacity between the media was observed when spore strips were heated for 5 min at 121 degrees C.

Colony Count, Microbial↗
PubMed1996

Effect on pH heating medium on the thermal resistance of Bacillus stearothermophilus spores.

The influence of the pH of heating medium on heat resistance of Bacillus stearothermophilus spores (ATCC 7953, 12980, 15951 and 15952) were studied. The pH values tested were: 4.0, 5.0, 6.0 and 7.0 at temperatures of 115, 120, 125, 130 and 135 degrees C. It was found that at low treatment temperatures (115 degrees C) D-values decreased between 7- and 10-fold with 7953, 12980 and 15951 strains and about 23-fold with 15952 strain when pH dropped from 7.0 to 4.0. At highest treatment temperatures (135 degrees C) D-values obtained with pH 6.0 and 7.0 did not show any significant statistical differences (p > 0.05). z-Values appeared to be higher when the medium was acidified, ranging from 7.58 to 8.20 and 9.43 10.0 for spores suspended in McIlvaine buffer pH 7.0 and pH 4.0, respectively, although the difference was not statistically significant. Heat resistance of strain ATCC 7953 at 120, 128, and 135 degrees C in asparagus purée and tomato purée at pH 5.0 under continuous monitoring of pH was also determined. D-values obtained in asparagus purée were similar to those obtained in buffer at the same pH, whereas those observed in tomato purée were found to be lower.

Culture Media↗
PubMed1996

Heat resistance of Bacillus stearothermophilus spores in alginate-mushroom puree mixture.

Thermal resistance of Bacillus stearothermophilus spores has been established inoculating spores in alginate-mushroom puree mixture (ungelled) and in alginate-mushroom puree mixture set in calcium chloride (gelled). Data are compared with those obtained suspending the spores in distilled water, mushroom extract and in calcium chloride. Results indicated that, in general, D values obtained in gelled mixture were higher than D values obtained in distilled water, mushroom extract or in ungelled mixture, while the D value in the ungelled mixture was similar to that obtained in distilled water. D121 value in gelled mixture was close to that obtained in 2% (w/v) calcium chloride.

Alginates↗
PubMed1996

Mathematical model for the combined effect of temperature and pH on the thermal resistance of Bacillus stearothermophilus and Clostridium sporogenes spores.

Two mathematical models have been studied to establish the relationship between the pH, treatment temperature and thermal destruction constant (k) of Bacillus stearothermophilus and Clostridium sporogenes spores. The study was carried out by heating the spores in mushroom extract acidified with two different acidulants (citric acid and glucono-delta-lactone). Among the models studied, the one that best described the inactivation was a second order polynomial equation, the precision of which depended on the microorganism studied.

Clostridium↗
PubMed1991

Production of 5-methyluridine by immobilized thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus JTS 859.

5-Methyluridine was produced continuously from thymine and inosine by immobilized enzymes, which consisted of thermostable purine nucleoside phosphorylase and thermostable pyrimidine nucleoside phosphorylase obtained from Bacillus stearothermophilus JTS 859. The process was carried out in a column reactor at 60 degrees C for 17 d without any bacterial contamination under non-aseptical conditions. Half-lives of the activity of the immobilized enzymes were 47 d and 4.5 d at 60 degrees C and 70 degrees C, respectively, although half-life of the crude enzyme was only 14 h at 70 degrees C.

Bacterial Proteins↗
PubMed1995

Aerobic thermophilic treatment of sewage sludge at pilot plant scale. 1. Operating conditions.

The aerobic thermophilic treatment process of sewage sludge was studied at different bioreactor scales in a pilot plant installation. Since, for a satisfactory sludge disinfection, the Swiss legislation requires minimal incubation times of all volume elements, the bioreactors were operated in repetitive batch mode (draw and fill). Different retention times and frequencies of the volume changes were applied in order to prove the capability of the particular operation modes in assuring high degradative potential. The main enzymatic activity involved during the aerobic treatment was proteolysis: the RQ values ranged between 0.8 and 0.9 depending on the applied operating conditions. Although not in a linear manner, the efficiency of the microflora decreased as the bioreactor scale increased, when this increase corresponded with a reduction of the specific power input. The sludge oxidation rates can be tuned by some process operating conditions such as the volume change frequency, the changed volume quantities and the retention times. It was possible to improve the microbial degradative efficiency by an increased frequency of the changes, while the mean retention time influenced in particular the ultimate product quality, described as residual organic matter content of the sludge. The microflora present was also satisfactorily active at mean hydraulic retention times of less than 10 h. The organic matter concentration of the inlet sewage sludge plays an important role: it influences the aerobic degradation process positively.

Aerobiosis↗
PubMed1989

The effect of air on the moist-heat resistance of Bacillus stearothermophilus spores.

The presence of air in an autoclave chamber and load is generally considered to reduce the lethal effect of the process on bacterial spores. In this study, the heat inactivation of spores of Bacillus stearothermophilus (NCIB 8224) was measured in the presence and absence of air at 100% relative humidity. The results confirm that air significantly enhances the lethality of moist-heat on this strain of B. stearothermophilus.

Air↗
PubMed1994

Feasibility of a combined carrier test for disinfectants: studies with a mixture of five types of microorganisms.

BACKGROUND: There is mounting concern regarding the efficacy of many germicides on the market because officially recognized germicidal tests for various classes of microorganisms vary widely and often lack reproducibility and proper quantitation. We report here a carrier method for simultaneously and quantitatively assessing the efficacy of liquid chemical germicides against a mixture of microorganisms of varying degrees of resistance. METHODS: In the test, each small glass cup (10 mm wide x 14 mm long) was contaminated with 10 microliters of a standardized mixture of Staphylococcus aureus, Mycobacterium bovis bacille Calmette-Guérin, Trichophyton mentagrophytes spores, Sabin poliovirus type 1, and Bacillus stearothermophilus spores in 5% fetal bovine serum. The inoculum was dried for 60 minutes under ambient conditions and covered with 60 microliters of the disinfectant under test or a balanced salt solution control for the desired contact time. The carrier was then placed in 2940 microliters of an eluent and the eluates assayed separately for the five microorganisms. Tap water was used to dilute the test product as needed. RESULTS: Of the 11 products tested, 2% alkaline glutaraldehyde, 0.6% sodium hypochlorite (about 5000 ppm free chlorine), and a 0.4% quarternary ammonium compound containing 23% hydrochloric acid were effective against all five challenge organisms. A hard-surface spray containing 0.1% o-phenylphenol with 79% ethanol was effective against all but bacterial spores; 70% (volume/volume) ethanol alone and povidone-iodine (1% available iodine) were effective against S. aureus, the mycobacterium, and the fungus; a 3% solution of peroxygen compounds was effective only against S. aureus and the poliovirus; 1.5% chlorhexidine gluconate, 0.06% quaternary ammonia compound, and 0.03% o-phenylphenol + 0.03% p-tertiary amylphenol could inactivate nothing but S. aureus; and 3% hydrogen peroxide was ineffective in all tests. CONCLUSIONS: This method shows promise for use with various classes of microorganisms, individually or as mixtures. Its application should enable the classification of germicides according to spectrum of activity.

Disinfectants↗
PubMed1989

Subchronic toxicity studies in dogs and in utero rats fed diets containing Bacillus stearothermophilus alpha-amylase from a natural or recombinant DNA host.

Beagle dogs and Fischer 344 rats were fed diets containing 0, 36 or 72 units Bacillus stearothermophilus alpha-amylase (Bsa)/g food or of Bacillus subtilis alpha-amylase (cBsa)/g food. The dogs (four/sex/group) received treated diets for 13 wk. For the rat studies, the parental (F0) generation (12 males and 24 females/group for the Bsa study, and 26 rats/sex/group for the cBsa study) received treated diets for 13 or 4 wk, respectively, before breeding and through weaning of the F1 pups; 20 F1 rats/sex/group received treated diets for at least 13 wk (from weaning until necropsy). There were no treatment-related antemortem observations, reproductive effects or ophthalmic, haematological, macroscopic or microscopic findings in treated dogs or rats, and no differences were noted in body weights for dogs or parental rats. Mean body weights of F1 pups from F0 rats exposed to 72 units cBsa/g were significantly lower than those of the control animals on lactation day 28. This effect may have been related to the slight reduction in body weights and significant reduction in food consumption (gestation days 14-20) of the F0 dams. However, this did not continue into the growth phase for the F1 generation. In the Bsa studies, there were no treatment-related effects in clinical pathology values, and organ-weight data did not correlate with macroscopic or microscopic findings. Male dogs given cBsa had significantly lower albumin (36 units/g), calcium (36 and 72 units/g) and inorganic phosphorus (72 units/g) values compared with those of the control males; there were no treatment-related changes in blood chemistry values in rats. Based on the results of these studies, the no-observable-effect level for alpha-amylase fed to dogs or rats is 36 units/g food.

Animals↗
PubMed1989

A study of the use of autoclave bags in non-vacuum autoclaves.

Two types of autoclave bags have been tested in five commonly used dental, non-vacuum autoclaves. The bags did not prevent the sterilization of instruments provided that most of the air was removed and the bags of instruments were not stacked. The use of a sterilization indicator in the bag is mandatory.

Dental Instruments↗