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| Appl Environ Microbiol, 2003 Dec, 69(12), 7124 - 9 Inactivation of Geobacillus stearothermophilus spores by high-pressure carbon dioxide treatment; Watanabe T et al.; High-pressure CO2 treatment has been studied as a promising method for inactivating bacterial spores . In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment . Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO2 treatment at 30 MPa and 35 degrees C, to high-hydrostatic-pressure treatment at 200 MPa and 65 degrees C, or to heat treatment at 0.1 MPa and 85 degrees C . All of the bacterial spores except the G . stearothermophilus spores were easily inactivated by the heat treatment . The highly heat- and pressure-resistant spores of G . stearothermophilus were not the most resistant to CO2 treatment . We also investigated the influence of temperature on CO2 inactivation of G . stearothermophilus . Treatment with CO2 and 30 MPa of pressure at 95 degrees C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95 degrees C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95 degrees C for 120 min had little effect . The activation energy required for CO2 treatment of G . stearothermophilus spores was lower than the activation energy for heat or pressure treatment . Although heat was not necessary for inactivationby CO2 treatment of G . stearothermophilus spores, CO2 treatment at 95 degrees C was more effective than treatment at 95 degrees C alone. J Biol Chem, 2004 Feb 13, 279(7), 5207 - 15 Epub 2003 Nov 17. Biophysical characterization of the entire bacterial surface layer protein SbsB and its two distinct functional domains; Runzler D et al.; The crystalline bacterial cell surface layer (S-layer) protein SbsB of Geobacillus stearothermophilus PV72/p2 was dissected into an N-terminal part defined by the three consecutive S-layer homologous motifs and the remaining large C-terminal part . Both parts of the mature protein were produced as separate recombinant proteins (rSbsB(1-178) and rSbsB(177-889)) and compared with the full-length form rSbsB(1-889) (rSbsB) . Evidence for functional and structural integrity of the two truncated forms was provided by optical spectroscopic methods and electron microscopy . In particular, binding of the secondary cell wall polymer revealed a high affinity dissociation constant of 3 nm and could be assigned solely to the soluble rSbsB(1-178), whereas rSbsB(177-889) self-assembled into the same lattice as the full-length protein . Furthermore, thermal as well as guanidinium hydrochloride induced equilibrium unfolding profiles monitored by intrinsic fluorescence, and circular dichroism spectroscopy allowed characterization of rSbsB(1-178) as an alpha-helical protein with a single cooperative unfolding transition yielding a DeltaG value of 26.5 kJ mol(-1) . The C-terminal rSbsB(177-889) could be characterized as a beta-sheet protein with typical multidomain unfolding, which is partially less stable as stand-alone protein . In general, the truncated forms showed identical properties compared with the full-length rSbsB with respect to structure and function . Consequently, rSbsB is characterized by its two functionally and structurally separated parts, the specific secondary cell wall polymer binding rSbsB(1-178) and the larger rSbsB(177-889) responsible for formation of the crystalline array. J Biol Chem, 2004 Jan 23, 279(4), 3014 - 24 Epub 2003 Oct 22. Crystal structures of Geobacillus stearothermophilus alpha-glucuronidase complexed with its substrate and products: mechanistic implications; Golan G et al.; Alpha-glucuronidases cleave the alpha-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and short xylooligomers as part of the hemicellulose degradation system . To date, all of the alpha-glucuronidases are classified as family 67 glycosidases, which catalyze the hydrolysis via the investing mechanism . Here we describe several high resolution crystal structures of the alpha-glucuronidase (AguA) from Geobacillus stearothermophilus, in complex with its substrate and products . In the complex of AguA with the intact substrate, the 4-O-methyl-d-glucuronic acid sugar ring is distorted into a half-chair conformation, which is closer to the planar conformation required for the oxocarbenium ion-like transition state structure . In the active site, a water molecule is coordinated between two carboxylic acids, in an appropriate position to act as a nucleophile . From the structural data it is likely that two carboxylic acids, Asp(364) and Glu(392), activate together the nucleophilic water molecule . The loop carrying the catalytic general acid Glu(285) cannot be resolved in some of the structures but could be visualized in its "open" and "closed" (catalytic) conformations in other structures . The protonated state of Glu(285) is presumably stabilized by its proximity to the negative charge of the substrate, representing a new variation of substrate-assisted catalysis mechanism. PDA J Pharm Sci Technol, 2003 Jul-Aug, 57(4), 249 - 62 A method of increasing test range and accuracy of bioindicators: Geobacillus stearothermophilus spores; Lundahl G; Spores of Geobacillus stearothermophilus are very sensitive to changes in temperature . When validating sterilizing processes, the most common bioindicator (BI) is spores of Geobacillus stearothermophilus ATCC12980 and ATCC7953 with about 10(6) spores /BI and a D121-value of about 2 minutes in water . Because these spores of Geobacillus stearothermophilus do not survive at a F0-value above 12 minutes, it has not been possible to evaluate the agreement between the biological F-value (F(BIO)) and physical measurements (time and temperature) when the physical F0-value exceeds that limit . However, it has been proven that glycerin substantially increases the heat resistance of the spores, and it is possible to utilize that property when manufacturing BIs suitable to use in processes with longer sterilization time or high temperature (above 121 degrees C) . By the method described, it is possible to make use of the sensitivity and durability of Geobacillus stearothermophilus' spores when glycerin has increased both test range and accuracy . Experience from years of development and validation work with the use of the highly sensitive glycerin-water-spore-suspension sensor (GWS-sensor) is reported . Validation of the steam sterilization process at high temperature has been possible with the use of GWS-sensors . It has also been shown that the spores in suspension keep their characteristics for a period of 19 months when stored cold (8 degrees C). EMBO J, 2003 Oct 1, 22(19), 4922 - 32 Crystal structure and snapshots along the reaction pathway of a family 51 alpha-L-arabinofuranosidase; Hovel K et al.; High-resolution crystal structures of alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycosidase, are described . The enzyme is a hexamer, and each monomer is organized into two domains: a (beta/alpha)8-barrel and a 12-stranded beta sandwich with jelly-roll topology . The structures of the Michaelis complexes with natural and synthetic substrates, and of the transient covalent arabinofuranosyl-enzyme intermediate represent two stable states in the double displacement mechanism, and allow thorough examination of the catalytic mechanism . The arabinofuranose sugar is tightly bound and distorted by an extensive network of hydrogen bonds . The two catalytic residues are 4.7 A apart, and together with other conserved residues contribute to the stabilization of the oxocarbenium ion-like transition state via charge delocalization and specific protein-substrate interactions . The enzyme is an anti-protonator, and a 1.7 A electrophilic migration of the anomeric carbon takes place during the hydrolysis. Biochemistry, 2003 Sep 9, 42(35), 10528 - 36 Detailed kinetic analysis of a family 52 glycoside hydrolase: a beta-xylosidase from Geobacillus stearothermophilus; Bravman T et al.; Geobacillus stearothermophilus T-6 encodes for a beta-xylosidase (XynB2) from family 52 of glycoside hydrolases that was previously shown to hydrolyze its substrate with net retention of the anomeric configuration . XynB2 significantly prefers substrates with xylose as the glycone moiety and exhibits a typical bell-shaped pH dependence curve . Binding properties of xylobiose and xylotriose to the active site were measured using isothermal titration calorimetry (ITC) . Binding reactions were enthalpy driven with xylobiose binding more tightly than xylotriose to the active site . The kinetic constants of XynB2 were measured for the hydrolysis of a variety of aryl beta-D-xylopyranoside substrates bearing different leaving groups . The Bronsted plot of log k(cat) versus the pK(a) value of the aglycon leaving group reveals a biphasic relationship, consistent with a double-displacement mechanism as expected for retaining glycoside hydrolases . Hydrolysis rates for substrates with poor leaving groups (pK(a) > 8) vary widely with the aglycon reactivity, indicating that, for these substrates, the bond cleavage is rate limiting . However, no such dependence is observed for more reactive substrates (pK(a) < 8), indicating that in this case hydrolysis of the xylosyl-enzyme intermediate is rate limiting . Secondary kinetic isotope effects suggest that the intermediate breakdown proceeds with modest oxocarbenium ion character at the transition state, and bond cleavage proceeds with even lower oxocarbenium ion character . Inhibition studies with several gluco analogue inhibitors could be measured since XynB2 has low, yet sufficient, activity toward 4-nitrophenyl beta-D-glucopyranose . As expected, inhibitors mimicking the proposed transition state structure, such as 1-deoxynojirimycin, bind with much higher affinity to XynB2 than ground state inhibitors. New Microbiol, 2003 Jul, 26(3), 249 - 56 Genetic polymorphism by RAPD-PCR and phenotypic characteristics of isolated thermotolerant Bacillus strains from hot spring sources; Hazem A et al.; The polymerase chain reaction (PCR) based random amplified polymorphic DNA (RAPD) assay, morphological, physiological, biochemical and antimicrobial susceptibility test methods have been evaluated for use in the taxonomy of isolated thermotolerant Bacillus from Jordanian hot springs, with specific reference to strains Geobacillus stearothermophilus (ATCC 12980), Bacillus circulans (ATCC 4513) and Bacillus sphaericus (ATCC 14577) . A RAPD assay has been optimized and is able to discriminate between numerous thermotolerant Bacillus strains . RAPD-PCR was found to give reproducible thermotolerant Bacillus strains classification of DNA fingerprints for 14 strains including 3 reference strains . A study of 14 isolates and 3 reference strains, analyzing 53 phenotypic characters, resulted in their allocation to five major clusters at 60% similarity . Whereas at 80% similarity, twelve taxonomically distinct groups were evident. Biotechnol Lett, 2003 Jun, 25(12), 963 - 7 Production of tagatose by a recombinant thermostable L-arabinose isomerase from Thermus sp . IM6501; Kim JW et al.; A gene (thaI) corresponding to L-arabinose isomerase from Thermus strain IM6501 was cloned by PCR . It comprised 1488 nucleotides and encoded a polypeptide of 496 residues with a predicted molecular weight of 56019 Da . The deduced amino acid sequence had 96.8% identity with the L-arabinose isomerase of Geobacillus stearothermophilus . Recombinant ThaI with N-terminal hexa-tistidine tags was over-expressed in Escherichia coli and purified by affinity chromatography using Ni-NTA resin . The purified ThaI was thermostable with maximal activity at 60 degrees C at pH 8 for 30 min of reaction . Zn2+ and Ni2+ inactivated the catalytic activity of ThaI, 5 mM Mn2+ enhanced the bioconversion yield by 90% . The bioconversion yield of 54% from D-galactose to D-tagatose was obtained by recombinant ThaI at 60 degrees C over 3 d. Acta Crystallogr D Biol Crystallogr, 2003 Aug, 59(Pt 8), 1466 - 8 Epub 2003 Jul 23. Crystallization and preliminary structure determination of the C-terminal truncated domain of the S-layer protein SbsC; Pavkov T et al.; The C-terminal truncated form of the S-layer protein SbsC from Geobacillus stearothermophilus, rSbsC(31-844), has been crystallized by the vapour-diffusion method using polyethylene glycol 6000 as a precipitating agent . The crystals diffract to 3 A resolution using synchrotron radiation and belong to space group P2(1), with unit-cell parameters a = 57.24, b = 98.91, c = 108.62 A, beta = 94.34 degrees . One molecule is present in the asymmetric unit, which corresponds to a solvent content of 65% . Native and heavy-atom derivative data have been collected . The Pt derivative yielded two high-occupancy sites per molecule. Acta Crystallogr D Biol Crystallogr, 2003 May, 59(Pt 5), 913 - 5 Epub 2003 Apr 25. Crystallization and preliminary X-ray analysis of a family 51 glycoside hydrolase, the alpha-l-arabinofuranosidase from Geobacillus stearothermophilus T-6; Hovel K et al.; Alpha-l-arabinofuranosidases (EC 3.2.1.55) are hemicellulases that cleave the glycosidic bond between l-arabinofuranoside side chains and various oligosaccharides . In this study, the first crystallization and preliminary X-ray analysis of the alpha-l-arabinofuranosidase from Geobacillus stearothermophilus T-6 (AbfA T-6), a family 51 glycoside hydrolase, is described . AbfA T-6 is a hexameric protein consisting of six identical subunits of 502 amino acids and with a calculated molecular mass of 57 218 Da . Purified recombinant native and selenomethionine-containing AbfA T-6 were crystallized by the sitting-drop method in two different space groups, P2(1) (unit-cell parameters a = 100.8, b = 178.1, c = 196.2 A, beta = 96.1 degrees ) and R3 (unit-cell parameters a = b = 179.3, c = 100.4 A) . The R3 crystals diffracted X-rays to a resolution of 1.8 A. J Biol Chem, 2003 Jul 18, 278(29), 26742 - 9 Epub 2003 May 08. Identification of the catalytic residues in family 52 glycoside hydrolase, a beta-xylosidase from Geobacillus stearothermophilus T-6; Bravman T et al.; beta-d-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units . The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism . Two catalytic mutants of a beta-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis . The activity of the E335G mutant decreased approximately 106-fold, and this activity was enhanced 103-fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration . These results are consistent with Glu335 as the nucleophile in this retaining enzyme . The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pKa) . The mutant exhibited 103-fold reduction in activity, and the Bronsted plot of log(kcat) versus pKa revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 103-fold . The rates of the glycosylation step, as reflected by the specificity constant (kcat/Km), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pKa) but decreased 102-fold for those that require strong acid catalysis (high pKa) . Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent . Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration . These results are consistent with Asp495 acting as the acid-base in XynB2. Lett Appl Microbiol, 2003, 36(4), 191 - 6 Enhanced secretion and low temperature stabilization of a hyperthermostable and Ca2+-independent alpha-amylase of Geobacillus thermoleovorans by surfactants; Uma Maheswar Rao JL et al.; AIMS: Selection of suitable surfactants for enhancing and stabilizing alpha-amylase of Geobacillus thermoleovorans . METHODS AND RESULTS: Geobacillus thermoleovorans was cultivated in shake flasks containing 50 ml of starch-yeast extract-tryptone (SYT) medium with/without surfactants . Titres of the enzyme in media were monitored . The enzyme was also preserved at 4 degrees C with/without surfactants and enzyme activities were determined . Among polyethylene glycol (PEGs) of different molecular weights, PEG 8000 (0.5%, w/v) caused a slight increase in the enzyme titre, while Tween-20, Tween-40 and Tween-60 (0.03%, w/v) exerted a significant stimulatory effect on enzyme secretion . In the presence of SDS, Tween-80 and cholic acid (0.03%, w/v), the enzyme production was nearly twofold higher than that in the control . The anionic (SDS, cholic acid) and non-ionic (Tweens) detergents increased the cell membrane permeability, and thus, enhanced alpha-amylase secretion . Furthermore, anionic surfactants exhibited stabilizing effect on the enzyme during preservation at 4 degrees C . CONCLUSIONS: PEG 8000 and the ionic detergents (SDS, cholic acid and Tween-80) were more effective in the solubilization of cell membrane components, and enhancing enzyme yields than the cationic detergents such as CTAB (N,Cetyl-N,N,N-trimethyl ammonium bromide) . Further, these surfactants were found to stabilize the enzyme at 4 degrees C . SIGNIFICANCE AND IMPACT OF THE STUDY: The secretion of Ca2+-independent hyperthermostable alpha-amylase was enhanced in the presence of certain anionic and non-ionic detergents in the medium . Furthermore, the surfactants stabilized the enzyme during preservation at 4 degrees C . The use of this enzyme in starch hydrolysis eliminates the addition of Ca2+ in starch liquefaction and its subsequent removal by ion exchange from sugar syrups. Appl Environ Microbiol, 2003 Jan, 69(1), 644 - 8 Distribution of microorganisms in the subsurface of the manus basin hydrothermal vent field in Papua New Guinea; Kimura H et al.; The distribution of microorganisms in the subsurfaces of hydrothermal vents was investigated by using subvent rock core samples . Microbial cells and ATP were detected from cores taken at depths of less than 99.4 and 44.8 m below the seafloor (mbsf), respectively . Cores from various depths were incubated anaerobically with a heterotrophic medium . Growth at 60 and 90 degrees C was ascribed to a Geobacillus sp . in the 448.6- to 99.4-mbsf cores and a Deinococcus sp . in the 64.8- to 128.9-mbsf cores, respectively, based on the 16S ribosomal DNA analysis. J Bacteriol, 2002 Dec, 184(23), 6709 - 13 Isolation of glucocardiolipins from Geobacillus stearothermophilus NRS 2004/3a; Schaffer C et al.; Glucose-substituted cardiolipins account for about 4 mol% of total phospholipid extracted from exponentially grown cells of Geobacillus stearothermophilus NRS 2004/3a . Individual glucocardiolipin species exhibited differences in fatty acid substitution, with iso-C(15:0) and anteiso-C(17:0) prevailing . The compounds were purified to homogeneity by a novel protocol and precharacterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. J Mass Spectrom, 2002 Oct, 37(10), 1086 - 94 Mapping and sequencing of cardiolipins from Geobacillus stearothermophilus NRS 2004/3a by positive and negative ion nanoESI-QTOF-MS and MS/MS; Beckedorf AI et al.; In the course of systematic studies on surface layer (S-layer) glycoproteins of bacilli, the chloroform/methanol extract from whole cells of Geobacillus stearothermophilus NRS 2004/3a has been submitted to MS analysis . Glucosylated cardiolipins were found as minor components of the total lipid and phospholipid mixture by de novo identification . After purification of the crude extract using a combined column chromatography/2D TLC protocol, structural investigations of components in the lipid fraction by high resolution ESI-QTOF MS analysis provided evidence about homologous molecules attributable to the cardiolipin species containing a glycosylated backbone, and about a diversity of ester-linked fatty acid substituents . In comparative studies by positive and negative ion nanoESI-QTOF-CID-MS, maps of cardiolipin molecular ions were obtained, followed by MS/MS of the most abundant species, to provide structural details of D-glucopyranosylcardiolipin and the fatty acid substituent patterns . Experiments of the parent ion scan type revealed the presence of fatty acid moieties as isobaric combinations, represented in single molecular ion species . J Biol Chem, 2002 Nov 15, 277(46), 43667 - 73 Epub 2002 Sep 06. Detailed kinetic analysis and identification of the nucleophile in alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase; Shallom D et al.; alpha-l-Arabinofuranosidases cleave the l-arabinofuranoside side chains of different hemicelluloses and are key enzymes in the complete degradation of the plant cell wall . The alpha-l-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study . Aryl-alpha-l-arabinofuranosides with various leaving groups were synthesized and used to verify the catalytic mechanism and catalytic residues of the enzyme . The steady-state constants and the resulting Bronsted plots for the E175A mutant are consistent with the role of Glu-175 as the acid-base catalytic residue . The proposed nucleophile residue, Glu-294, was replaced to Ala by a double-base pairs substitution . The resulting E294A mutant, with 4-nitrophenyl alpha-l-arabinofuranoside as the substrate, exhibited eight orders of magnitude lower activity and a 10-fold higher K(m) value compared with the wild type enzyme . Sodium azide accelerated by more than 40-fold the rate of the hydrolysis of 2',4',6'-trichlorophenyl alpha-l-arabinofuranoside by the E294A mutant . The glycosyl-azide product formed during this reaction was isolated and characterized as beta-l-arabinofuranosyl-azide by (1)H NMR, (13)C NMR, mass spectrometry, and Fourier transform infrared analysis . The anomeric configuration of this product supports the assignment of Glu-294 as the catalytic nucleophile residue of the alpha-l-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51. FEBS Lett, 2002 Mar 13, 514(2-3), 163 - 7 The identification of the acid-base catalyst of alpha-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase; Shallom D et al.; The alpha-L-arabinofuranosidase from Geobacillus stearothermophilus T-6 (AbfA T-6) belongs to the retaining family 51 glycoside hydrolases . The conserved Glu175 was proposed to be the acid-base catalytic residue . AbfA T-6 exhibits residual activity towards aryl beta-D-xylopyranosides . This phenomenon was used to examine the catalytic properties of the putative acid-base mutant E175A . Data from kinetic experiments, pH profiles, azide rescue, and the identification of the xylopyranosyl azide product provide firm support to the assignment of Glu175 as the acid-base catalyst of AbfA T-6.
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