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PubMed2006

Improving production of hyperthermostable and high maltose-forming alpha-amylase by an extreme thermophile Geobacillus thermoleovorans using response surface methodology and its applications.

By cultivating Geobacillus thermoleovorans in shake flasks containing cane molasses medium at 70 degrees C, the fermentation variables were optimized by 'one variable at a time' approach followed by response surface methodology (RSM). The statistical model was obtained by central composite design (CCD) using three variables (cane-molasses, urea and inoculum density). An overall 1.6- and 2.1-fold increase in enzyme production was achieved in the optimized medium in shake flasks and fermenter, respectively. The alpha-amylase titre increased significantly in cane-molasses medium (60 U ml(-1)) as compared to that in the synthetic medium (26 U ml(-1)). Thus the cost of enzyme produced in cane molasses medium (0.823 euros per million U) was much lower than that produced in the synthetic starch-yeast extract-tryptone medium (18.52 euros per million U). The shelf life of bread was improved by supplementing dough with alpha-amylase, and thus, the enzyme was found to be useful in preventing the staling of bread. Reducing sugars liberated from 20% and 30% raw pearl millet starch were fermented to ethanol; ethanol production levels attained were 35.40 and 28.0 g l(-1), respectively.

Bacillaceae↗
PubMed2005

The secondary cell wall polymer of Geobacillus tepidamans GS5-97T: structure of different glycoforms.

Nuclear magnetic resonance spectroscopic studies of the strain-specific secondary cell wall polymer (SCWP) of the Gram-positive, moderately thermophilic organism Geobacillus tepidamans GS5-97T reveal two glycoforms consisting of identical tetrasaccharide repeating units with different chemical modifications of the amide moieties. On the basis of sugar analyses along with 1D and 2D 1H, 13C, 15N, and 31P NMR spectroscopy at natural isotope abundance, the basic backbone structure of the SCWP was established to be [beta-D-Manp-2,3-diNAcANH2-(1-->6)-alpha-D-Glcp-(1-->4)-beta-D-Manp-2,3-diNAcANH2-(1-->3)-alpha-D-GlcpNAc-(1-->]6-(1-->O)-PO2-(O-->6)-MurNAc-, with modifications of the amide groups. In one glycoform, all beta-D-Manp-2,3-diNAcANH2 (2,3-diacetamido-2,3-dideoxy-beta-D-mannopyranuronamide, ManpANH2) residues are substituted with two acetyl groups (glycoform I) at the amide group at C-6; in the other glycoform (glycoform II), only one proton of this amide group is substituted by an acetyl group. The ratio between both the glycoforms approximates 1:1.

Bacillaceae↗
PubMed2006

The structures of the cell wall teichoic acids from the thermophilic microorganism Geobacillus thermoleovorans strain Fango.

The structures of two teichoic acid fractions (TA1 and TA2) isolated from the thermophilic gram-positive bacterium Geobacillus thermoleovorans strain Fango were investigated by means of chemical and NMR spectroscopic methods. The most abundant species (TA1) exhibited a rather regular structure comprising two different repeating units of 1,3-glycerol phosphate nonstoichiometrically substituted by terminal-alpha-D-Gal p (t-alpha-D-Gal p). The second molecular species (TA2) presented a higher structural variability and t-alpha-D-Glc p and the disaccharides t-alpha-D-Glc pNAc-(1-->2)-alpha-D-Glc p and t-alpha-D-Glc pNAc-(1-->3)-alpha-D-Glc p were also present as minor substituents at O-2 of the glycerol phosphate residues. Minor substitution by alanine could also be detected.

Bacillaceae↗
PubMed2005

Zinc in lipase L1 from Geobacillus stearothermophilus L1 and structural implications on thermal stability.

Lipase L1 from Geobacillus stearothermophilus L1 contains an unusual extra domain, making a tight intramolecular interaction with the main catalytic domain through a Zn2+-binding coordination. To elucidate the role of the Zn2+, we disrupted the Zn2+-binding site by mutating the zinc-ligand residues (H87A, D61A/H87A, and D61A/H81A/H87A/D238A). The activity vs. temperature profiles of the mutant enzymes showed that the disruption of the Zn2+-binding site resulted in a notable decrease in the optimal temperature for maximal activity from 60 to 45-50 degrees C. The mutations also abolished the Zn2+-induced thermal stabilization. The wild-type enzyme revealed a 34.6-fold increase in stabilization with the addition of Zn2+ at 60 degrees C, whereas the mutant enzymes exhibited no response to Zn2+. Additional circular dichroism spectroscopy studies also confirmed the structural stabilizing role of Zn2+ on lipase L1 at elevated temperatures.

Amino Acid Sequence↗
PubMed2005

Housekeeping recA gene interrupted by group II intron in the thermophilic Geobacillus kaustophilus.

Most of group II introns are found in intergenes and CDSs with unknown functions, but not in housekeeping genes. In particular, no group II intron within the housekeeping recA gene has been reported either in eukaryotic genomes or in prokaryotic genomes. In this study, we found that the recA gene of the thermophilic Geobacillus kaustophilus genome is interrupted by a group II intron (Gk. Int1), and that Gk.Int1 can splice in temperatures above 70 degrees C in vivo. Here, we report the first prokaryotic group II intron to be found in a housekeeping gene, the characteristics of its self-splicing in vivo and in vitro, and our conclusion that the recA gene functions through the self-splicing of Gk.Int1. It is suggested that the amelioration of Gk.Int1 intron has occurred recently, and that it is still in the process of evolution to the recipient genome.

Amino Acid Sequence↗
PubMed2005

Purification and characterization of an L-arabinose isomerase from an isolated strain of Geobacillus thermodenitrificans producing D-tagatose.

The araA gene, encoding l-arabinose isomerase (AI), from the thermophilic bacterium Geobacillus thermodenitrificans was cloned and expressed in Escherichia coli. Recombinant AI was isolated with a final purity of about 97% and a final specific activity of 2.10 U/mg. The molecular mass of the purified AI was estimated to be about 230 kDa to be a tetramer composed of identical subunits. The AI exhibited maximum activity at 70 degrees C and pH 8.5 in the presence of Mn2+. The enzyme was stable at temperatures below 60 degrees C and within the pH range 7.5-8.0. d-Galactose and l-arabinose as substrate were isomerized with high activities. Ribitol was the strongest competitive inhibitor of AI with a Ki of 5.5mM. The apparent Km and Vmax for L-arabinose were 142 mM and 86 U/mg, respectively, whereas those for d-galactose were 408 mM and 6.9 U/mg, respectively. The catalytic efficiency (kcat/Km) was 48 mM(-1)min(-1) for L-arabinose and 0.5mM(-1)min(-1) for D-galactose. Mn2+ was a competitive activator and increased the thermal stability of the AI. The D-tagatose yield produced by AI from d-galactose was 46% without the addition of Mn2+ and 48% with Mn2+ after 300 min at 65 degrees C.

Aldose-Ketose Isomerases↗
PubMed2006

Purification, characterization, and synergistic action of phytate-resistant alpha-amylase and alpha-glucosidase from Geobacillus thermodenitrificans HRO10.

The alpha-amylase (1, 4-alpha-d-glucanohydrolase; EC 3.2.1.1) and alpha-glucosidase (alpha-d-glucoside glucohydrolase; EC 3.2.1.20) secreted by Geobacillus thermodenitrificans HRO10 were purified to homogeneity (13.6-fold; 11.5% yield and 25.4-fold; 32.0% yield, respectively) through a series of steps. The molecular weight of alpha-amylase was 58kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The alpha-amylase activity on potato starch was optimal at pH 5.5 and 80 degrees Celsius. In the presence of Ca(2+), the alpha-amylase had residual activity of more than 92% after 1h of incubation at 70 degrees Celsius. The alpha-amylase did not lose any activity in the presence of phytate (a selective alpha-amylase inhibitor) at concentrations as high as 10mM, rather it retained 90% maximal activity after 1h of incubation at 70 degrees Celsius. EGTA and EDTA were strong inhibitory substances of the enzyme. The alpha-amylase hydrolyzed soluble starch at 80 degrees Celsius, with a K(m) of 3.05mgml(-1) and a V(max) of 7.35Uml(-1). The molecular weight of alpha-glucosidase was approximately 45kDa, as determined by SDS-PAGE. The enzyme activity was optimal at pH 6.5-7.5 and 55 degrees Celsius. Phytate did not inhibit G. thermodenitrificans HRO10 alpha-glucosidase activity, whereas pCMB was a potent inhibitor of the enzyme. The alpha-glucosidase exhibited Michaelis-Menten kinetics with maltose at 55 degrees Celsius (K(m): 17mM; V(max): 23micromolmin(-1)mg(-1)). Thin-layer chromatography studies with G. thermodenitrificans HRO10 alpha-amylase and alpha-glucosidase showed an excellent synergistic action and did not reveal any transglycosylation catalyzed reaction by the alpha-glucosidase.

Bacillaceae↗
PubMed2006

Analysis of reduction of Geobacillus stearothermophilus spores treated with high hydrostatic pressure and mild heat in milk buffer.

Our unpublished experimental results of fractional factorial experiments showed that the significant external factors affecting high pressure processing (HPP) inactivation were pressure, temperature and pressure holding time. Based on these results, response surface methodology (RSM) was employed in the present work and a quadratic equation for HPP inactivation was built. By analyzing the response surface plots and their corresponding contour plots as well as solving the quadratic equation, the experimental values were shown to be significantly in good agreement with predicted values since the adjusted determination coefficient (R(Adj)(2)) was 0.9747. The optimum process parameters for six log-cycles reduction of Geobacillus stearothermophilus spores were obtained as: temperature, 86 degrees C; pressure, 625.0 MPa and pressure holding time, 14.0 min. The adequacy of the model equation for predicting the optimum response values was verified effectively by the validation data.

Bacillaceae↗
PubMed2004

Covalent reaction intermediate revealed in crystal structure of the Geobacillus stearothermophilus carboxylesterase Est30.

Est30 is a thermophilic carboxylesterase cloned from Geobacillus stearothermophilus that showed optimal hydrolysis of esters with short acyl chains at 70 degrees C. Est30 is a member of a new family of carboxylesterases with representatives in other Gram-positive bacteria. The crystal structure has been determined at 1.63A resolution using multiple anomalous dispersion data. The two-domain crystal structure showed a large domain with a modified alpha/beta hydrolase core including a seven, rather than an eight-stranded beta sheet, and a smaller cap domain comprising three alpha helices. The catalytic triad consists of residues Ser94, Asp193, and His223. A 100Da tetrahedral ligand was observed to be covalently bound to the side-chain of Ser94. The propyl acetate ligand represents the first tetrahedral intermediate in the reaction mechanism. Therefore, this Est30 crystal structure will help understand the mode of action of all enzymes in the serine hydrolase superfamily.

Amino Acid Sequence↗
PubMed2005

Enzyme-substrate complex structures of a GH39 beta-xylosidase from Geobacillus stearothermophilus.

Beta-D-Xylosidases are glycoside hydrolases that catalyse the release of xylose units from short xylooligosaccharides and are engaged in the final breakdown of plant cell-wall hemicelluloses. beta-D-Xylosidases are found in glycoside hydrolase families 3, 39, 43, 52 and 54. The first crystal structure of a GH39 beta-xylosidase revealed a multi-domain organization with the catalytic domain having the canonical (beta/alpha)8 barrel fold. Here, we report the crystal structure of the GH39 Geobacillus stearothermophilus beta-D-xylosidase, inactivated by a point mutation of the general acid-base residue E160A, in complex with the chromogenic substrate molecule 2,5-dinitrophenyl-beta-D-xyloside. Surprisingly, six of the eight active sites present in the crystallographic asymmetric unit contain the trapped covalent glycosyl-enzyme intermediate, while two of them still contain the uncleaved substrate. The structural characterization of these two critical species along the reaction coordinate of this enzyme identifies the residues forming its xyloside-binding pocket as well as those essential for its aglycone recognition.

Bacillaceae↗
PubMed2006

Identification and over-expression of a thermostable lipase from Geobacillus thermoleovorans Toshki in Escherichia coli.

A newly isolated thermophilic strain producing thermostable lipase was identified based on 16S rRNA sequencing, where phylogenetic analysis revealed its closeness to Geobacillus thermoleovorans. Thermostable lipase from this bacterium was cloned using consensus degenerate PCR primers. For over-expression in Escherichia coli, the lipase gene was sub-cloned in pET 15b vector with a strong T7 promotor. Lipase activity was approximately 4.5-fold higher than in the wild-type strain. The lipase enzyme was thermostable at 60 degrees C and pH 8, whereas a 30% residual activity was retained when incubated for 1h at 100 degrees C. Optimum lipase expression was obtained in 2 x YT medium after 70min of induction by IPTG.

Bacillaceae↗
PubMed2005

Characterization of thermostable lipase from thermophilic Geobacillus sp. TW1.

A novel lipase-producing thermophilic strain TW1, assigned to Geobacillus sp. TW1 based on 16S rRNA sequence, was isolated from a hot spring in China. Based on this strain, a lipase gene encoding 417 amino acids was cloned. Subsequently, the lipase gene was expressed in Escherichia coli and purified as a fusion protein with glutathione S-transferase. The results showed that the recombinant lipase had an activity optimum at 40 degrees C and pH at 7.0-8.0. It was active up to 90 degrees C at pH 7.5, and stable over a wide pH ranging from 6.0 to 9.0. The recombinant lipase was stable in 1 mM enzyme inhibitors (EDTA, 2-ME, SDS, PMSF or DTT), as well as in 0.1% detergents (Tween 20, Chaps or Triton X-100). Its catalytic function was enhanced in the presence of Ca(2+), Mg(2+), Zn(2+), Fe(2+) or Fe(3+), but inhibited by Cu(2+), Mn(2+), and Li(+). By comparison with the crude lipase, the recombinant lipase had similar properties and was characteristic of thermostable enzymes. Our study presented a rapid overexpression and purification of the lipase gene from thermophile, aimed at improving the enzyme yield for industrial applications.

Amino Acid Sequence↗
PubMed2002

The identification of the acid-base catalyst of alpha-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase.

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.

Acids↗
PubMed2003

Detailed kinetic analysis of a family 52 glycoside hydrolase: a beta-xylosidase from Geobacillus stearothermophilus.

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 Brønsted 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.

Bacillaceae↗
PubMed2005

Biochemical characterization and identification of the catalytic residues of a family 43 beta-D-xylosidase from Geobacillus stearothermophilus T-6.

Beta-D-xylosidases are hemilcellulases that hydrolyze short xylooligosaccharides into xylose units. Here, we describe the characterization and kinetic analysis of a family 43 beta-xylosidase from Geobacillus stearothermophilus T-6 (XynB3). Enzymes in this family use an inverting single-displacement mechanism with two conserved carboxylic acids, a general acid, and a general base. XynB3 was most active at 65 degrees C and pH 6.5, with clear preference to xylose-based substrates. Products analysis indicated that XynB3 is an exoglycosidase that cleaves single xylose units from the nonreducing end of xylooligomers. On the basis of sequence homology, amino acids Asp15 and Glu187 were suggested to act as the general-base and general-acid catalytic residues, respectively. Kinetic analysis with substrates bearing different leaving groups showed that, for the wild-type enzyme, the k(cat) and k(cat)/K(m) values were only marginally affected by the leaving-group reactivity, whereas for the E187G mutant, both values exhibited significantly greater dependency on the pK(a) of the leaving group. The pH-dependence activity profile of the putative general-acid mutant (E187G) revealed that the protonated catalytic residue was removed. Addition of the exogenous nucleophile azide did not affect the activities of the wild type or the E187G mutant but rescued the activity of the D15G mutant. On the basis of thin-layer chromatography and (1)H NMR analyses, xylose and not xylose azide was the only product of the accelerated reaction, suggesting that the azide ion does not attack the anomeric carbon directly but presumably activates a water molecule. Together, these results confirm the suggested catalytic role of Glu187 and Asp15 in XynB3 and provide the first unequivocal evidence regarding the exact roles of the catalytic residues in an inverting GH43 glycosidase.

Amino Acid Substitution↗
PubMed2006

Top-down proteomic analysis of the soluble sub-proteome of the obligate thermophile, Geobacillus thermoleovorans T80: insights into its cellular processes.

We report the first analysis of the soluble sub-proteome of the obligate thermophile, Geobacillus thermoleovorans T80, utilizing a robust multidimensional protein identification protocol. A total of 1,336 proteins were initially identified utilizing automated MS/MS identification software. Intensive manual curation resulted in a final list containing a total of 294 unique proteins. Physiochemical characterization and functional classification of the soluble sub-proteome was carried out. The strategy has allowed us to gain an insight into the cellular processes of this obligate thermophile, identifying a variety of proteins known to play a role in stress response. Included within these were a number of sigma factors such as sigma(A) that initiate transcription of the heat shock operons controlled by the HrcA-CIRCE complex within gram positive bacteria. In addition, it has enabled us to assign a degree of functionality to 29 out of 36 gene products detected in this study that were hitherto described as being only hypothetical conserved proteins.

Bacillaceae↗
PubMed2006

A combined shotgun and multidimensional proteomic analysis of the insoluble subproteome of the obligate thermophile, Geobacillus thermoleovorans T80.

To further our understanding of the biology of the thermophilic bacterium Geobacillus thermoleovorans T80, we now report the first proteomic analysis of the insoluble subproteome of this isolate. A combination of both shotgun and multidimensional methodologies were utilized, and a total of 8628 peptides was initially identified by automated MS/MS identification software. Curation of these peptides led to a final list of 184 positive protein identifications. The proteins from this insoluble subproteome were functionally classified, and physiochemical characterization was carried out. Of 15 hypothetical conserved proteins identified, we have assigned function to all but four. A total of 31 proteins were predicted to possess signal peptides. In silico investigation of these proteins allowed us to identify four of the five bacterial classes of signal peptide, namely, (i) twin-arginine translocation; (ii) Sec-type; (iii) lipoprotein, and (iv) ABC transport. In addition, a number of proteins were identified that are known to be involved in the transport of compatible solutes, known to be important in microbial stress responses.

Amino Acid Sequence↗
PubMed2003

Isolation and characterization of a thermophilic bacterium, Geobacillus thermocatenulatus, degrading nylon 12 and nylon 66.

A thermophilic bacterium, identified as a neighboring species to Geobacillus thermocatenulatus, having a growth optimum at 55 degrees C and, capable of degrading nylon 12, was isolated from soil by enrichment culture technique at 60 degrees C. At this temperature, the strain grew on 5 g nylon 12 l(-1) with a decrease in its molecular weight from 41000 to 11000 over 20 d. The degradation was assumed to be due to endogenous hydrolysis of amide bond in nylon 12. The strain degraded also nylon 66 with a decrease in its molecular weight from 43000 to 17000 in 20 d at 60 degrees C. Nylon 6 was not degraded.

Bacillaceae↗