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In this study, a thermophilic facultative anaerobic strain Geobacillus thermoglucosidasius W-2 was found to degrade nitroalkane under both aerobic and anaerobic conditions. Bioinformatical analysis revealed three putative nitroalkane-oxidizing enzymes (Gt-NOEs) genes from the W-2 genome. The three identified proteins Gt2929, Gt1378, and Gt1208 displayed optimal activities at high temperatures (70, 70, and 80 °C, respectively). Among these, Gt2929 exhibited excellent degradation capability, pH stability, and metal ion tolerance for nitronates under aerobic condition. Interestingly, under anaerobic condition, only Gt1378 still maintained high activity for 2-nitropropane and nitroethane, indicating that the W-2 strain utilized various pathways to degrade nitronates under aerobic and anaerobic conditions, respectively. Taken together, the first revelation of thermophilic nitroalkane-degrading mechanism under both aerobic and anaerobic conditions provides guidance and platform for biotechnological and industrial applications., (Copyright © 2019 Elsevier Ltd. All rights reserved.) more...

The β-xylosidase encoding gene (XsidB) of the extremely thermophilic bacterium Geobacillus thermodenitrificans has been cloned and expressed in Escherichia coli. The homotrimeric recombinant XsidB is of 204.0kDa, which is optimally active at 60°C and pH 7.0 with T1/2 of 58min at 70°C. The β-xylosidase remains unaffected in the presence of most metal ions and organic solvents. The Km [p-nitrophenyl β-xyloside (pNPX)], Vmax and kcat values of the enzyme are 2×10(-3)M, 1250μmolesmg(-1)min(-1) and 13.20×10(5)min(-1), respectively. The enzyme catalyzes transxylosylation reactions in the presence of alcohols as acceptors. The pharmaceutically important β-methyl-d-xylosides could be produced using pNPX as the donor and methanol as acceptor. The products of transxylosylation were identified by TLC and HPLC, and the structure was confirmed by (1)H NMR analysis. The enzyme is also useful in synthesizing transxylosylation products from the wheat bran hydrolysate., (Copyright © 2014 Elsevier Ltd. All rights reserved.) more...

A gene encoding a GH10 endo-xylanase from Geobacillus sp. WSUCF1 was cloned and expressed in Escherichia coli. Recombinant endo-xylanase (37kDa) exhibited high specific activity of 461.0U/mg of protein. Endo-xylanase was optimally active on birchwood xylan at 70°C and pH 6.5. The endo-xylanase was found to be highly thermostable at 50 and 60°C, retaining 82% and 50% of its original activity, respectively, after 60h. High xylan conversions (92%) were obtained with oat-spelt xylan hydrolysis. Higher glucan and xylan conversions were obtained on AFEX-treated corn stover with an enzyme cocktail containing WSUCF1 endo-xylanase (71% and 47%) as compared to enzyme cocktail containing commercial fungal endo-xylanase (64% and 41%). High specific activity, active at high pH's, wide substrate specificity, and higher hydrolytic activity on recalcitrant lignocellulose, make this endo-xylanase a suitable candidate for biofuel and bioprocess industries., (Copyright © 2014 Elsevier Ltd. All rights reserved.) more...

A thermophile cellulase-producing bacterium was isolated and identified as closely related to Geobacillus subterraneus. The strain, named Geobacillus sp. T1, was able to grow and produce cellulase on cellobiose, microcrystalline cellulose, carboxymethylcellulose (CMC), barley straw, wheat straw and Whatman No. 1 filter paper. However, barley and wheat straws were significantly better substrates for cellulase production. When Geobacillus sp. T1 was cultivated in the presence of 0.5% barley straw, 0.1% Tween 80 and pH 6.5 at 50°C, the maximum level of free cellulase up to 143.50 U/mL was produced after 24h. This cellulase (≈ 54 kDa) was most active at pH 6.5 and 70°C. The enzyme in citrate phosphate buffer (10mM) was stable at 60°C for at least 1h. Geobacillus sp. T1 with efficient growth and cellulase production on straws seems a potential candidate for conversion of agricultural biomass to fuels., (Copyright © 2012 Elsevier Ltd. All rights reserved.) more...

A xylanase gene (xyl-gt) of 1.224 kbp was cloned from the extremely thermophilic bacterium Geobacillus thermoleovorans that encodes a protein containing 408 amino acid residues. Eight conserved regions (signature sequences) of GH family 10 xylanases have been found in the xylanase. When the xylanase gene was cloned and expressed in Escherichia coli BL21 (DE3), the recombinant strain produced xylanase titer of 270 U mg(-1) which is 27-fold higher than the wild strain. It is optimally active at 80°C and pH 8.5 with a high thermostability over broad range of pH (6-12) and temperature (40-100°C). The end products of the hydrolysis of birch wood xylan and agro-residues included xylobiose, xylotriose, xylotetraose and xylopentaose. The xylanase of G. thermoleovorans is one of the rare xylanases that exhibits thermo-alkali-stability, and thus, it is a suitable candidate for pre-bleaching of paper pulps and generating xylooligosaccharides from agro-residues for use as prebiotics., (Copyright © 2011 Elsevier Ltd. All rights reserved.) more...

The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg(-1)), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65°C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50°C for more than 150 min., (Copyright © 2011 Elsevier Ltd. All rights reserved.) more...

The composition of thermophilic (60 degrees C) mixed cellulose-degrading enrichment culture initiated from compost samples was examined by constructing a 16S rRNA gene clone library and the presence of sequences related to Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Proteobacteria were identified. Eight isolates capable of degrading cellulose, carboxymethyl cellulose (CMC), or ponderosa pine sawdust were identified as belonging to the genera Geobacillus, Thermobacillus, Cohnella, and Thermus. A compost isolate WSUCF1 (Geobacillus sp.) was selected based on its higher growth rate and cellulase activity compared to others in liquid minimal medium containing cellulose as a source of carbon and energy. Strain WSUCF1 and a previously isolated thermophilic cellulose-degrading deep gold mine strain DUSELR13 (Bacillus sp.) were examined for their enzyme properties and kinetics. The optimal pH for carboxymethyl cellulase (CMCase) activity was 5.0 for both isolates. The optimum temperatures for CMCase of WSUCFI and DUSELR13 were 70 and 75 degrees C, respectively. For CMC, the DUSELR13 and WSUCF1 CMCases had K(m) values of 3.11 and 1.08mg/ml, respectively. Most remarkably, WSUCF1 and DUSELR13 retained 89% and 78% of the initial CMCase activities, respectively, after incubation at 70 degrees C for 1day. These thermostable enzymes would facilitate development of more efficient and cost-effective forms of the simultaneous saccharification and fermentation process to convert lignocellulosic biomass into biofuels. more...