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The xyn10B gene, encoding the endo-1,4-β-xylanase Xyn10B from Thermotoga thermarum, was cloned and expressed in Escherichia coli. The ORF of the xyn10B was 1,095 bp and encoded to mature peptide of 344 amino acids with a calculated MW of 40,531 Da. The recombinant xylanase was optimally active at 80 °C, pH 6.0 and retained approx. 60 % of its activity after 2 h at 75 °C. Apparent K m , k cat and k cat /K m values of the xylanase for beechwood xylan were 1.8 mg ml(-1), 520 s(-1) and 289 ml mg(-1) s(-1), respectively. The end products of the hydrolysis of beechwood xylan were mainly oligosaccharides but without xylose after 2 h hydrolysis.

Arabinan is an important plant polysaccharide degraded mainly by two hydrolytic enzymes, endo-arabinanase and α-L-arabinofuranosidase. In this study, the characterization and application in arabinan degradation of an endo-arabinanase from Thermotoga thermarum were investigated. The recombinant endo-arabinanase was expressed in Escherichia coli BL21 (DE3) and purified by heat treatment followed by purification on a nickel affinity column chromatography. The purified endo-arabinanase exhibited optimal activity at pH 6.5 and 75°C and its residual activity retained more than 80% of its initial activity after being incubated at 80°C for 2 h. The results showed that the endo-arabinanase was very effective for arabinan degradation at higher temperature. When linear arabinan was used as the substrate, the apparent K m and V max values were determined to be 12.3 ± 0.15 mg ml−1 and 1,052.1 ± 12.7 μmol ml−1 min−1, respectively (at pH 6.5, 75°C), and the calculated k cat value was 349.3 ± 4.2 s−1. This work provides a useful endo-arabinanase with high thermostability andcatalytic efficiency, and these characteristics exhibit a great potential for enzymatic conversion of arabinan.

Background Mannan is one of the primary polysaccharides in hemicellulose and is widely distributed in plants. β-Mannosidase is an important constituent of the mannan-degrading enzyme system and it plays an important role in many industrial applications, such as food, feed and pulp/paper industries as well as the production of second generation bio-fuel. Therefore, the mannose-tolerant β-mannosidase with high catalytic efficiency for bioconversion of mannan has a great potential in the fields as above. Results A β-mannosidase gene (Tth man5) of 1,827 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 608 amino acid residues, and was over-expressed in Escherichia coli BL21 (DE3). The results of phylogenetic analysis, amino acid alignment and biochemical properties indicate that the Tth Man5 is a novel β-mannosidase of glycoside hydrolase family 5. The optimal activity of the Tth Man5 β-mannosidase was obtained at pH 5.5 and 85°C and was stable over a pH range of 5.0 to 8.5 and exhibited 2 h half-life at 90°C. The kinetic parameters K m and V max values for p-nitrophenyl-β-D-mannopyranoside and 1,4-β-D-mannan were 4.36±0.5 mM and 227.27±1.59 μmol min-1 mg-1, 58.34±1.75 mg mL-1 and 285.71±10.86 μmol min-1 mg-1, respectively. The k cat /K m values for p-nitrophenyl-β-D-mannopyranoside and 1,4-β-D-mannan were 441.35±0.04 mM-1 s-1 and 41.47±1.58 s-1 mg-1 mL, respectively. It displayed high tolerance to mannose, with a K i value of approximately 900 mM. Conclusions This work provides a novel and useful β-mannosidase with high mannose tolerance, thermostability and catalytic efficiency, and these characteristics constitute a powerful tool for improving the enzymatic conversion of mannan through synergetic action with other mannan-degrading enzymes.