Abstract
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.
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Acknowledgments
This work was financially supported by the Jiangsu Education (11KJA48001, CXZZ11_0526), the National Natural Science Foundation of China (Nos. 31370572, 31200564) and the Doctorate Fellowship Foundation of Nanjing Forestry University as well as the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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Hao Shi and Yu Zhang contributed equally to this work.
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10529_2013_1392_MOESM1_ESM.tif
Supplementary material 1 Neighbor-Joining tree resulted from analysis of xylanases with 28 amino acid sequences. All these 28 amino acid sequences belong to GH10 family. Numbers on nodes correspond to percentage bootstrap values for 1000 replicates. (TIFF 4942 kb)
10529_2013_1392_MOESM2_ESM.tif
Supplementary material 2 Analysis of beechwood xylan hydrolyzed by Xyn10B. Supernatants of the reaction mixtures were applied on TLC silica gel plates (60 RP18 F254S, Merck KGaA). The plates were subsequently developed with three runs of n-butanol/acetic acid/water (2:1:1, v/v) followed by heating for 20 min at 100oC in an oven after spraying the plates with a methanol/sulfuric acid reagent (9:1, v/v). M, mixture of xylose, xylobiose, xylotriose, xylotetraose and xylopentaose, lane 1, 2, 3, 4: samples of beechwood xylan (5%, wt/vol) incubated with Xyn10B xylanase (1.5 U) for 0.5 h, 1 h, 2 h and 3 h, respectively. (TIFF 3864 kb)
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Shi, H., Zhang, Y., Zhong, H. et al. Cloning, over-expression and characterization of a thermo-tolerant xylanase from Thermotoga thermarum . Biotechnol Lett 36, 587–593 (2014). https://doi.org/10.1007/s10529-013-1392-2
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DOI: https://doi.org/10.1007/s10529-013-1392-2