Abstract
To obtain extracellular and high-level expression of the Dictyoglomus thermophilum Rt46B.1 xylanase B gene, this gene was integrated into the α-amylase gene site of a host strain of Bacillus subtilis WB800. The extreme thermophile xylanase gene was successfully integrated and expressed in the host, measured at 24 ± 0.4 XUs/mL in the Luria broth medium supernatant. The recombinant enzyme was purified by ammonium sulfate precipitation, anion exchange chromatography, and gel filtration. The molecular mass and pI value of xylanase were estimated to be 24 kDa and 4.3, respectively. The optimal pH level and temperature of the purified enzyme were 6.5 and 85 °C, respectively. Xylanase showed reasonable activity at temperatures up to 95 °C and remained stable at 4 °C for 1 week. The purified enzyme retained most of its activity in 1 mM ethylenediaminetetraacetic acid or dithiothreitol and 0.1% Tween-20 or Triton X-100. However, strong inhibition was observed in the presence of 5 mM Mn2+, 0.5% sodium dodecyl sulfate, Tween-20, or Triton X-100; a strong stimulating effect was also observed in the presence of Fe2+. The K m and V max values of the recombinant xylanase for birchwood xylan were calculated to be 2.417 ± 0.36 mg/mL and 325 ± 41 µmol/min mg, respectively. Xylanase was found to be useful in the prebleaching process of paper pulps.
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Acknowledgements
This work was financially supported by the High Technology Foundation (200515124) of Xinjiang Province, People's Republic of China. We thank Dr Satoshi Nakamula and Dr. Xing Xin-hui for providing the material of integration vector pDG364 and the plasmid of pET-xynB.
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Zhang, W., Lou, K. & Li, G. Expression and Characterization of the Dictyoglomus thermophilum Rt46B.1 Xylanase Gene (xynB) in Bacillus subtilis . Appl Biochem Biotechnol 160, 1484–1495 (2010). https://doi.org/10.1007/s12010-009-8634-8
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DOI: https://doi.org/10.1007/s12010-009-8634-8