Expression and characterization of the Dictyoglomus thermophilum Rt46B.1 xylanase gene (xynB) in Bacillus subtilis.
To obtain extracellular and high-level expression of the Dictyoglomus thermophilum Rt46B.1 xylanase B gene, this gene was integrated into the alpha-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 + or - 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 degrees Celsius, respectively. Xylanase showed reasonable activity at temperatures up to 95 degrees Celsius and remained stable at 4 degrees Celsius 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 Mn(2+), 0.5% sodium dodecyl sulfate, Tween-20, or Triton X-100; a strong stimulating effect was also observed in the presence of Fe(2+). The K(m) and V(max) values of the recombinant xylanase for birchwood xylan were calculated to be 2.417 + or - 0.36 mg/mL and 325 + or - 41 micromol/min mg, respectively. Xylanase was found to be useful in the prebleaching process of paper pulps.
Zhang
Wei
W
College of Life Sciences and Technology, Xinjiang University, No. 14 Shengli Road, Urumqi, 830046, People's Republic of China.
Lou
Kai
K
Li
Guan
G
D001412:Bacillus subtilis / Q000187:drug effects / Q000378:metabolism*; D001419:Bacteria / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000302:isolation & purification; D004795:Enzyme Stability / Q000187:drug effects; D005798:Genes, Bacterial / Q000235:genetics*; D056426:Genetic Loci / Q000235:genetics; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007700:Kinetics; D010957:Plasmids / Q000235:genetics; D012492:Salts / Q000494:pharmacology; D013696:Temperature
D012492:Salts; D004492:Edetic Acid; D043364:Endo-1,4-beta Xylanases; D004229:Dithiothreitol