Purification, Characterization And Mechanism Of Trichoderma Reesei Xylanase

The applications of xylanase in saccharification of lignocellulosics, pulp and paper industry, feed industry, xylooligosaccharide preparation and brewing industry were attracted more interesting last decades. The purification, characterization and mechanism of Trichoderma reesei Rut C30 xylanase were investigated in this paper. The main results are as follows:β-xylosidase (XYND), endoxylanaseⅠ(XYNⅠ) and endoxylanaseⅡ(XYNⅡ) were isolated and purified from Trichoderma reesei Rut C30 xylanase. The specific activity of XYND, XYNⅠand XYNⅡwere 23942.86, 4523.61 and 61.99IU/mg, respectively.Both endoxylanases andβ-xylosidase showed apparent homogeneity in SDS-PAGE. The estimated molecular masses of XYNⅠ, XYNⅡand XYND were 19.9, 20.3 and 110.8kDa, whereas the molecular masses of XYNⅠ, XYNⅡwere 6.2 and 9.1kDa determined with HiPrep Sephacryl S-100 HR gel filtration.The optimal reaction conditions were at 50℃, pH5.5 for XYNⅠ, at 50℃, pH4.0 for XYNⅡ, and at 60℃, pH3.5 for XYND. XYNⅠwas stable over the range of pH5.5-8.0, while XYNⅡover the range of pH3.0-5.0. XYND showed stability over the range of pH3.0-8.0 at 50℃. Two endoxylanases were stable at 50℃for 30min, andβ-xylosidase, which was stable up to 60℃, showed more thermal stability than that of two endoxylanases. The Km and Vmax values of XYNⅠwere 12.28mg/mL and 8142.42IU/mg protein for brich xylan as substrate, the Km and Vmax values of XYNⅡwere 25.42mg/mL and 2513.93IU/mg protein in the same condition. XYND had a Km of 0.29μmol/mL and Vmax of 169.99IU/mg protein for p-nitrophenyl-β-D- xylopyranoside as substrate.XYNⅠand XYNⅡwere endo-β-1,4-xylanases which catalyzed the hydrolysis of xylan radomly to xylooligosaccharides. The products of xylan hydrolysis with XYNⅠwere xylo- oligosccharide of DP2-7, besides xylooligosaccharides, a substantial amount of xylose was formation when xylan was hydrolyzed with XYNⅡ. The best substrate of XYNⅠwas long- chain xylan, while short-chain xylan and xylooligosaccharides were facilitated to be catalyzed by XYNⅡ. XYND was aβ-xylosidase which released xylose residues from nonreducing ends...