Thermoanaerobacterium saccharolyticum JW/SL-YS485来源的GH11家族木聚糖酶的克隆表达及其应用

Expression and characterization of a GH11 xylanase from Thermoanaerobacterium saccharolyticum JW/SL-YS485 and its application in xylooligosaccharide production

对Thermoanaerobacterium saccharolyticum JW/SL-YS485来源的GH11家族木聚糖酶基因xyn11进行了克隆表达研究。xyn11基因全长636bp,编码211氨基酸。通过构建重组质粒,使其在大肠杆菌中实现了活性表达,酶活达到13.8U/mL。重组酶通过热处理和Ni亲和层析达到电泳纯,SDS-PAGE显示其分子量为20000,与理论分子量吻合。重组酶的最适反应温度为65℃,最适反应pH为4.5,在pH 3.5~6.0范围内保持较高的稳定性,60℃保温1h,酶活还残存60%,Cu^(2+)对该酶有激活作用。重组酶底物特异性强,且不能降解木二糖和木三糖。重组酶以榉木木聚糖为底物,其V_(max)和K_m分别为9809U/mg和5.9mg/mL。榉木木聚糖质量浓度为25g/L,重组木聚糖酶用量为20U/g,在50℃、pH 4.5条件下水解12h低聚木糖的得率为27.8%,水解产物主要由木二糖和木三糖组成,且不产生任何木糖。结果表明,该木聚糖酶催化特性优异,可用于低聚木糖的制备。

The GH11 xylanase gene,xyn11,from Thermoanaerobacterium saccharolyticum JW/SL-YS485 was cloned and expressed in Escherichia coli.The protein(AFK85913.1)consists of 636bp fragment encoding 211 amino acids.The activity of recombinant xylanase was 13.8U/mL in LB medium after IPTG induction.The recombinant xylanase was purified by heat treatment followed by Ni-NTA affinity,and the protein’s molecular weight was approximately 20000.The optimal activity occurred at pH 4.5 and 65℃.The enzyme was stable over the pH range of 3.5 to 6.0 and had a 1-h half-life at 60℃.The activity of recombinant xylanase was significantly enhanced by Cu2+.The Vmax and Km for beechwood xylan were 9809U/mg and 5.9mg/mL,respectively.When 25g/L beechwood xylan was treated with 20U/g xylanase for hydrolysis 12h,the xylooligosaccharides ranging from xylobiose(X2)to xylohexaose(X6)yield was 27.8%without the production of xylose.All these favorable enzymatic properties make xyn11 attractive for potential applications in the xylooligosaccharide production.