Chemical Industry and Engineering Progress ›› 2018, Vol. 37 ›› Issue (04): 1544-1551.DOI: 10.16085/j.issn.1000-6613.2017-1273

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

PEI Jianjun1,2, LI Jie1,2, LI Qi1,2, ZHAO Linguo1,2   

  1. 1 College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, Jiangsu, China;
    2 Jiangsu Key Lab for the Chemistry & Utilization of Agricultural and Forest, Nanjing 210037, Jiangsu, China
  • Received:2017-06-25 Revised:2017-07-17 Online:2018-04-05 Published:2018-04-05

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

裴建军1,2, 李杰1,2, 李琦1,2, 赵林果1,2   

  1. 1 南京林业大学化学工程学院, 江苏 南京 210037;
    2 江苏省农林生物质化学与利用国家重点实验室培育点, 江苏 南京 210037
  • 通讯作者: 赵林果,教授。
  • 作者简介:裴建军(1977-),男,副教授,研究方向为代谢工程。E-mail:peijj2000@sina.com.cn。
  • 基金资助:
    国家重点研发计划(2017YFD0601001)、江苏省高校自然科学研究重大项目(13KJA220004)、江苏省"青蓝工程"项目及江苏高校优势学科建设工程项目(PAPD)。

Abstract: 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.

Key words: xylanase, Thermoanaerobacterium saccharolyticum, hydrolysis, xylooligosaccharide

摘要: 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%,Cu2+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VmaxKm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。

关键词: 木聚糖酶, 嗜热糖杆菌, 水解, 低聚木糖

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