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

doi:10.16085/j.issn.1000-6613.2017-1273

化工进展 ›› 2018, Vol. 37 ›› Issue (04): 1544-1551.DOI: 10.16085/j.issn.1000-6613.2017-1273

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 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

收稿日期:2017-06-25 修回日期:2017-07-17 出版日期:2018-04-05 发布日期:2018-04-05
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）。

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

PEI Jianjun^1,2, LI Jie^1,2, LI Qi^1,2, ZHAO Linguo^1,2

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

摘要/Abstract

摘要： 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%，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。

关键词: 木聚糖酶, 嗜热糖杆菌, 水解, 低聚木糖

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 V_max and K_m 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

中图分类号:

Q814

裴建军, 李杰, 李琦, 赵林果. Thermoanaerobacterium saccharolyticum JW/SL-YS485来源的GH11家族木聚糖酶的克隆表达及其应用[J]. 化工进展, 2018, 37(04): 1544-1551.

PEI Jianjun, LI Jie, LI Qi, ZHAO Linguo. Expression and characterization of a GH11 xylanase from Thermoanaerobacterium saccharolyticum JW/SL-YS485 and its application in xylooligosaccharide production[J]. Chemical Industry and Engineering Progress, 2018, 37(04): 1544-1551.

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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

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