解糖热纤维菌木糖苷酶的分离纯化及其酶学性质

doi:10.16085/j.issn.1000-6613.2017.03.036

化工进展 ›› 2017, Vol. 36 ›› Issue (03): 1041-1046.DOI: 10.16085/j.issn.1000-6613.2017.03.036

解糖热纤维菌木糖苷酶的分离纯化及其酶学性质

王月皎, 夏乾竣, 周佩, 张瑜, 王飞, 李迅

南京林业大学江苏省林业资源高效加工利用协同创新中心, 江苏省生物质绿色燃料与化学品重点实验室(南京林业大学), 江苏南京 210037

收稿日期:2016-09-14 修回日期:2016-09-28 出版日期:2017-03-05 发布日期:2017-03-05
通讯作者: 李迅,教授,生物质能源与化学品。
作者简介:王月皎(1993-),女,硕士研究生。E-mail:wyj_audi@163.com。
基金资助:
国家林业局“948”项目（2014-4-37），国家自然科学基金（31270612，31370572）及江苏高校品牌专业建设工程项目（PPZY2015C22

Purification and characterization of a thermostable β-xylosidase from Caldicellulosiruptor saccharolyticus

WANG Yuejiao, XIA Qianjun, ZHOU Pei, ZHANG Yu, WANG Fei, LI Xun

Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources of Nanjing Forestry University, College of Chemical Engineering, Jiangsu Key Laboratory of Biomass-Based Green Fuels and Chemicals(Nanjing Forestry University), Nanjing 210037, Jiangsu, China

Received:2016-09-14 Revised:2016-09-28 Online:2017-03-05 Published:2017-03-05

摘要/Abstract

摘要：

通过生物信息学分析，确定厌氧孢子菌Caldicellulosiruptor saccharolyticus编码的木糖苷酶（XYL39B）属于糖苷水解酶第2家族，是极端耐热的水解低聚木糖为木糖的关键酶。将XYL39B基因（xyl39B）导入大肠杆菌BL21（DE3）中表达，通过热处理和镍柱亲和层析获得纯酶。对纯化的重组XYL39B进行酶学性质的表征，结果表明该酶的最适反应温度为70℃，最适反应pH为6.0。在pH 6.0，70℃条件下保温2h后残留相对酶活仍达90%以上，在55～75℃范围内酶活较高且比较稳定。Zn²⁺、Cu²⁺、SDS对酶活力的抑制作用较明显，其他金属离子和化学试剂对该酶的酶活力影响不显著。在以4-对硝基苯酚-β-D-木糖苷（pNPX）为底物，该酶的表观活化能为14.52kJ/mol，其K_m值为11.47mmol/L，V_max为29.4U/g，K_cat为20.60s^-1，木糖对其反馈抑制较弱（抑制常数K_i为306.87mmol/L），综上结果显示该酶是活性较高且抑耐木糖抑制的细菌源极端耐热木糖苷酶。研究结果为其后续的基因改造和实际应用奠定理论基础。

关键词: 极端耐热, β-木糖苷酶, 解糖热纤维菌, 亲和层析, 酶学性质

Abstract:

The xylosidase from the anaerobic spore bacteria Caldicellulosiruptor saccharolyticus (XYL39B),which hydrolyzes xylooligosaccharides to xyloses,was classified into the glycoside hydrolase family 2(GH₂) by the bioinformatics analyses. The XYL39B gene (xyl39B) ression was transformed into Escherichia coli BL21(DE3). The recombinant XYL39B was purified by heat treatment and the Ni-NTA chromatography. The purified XYL39B reached its maximum activity at 70℃ and pH 6.0; and it retained over 90% of its initial activity at 70℃ and pH 6.0 for 2 hours. The enzyme had a broad temperature optima,and it was quite stable at temperatures ranging from 55 to 75℃. The activities of XYL39B were significantly inhibited by Zn²⁺、Cu²⁺ and SDS. The XYL39B had a Km of 11.47mmol/L,a V_max of 29.40U/g,a K_cat of 20.60s^-1 and had an apparent activation energy(E_a) of 14.52kJ/mol for p-nitrophenyl-β-D-xyloside(pNPX). All results indicated that the recombinant enzyme was an extreme thermophilic bacterial xylosidase which exhibited efficient catalytic activities and highly tolerant to xylose inhibition(inhibition constant Ki=306.87mmol/L). This work established a foundation for the subsequent modification and application of XYL39B.

Key words: thermostable, β-xylosidase, Caldicellulosiruptor saccharolyticus, affinity chromatography, enzyme characterization

中图分类号:

Q556.2

王月皎, 夏乾竣, 周佩, 张瑜, 王飞, 李迅. 解糖热纤维菌木糖苷酶的分离纯化及其酶学性质[J]. 化工进展, 2017, 36(03): 1041-1046.

WANG Yuejiao, XIA Qianjun, ZHOU Pei, ZHANG Yu, WANG Fei, LI Xun. Purification and characterization of a thermostable β-xylosidase from Caldicellulosiruptor saccharolyticus[J]. Chemical Industry and Engineering Progress, 2017, 36(03): 1041-1046.

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解糖热纤维菌木糖苷酶的分离纯化及其酶学性质

Purification and characterization of a thermostable β-xylosidase from Caldicellulosiruptor saccharolyticus

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