[1]雒军,宫晓燕,魏炜,等.瑞氏木霉内切葡聚糖酶II在大肠杆菌中的重组表达及重组酶性质测定[J].南京师大学报(自然科学版),2009,32(03):92-98.
 Luo Jun,Gong Xiaoyan,Wei Wei,et al.Expression of Recombinant Trichoderma Reesei Endoglucanase II in Escherichia coli and Properties of the Recombinant Enzyme[J].Journal of Nanjing Normal University(Natural Science Edition),2009,32(03):92-98.
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瑞氏木霉内切葡聚糖酶II在大肠杆菌中的重组表达及重组酶性质测定()
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《南京师大学报(自然科学版)》[ISSN:1001-4616/CN:32-1239/N]

卷:
第32卷
期数:
2009年03期
页码:
92-98
栏目:
生命科学
出版日期:
2009-09-30

文章信息/Info

Title:
Expression of Recombinant Trichoderma Reesei Endoglucanase II in Escherichia coli and Properties of the Recombinant Enzyme
作者:
雒军;宫晓燕;魏炜;杨纯;方何建;戴亦军;袁生;
江苏省生物多样性和生物技术重点实验室, 南京师范大学生命科学学院, 江苏南京210046
Author(s):
Luo JunGong XiaoyanWei WeiYang ChunFang HejianDai YijunYuan Sheng
Jiangsu Engineering and Technology Research Center for Industralization of Microbial Resources,School of Life Science,Nanjing Normal University,Nanjing 210046,China
关键词:
羧甲基纤维素钠盐 内切葡聚糖酶 大肠杆菌β葡聚糖 瑞氏木霉
Keywords:
CM C-Na endog lucanase E scher ichia coli βg lucan Trichod erm a reesei
分类号:
Q78
摘要:
将不含信号肽的瑞氏木霉内切葡聚糖酶II( Ce l5A)的cDNA克隆到pET- 28a( + )表达质粒上, 与其N 末端6个组氨酸标签序列融合, 构建成pET- 28a( + ) - eg l2表达质粒, 在大肠杆菌BL21 ( DE3)中诱导表达. 利用低温诱导策略, 成功表达出活性重组蛋白. W este rn b lo t结果显示重组Ce l5A相对分子分量大约为43 000. 进一步对重组Ce l5A进行N i- NTA 亲和层析柱纯化并对其进行酶学性质测定, 结果显示以羧甲基纤维素钠为作用底物时重组酶最适作用pH 为4.5, 最适作用温度为60℃ ; 重组酶热稳定性较好, 在65℃及以下保温1.5 h, 活性仍相当稳定. M n 2+ 对Ce l5A 的酶活有促进作用, Fe 3+ 和Cu 2+ 有抑制作用, 其它金属离子和EDTA对酶活没有明显影响. 底物特异性研究表明, 重组Ce l5A只能水解混合键葡聚糖和羧甲基纤维素钠, 对混合键葡聚糖的水解能力是其对羧甲基纤维素钠水解能力的6.7倍, 但对微晶纤维素、G lass m icro fibe r filters、木聚糖、阿拉伯木聚糖和木葡聚糖没有水解作用.
Abstract:
Ce l5A ofT richoderma reesei w as fused w ith h istid ine( 6) tag and cloned in pET- 28a( + ) p lasm id. W ith a low tem pera ture induction stra tegy, the enzym ew as successfu lly expressed in E. co li host. W este rn b lot analysis show ed tha t the recomb inan tC el5A had am o lecu larw e ight of 43 000. The recom binant Ce l5A had m ax ima l ac tiv ities at pH 4. 5, 60℃ and w as stab lew hen it was heated fo r 1.5 h a t 65℃ . The enzym atic ac tiv ityw as enhanced by 1mmo l/LM n 2+ and inh ibited by Fe 3+ and Cu 2+ wh ile o ther m eta l ions and EDTA had no effec t. The recom binant Ce l5A was ab le to hydro-lyze βg lucan and CM C- Na, but unab le to hydro ly zeAv ice,l G lassm ic ro fiber filte rs, Xy lan, A rabiroxy lan and Xy log lu-can. The ac tiv ity of Cel5A to digest βg lucan is 6. 7-fo ld h igher than to d igest CMC-Na.

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备注/Memo

备注/Memo:
基金项目: 国家自然科学基金( 211100B386) .
通讯联系人: 袁 生, 教授, 博士生导师, 研究方向: 微生物生长代谢调控. E-m ailyu ansheng@ n jnu. edu. cn
更新日期/Last Update: 2013-04-23