[1]王　军,康利琴,刘中华,等.一种灰盖鬼伞外切β-1，3葡聚糖酶基因的克隆与表达[J].南京师范大学学报(自然科学版),2016,39(03):96.[doi:10.3969/j.issn.1001-4616.2016.03.016]
　Wang Jun,Kang Liqin,Liu Zhonghua,et al.Cloning and Expression of an Exo-β-1，3 glucanaseGene from Coprinopsis cinerea[J].Journal of Nanjing Normal University(Natural Science Edition),2016,39(03):96.[doi:10.3969/j.issn.1001-4616.2016.03.016]
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一种灰盖鬼伞外切β-1，3葡聚糖酶基因的克隆与表达()
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《南京师范大学学报》（自然科学版）[ISSN:1001-4616/CN:32-1239/N]

卷:
第39卷
期数:
2016年03期
页码:
96
栏目:
·生命科学·
出版日期:
2016-09-30

文章信息/Info

Title:
Cloning and Expression of an Exo-β-1，3 glucanaseGene from Coprinopsis cinerea
文章编号:
1001-4616（2016）03-0096-06
作者:
王　军; 康利琴; 刘中华; 袁　生
南京师范大学生命科学学院，江苏省微生物与微生物功能基因组学重点实验室，江苏省微生物资源产业化工程技术研究中心，江苏 南京 210023
Author(s):
Wang Jun; Kang Liqin; Liu Zhonghua; Yuan Sheng
School of Life Sciences，Nanjing Normal University，Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics，Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources，Nanjing 210023，China
关键词:
外切β-1; 3葡聚糖酶; 基因克隆; 异源表达; 生化特性; 灰盖鬼伞
Keywords:
exo-β-1; 3 glucanase; gene cloning; heterologous expression; biochemical characterization; Coprinopsis cinerea
分类号:
Q786
DOI:
10.3969/j.issn.1001-4616.2016.03.016
文献标志码:
A
摘要:
为了简单快速地获取大量的β-1，3葡聚糖酶以研究其在真菌形态发生过程中所发挥的作用，本研究以灰盖鬼伞的cDNA为模板克隆了一种编码外切β-1，3葡聚糖酶的基因，并将该基因插入表达质粒pET28A（+）中，获得了重组表达质粒pET28A（+）-exg，转化到E. coli Rosetta菌株中并进行异源重组表达. 结果显示，克隆基因的cDNA全长2 415 bp，共编码786个氨基酸；SDS-PAGE电泳表明该基因在E.coli Rosetta菌株中得到了高效表达，重组表达的酶蛋白表观分子量为85 kDa；纯化后获得的表达酶经DNS法测得比活力为45 U/mg. 酶学性质测定结果表明，该酶具有β-1，3葡聚糖外切酶活性，以昆布多糖为底物时，最适反应条件为pH 6.0、温度60 ℃，且有一定的耐热能力和较好的pH稳定性，具有较好的应用前景.
Abstract:
To acquire enough β-1，3 glucanase for investigating its role in morphological changes of fungus，an exo-β-1，3 glucanase gene（exg）was cloned from cDNA of Coprinopsis cinerea okayama 7#130 and inserted into plasmid pET28A（+）to generate the recombinant expression plasmid pET28A（+）-exg. pET28A（+）-exg was transformed into Escherichia coli Rosetta strain for heterologous expression. The result showed that the length of exg gene is 2 415 bp，which encodes 786 amino acids. SDS-PAGE analysis showed that the exo-β-1，3 glucanase was effectively expressed in E. coli Rosetta with an apparent molecular masses of 85 kDa. The specific enzyme activity was 45 U/mg after purification. Characterization of the recombinant enzyme showed that it had an exo-hydrolases activity and the optimum pH value was 6.0，and the optimum reaction temperature was at 60 ℃ when laminarin as substrate. Besides，the thermostability and pH stability give it some prospect in the future.

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备注/Memo

备注/Memo:
收稿日期：2015-09-08.　
基金项目：国家自然科学基金面上项目（31170028）、江苏省自然科学基金青年基金（BK20140918）、江苏省高校自然科学研究项目（14KJB180013）.　
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和酶工程研究. E-mail：yuansheng@njnu.edu.cn
更新日期/Last Update: 2016-09-30