|本期目录/Table of Contents|

[1]赵陈阁 柴树茂 冯治洋**.土壤宏基因组文库中纤维素酶的筛选与鉴定*[J].应用与环境生物学报,2020,26(01):1-12.[doi:10.19675/j.cnki.1006-687x.2019.05023]
 ZHAO Chenge,CHAI Shumao & FENG Zhiyang.Screening and identification of cellulases in soil metagenomic library[J].Chinese Journal of Applied & Environmental Biology,2020,26(01):1-12.[doi:10.19675/j.cnki.1006-687x.2019.05023]
点击复制

土壤宏基因组文库中纤维素酶的筛选与鉴定*()
分享到:

《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
26卷
期数:
2020年01期
页码:
1-12
栏目:
研究论文
出版日期:
2020-01-25

文章信息/Info

Title:
Screening and identification of cellulases in soil metagenomic library
作者:
赵陈阁 柴树茂 冯治洋**
南京农业大学食品科技学院 南京 210095
Author(s):
ZHAO Chenge CHAI Shumao & FENG Zhiyang
College of Food Science and Technology, Nanjing Agricultutal University , Nanjing 210095, China
关键词:
功能宏基因组学土壤宏基因组文库纤维素酶酶学性质
Keywords:
functional metagenomics soil metagenomic library cellulase enzymatic properties
DOI:
10.19675/j.cnki.1006-687x.2019.05023
摘要:
纤维素酶是最重要的工业用酶之一,已广泛应用于食品、纺织、造纸、洗涤和生物燃料生产等多个领域中。为挖掘微生物来源的纤维素酶,基于功能宏基因组学的方法,筛选云南土壤宏基因组文库获得具有纤维素酶活性的阳性克隆;通过构建亚克隆和测序分析确定纤维素酶的编码基因,将其克隆到表达载体上并转化到大肠杆菌中构建重组表达系统,诱导蛋白表达后对纤维素酶酶学性质进行分析。通过筛选约65万个文库克隆,获得一个有纤维素酶水解活性的克隆,预测该纤维素酶基因全长为1419 bp,编码蛋白分子量为50.99 kDa,蛋白的氨基酸序列与Rhizobacter sp. S-16中的内切葡聚糖酶有88.66%的相似性,蛋白同源比对结果表明该酶属于糖苷水解酶第五家族(GH5),将其命名为YNEG5;对YNEG5的生化特性进行分析,确定其最佳反应温度为66 °C,最适pH为4.8,该酶热稳定性良好且对工业试剂尿素具有较强的耐受性。本研究基于功能宏基因组学技术获得一个具有工业应用潜能的纤维素酶,可为不可培养微生物中纤维素酶的挖掘提供参考。
Abstract:
Cellulase, one of the most important industrial enzymes, has been widely applied in various fields including food, textile, paper, laundry, and in biofuel production industries . This study aimed to discovery microbe derived cellulases using metagenomic approach. A positive clone with cellulase hydrolysis activity was obtained by screening about 650,000 clones of a soil metagenomic library on LB agar plates containing 0.5% carboxymethylcellulose sodium . The putative cellulase gene in the positive clones was identifed by subcloning and sequencing analysis. Its full open reading frame is 1419 bp. Sequence alignments and phylogenetic tree analysis revealed that the deduced protein, which was named YNEG5, belong ed to the fifth family of glycoside hydrolase (GH5), and shared 88.66% similarity with an endoglucanase of Rhizobacter sp. S-16. The cellulase gene was amplified by PCR and ligated with pET-30a(+) expression vector and transformed into Escherichia coli BL21(DE3) for overexpression. The recombinant protein was studied for its enzymatic properties. Characterization of the enzyme indicated that its optimum temperature and optimum pH were 66 °C and 4.8, respectively, and the enzyme was tolerant to high temperature and urea. In conclusion, a cellulase with industrial potential was obtain ed using functional metagenomics technology, which provided a reference for the excavation of cellulases from uncultured microorganisms.

备注/Memo

备注/Memo:
收稿日期 Received: 2019-05-14 接受日期 Accepted: 2019-06-19
*国家自然科学基金项目(31370088, 31770049)资助
**通讯作者 Corresponding author (E-mail: zfeng@njau.edu.cn)
点击摘要页题目后的“PDF”可下载阅读全文;本文为已录用的作者修定稿,尚未经编辑全面修改。
引用本文请注明出处本刊;发表刊期和页码将以正式出版时的安排为准,但DOI确定不变。
更新日期/Last Update: 2019-06-19