土壤宏基因组文库中纤维素酶的筛选与鉴定

Screening and identification of cellulases in a soil metagenomic library

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1 419 bp,编码蛋白分子量(Mr)为50.99×103,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℃,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.(图10表1参36)

Cellulase,one of the most important industrial enzymes,has been widely applied in various fields including food,textile,paper,laundry,and biofuel production industries.This study aimed to discover microbederived cellulases using the metagenomic approach.A positive clone with cellulase hydrolysis activity was obtained by screening approximately 650000 clones from a soil metagenomic library on LB agar plates containing 0.5%carboxymethylcellulose sodium.The putative cellulase gene in the positive clones was identified by subcloning and sequencing analysis.Its full open reading frame was 1419 base pairs.Sequence alignment and phylogenetic tree analysis revealed that the deduced protein,which was named YNEG5,belonged to the fifth family of glycoside hydrolase and shared 88.66%similarity with an endoglucanase of Rhizobacter sp.S-16.The cellulase gene was amplified by polymerase chain reaction and ligated with a 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 pH were 66℃and 4.8,respectively,and the enzyme was tolerant to high temperature and urea.In conclusion,a cellulase with industrial potential was obtained using functional metagenomics technology,which provided a reference for the excavation of cellulases from uncultured microorganisms.