绿色木霉内切葡聚糖酶基因Eg Ⅶ的克隆及其表达

Cloning and expression of Trichoderma viride Eg Ⅶ

【目的】在枯草芽孢杆菌中表达绿色木霉(Trichodoma vivide,T.viride)内切葡聚糖酶基因EgⅦ。研究内切葡聚糖酶基因EgⅦ的生物信息、基因克隆与表达及重组酶学性质。【方法】提取纤维素酶生产菌株绿色木霉AS3.3711的总RNA,通过反转录合成cDNA,并以之为模板利用PCR技术扩增获得内切葡聚糖酶基因EgⅦ,并进行生物信息学分析;构建了重组表达载体pMA5-EgⅦ,并利用热激转化法将其转入枯草芽孢杆菌(Bacillus subtilis,B.subtilis)WB600感受态细胞中,成功进行表达。【结果】生物信息学分析表明,内切葡聚糖酶基因EgⅦ编码249个氨基酸,蛋白质的分子量为26.8002 KD,理论等电点7.62,属于胞外蛋白;预测蛋白质三级结构与二级结构一致,由ɑ-螺旋、延伸链区、β-转角和无规则卷曲构成。电泳结果表示,重组枯草芽孢杆菌在EgⅦ基因片段大小符合处出现条带,表明成功将EgⅦ-pMA5转化到B.subtilis WB600中。刚果红染色结果显示重组枯草芽孢杆菌在CMC培养基上出现较大直径的透明圈,重组菌株上清粗酶液DNS酶活力达到22.71 U/mL,表明绿色木霉内切葡聚糖酶基因EgⅦ可以在枯草芽孢杆菌中表达,并具有较高活性。重组酶酶学性质分析表明,该酶的最适温度是60℃,最适pH值为6.4,且温度在50℃以下,pH值在5.6~8.0范围内,酶活力具有良好的稳定性。【结论】通过对内切葡聚糖酶基因EgⅦ克隆及分析,获得了表达内切葡聚糖酶基因EgⅦ的枯草芽孢杆菌基因工程菌,了解了重组酶酶学性质,为纤维素酶的广泛应用提供了理论基础。

【Objective】In this study,the endoglucanase gene EgⅦfrom Trichodoma viride(T.viride)was expressed in Bacillus subtilis(B.subtilis).Meanwhile,the biological information of the gene was studied,and the gene was cloned,expressed and enzymatically analyzed.【Method】By extracting the total RNA of T.viride AS3.3711,the cDNA was synthesized by reverse transcription.Then the cDNA was used as a template to amplify the endoglucanase gene EgⅦ,and the bioinformatics analysis of the gene was conducted.The recombinant vector pMA5-EgⅦwas constructed and transformed into B.subtilis WB600 competent cells by heat shock method.The EgⅦgene was successfully expressed in B.subtilis.【Result】Bioinformatics analysis showed that the EgⅦgene encodes 249 amino acids and the molecular weight of the protein is 26.8002 KD with a theoretical isoelectric point of 7.62,which is an extracellular protein.The predicted protein tertiary structure was consistent with the secondary structure,consisting ofα-helix,extended chain region,β-turn and random coil.The electrophoresis results showed that the PCR product of the transformed B.subtilis exhibited a band at the size of EgⅦ,indicating that the pMA5-EgⅦplasmid had been successfully transformed into B.subtilis WB600.After Congo red staining,the recombinant strain had a large diameter transparent circle on sodium carboxymethyl cellulose medium.The DNS enzyme activity of the supernatant of the recombinant strain reached 22.71U/mL,which indicated that T.viride EgⅦcould be expressed in B.subtilis and its protein product had high activity.The analysis of the enzymatic properties of the recombinant enzyme showed that the optimum temperature was 60℃,and the optimum pH value was 6.4.When the temperature was below 50℃,the enzyme activity had good stability in a pH range of 5.6~8.0.【Conclusion】Through the cloning and analysis of EgⅦ,the transformed B.subtilis strains expressing EgⅦhave been obtained.Meanwhile,the enzymatic properties of the recombinant enzyme have been characterized,which provides a theoretical basis for the wide application of cellulase.