热纤梭菌内切葡聚糖酶在酿酒酵母细胞表面上展示研究

Yeast Surface Display of Endoglucanase of Clostridium thermocellum

[目的]对热纤梭菌内切葡聚糖酶进行酿酒酵母细胞表面展示研究,探索降低纤维素酶成本和提高酶活的方法。[方法]通过提取产内切葡聚糖酶的热纤梭菌总DNA,根据热纤梭菌内切葡聚糖酶基因序列和酿酒酵母表面展示质粒载体pYD1上的多克隆位点设计引物,克隆内切葡聚糖酶目的基因,将其连接到酵母表面展示质粒载体pYD1上,构建重组质粒pYD1-CelA,并将其转化入酿酒酵母菌株EBY100中,经诱导表达后,测定表达产物的最适温度和pH,并分析影响其活性的金属离子种类和浓度。[结果]PCR扩增获得1 400bp左右的内切葡聚糖酶CelA基因片段,并成功构建了该基因与酵母表面展示质粒载体pYD1的重组质粒pYD1-CelA。重组质粒转化酿酒酵母菌株EBY100后,在鉴定培养基上测定透明圈的大小得出最佳诱导时间为60 h。表达产物性质的研究结果显示其最适反应温度为50℃,在pH 4.6时酶活性相对较高。[结论]该研究结果为后续对纤维素酶的进一步研究、改造、大量生产及应用奠定了一定的基础。

[ Objective ] The aim was to study the yeast surface display of endoglucanase of Clostridium the^llum, and explore the method to reduce the cellulose costs and improve the enzyme activity. [ Method] The total DNA in Clostridium thermocellum which could produce endoglu- canase was extracted. According to the gene sequence of endoglucanase and the multiple cloning site of the plasmid pYD1, the target gene of en- doglucanase was cloned and connected to the yeast surface display plasmid pYD1 to construct a recombinant plasmid pYD1-CelA, which was then transferred into the yeast strain EBY100. After being induction expressed, the optimal temperature and pH of the products were determined, and the type and concentration of metal ions influencing the enzyme activity were analyzed. [ Result ] A 1 400 bp fragment of endoglucanase CelA gene was amplified by PCR, and a recombinant plasmid pYD1-CelA was successfully constructed to transform the yeast strain EBY100, the size of transparent circle was determined and the best induction time was 60 h. The optimal reaction temperature and pH of the enzyme were about 50 ℃ and 4.6, respectively. [ Conclusion] The study results lay foundation for the further study, transformation, large quantity production and ap- plication of cellulose.