作　　者： (秦慧彬）; (李梦菲）; (杨洪江）;

机构地区： 工业微生物教育部重点实验室天津市工业微生物重点实验室天津科技大学生物工程学院,天津300457 山西省农业科学院农作物品种资源研究所、农业部黄土高原基因资源与种质创制重点实验室、杂粮种质资源发掘与遗传改良山西省重点实验,山西太原030031

出　　处： 《生物技术》 2017年第4期330-336,共7页

摘　　要： [目的]对黑曲霉纤维二糖水解酶cbhA基因进行了克隆和在毕赤酵母中的真核表达。[方法]采用PCR方法扩增黑曲霉纤维二糖水解酶A(Cellobiohydrolase A,CBHA)基因,获得的DNA序列与cbhA基因表现出高度相似,推导出的氨基酸序列与真菌CBHA酶也高度相似,属于糖基水解酶第7家族。将扩增得到的cbhA基因克隆到毕赤酵母表达载体pPIC9K上,与α-因子信号肽序列形成融合蛋白,进一步通过电转化方法将线性化质粒p PIC9K-cbhA转化至毕赤酵母GS115菌株进行表达。[结果]在甲醇诱导下,重组菌株CMC比酶活力是对照的2.5倍,SDS-PAGE分析结果也确认了cbhA基因在重组菌株GS115/p PIC9K-cbhA中的表达。对该酶性质的分析表明,重组CBHA酶水解CMC底物最适p H值为5.0,最适温度为55℃。[结论]黑曲霉纤维二糖水解酶基因cbhA的克隆和其真核表达工程菌株的构建,为获得纤维二糖水解酶A高产菌株,实现纤维素酶多组分的人工组装奠定了基础。 [Objective]Aspergillus niger Cellobiohydrolysis enzyme gene cbhA was cloned and expressed in Pichia pastoris.[Methods]Using polymerase chain reaction（ PCR） the cloned the cellobiohydrolase A gene from Asprgillus niger was similar to cbhA gene highly,and the deduced amino acid sequence was highly similar to fungi cellobiohydrolysis enzyme A,belonging to glycosyl hydrolase family 7. The cloned gene was subcloned into Pichia expression vector p PIC9 K. Introduced gene sequence and the α-factor signal peptide sequence formed fusion protein. The plasmid p PIC9K-cbhA was linearized and then electrotransformed into Pichia pastoris GS115. [Results]Under methanol introduction,the CMC enzyme activity of recombinant strain was 2. 5 times of that in control. SDS-PAGE analysis confirmed the expression of cbhA gene in the recombinant strain GS115/p PIC9K-cbhA. Nature of this enzyme analysis showed that with CMC as the substrate the optimum p H value of recombinant enzyme CBHA was 5. 0,and the optimum temperature was 55 ℃. [Conclusion] The cbhA gene cloned from Aspergillus niger laid a foundation of assemble the cellulase multicomponent,of which construction of eukaryotic expression engineering strain laid the foundation of obtaining the Cellobiohydrolase A high-yield strains.

关 键 词： 黑曲霉 纤维二糖水解酶 毕赤酵母 异源表达