Pyrococcus furiosus耐热淀粉酶工程菌的构建及直链麦芽寡糖的制备  

Construction of genetic engineered strain expressing Pyrococcus furiosus thermostable amylase and preparation of linear malto oligosaccharides

作　　者：凡建刚[1] 褚仲梅[2,3] 张毅[2,3] 

机构地区：[1]华东理工大学生物工程学院,上海200237 [2]中科院上海生命科学研究院,上海200233 [3]中科院合成生物学重点实验室,上海200032

出　　处：《工业微生物》2013年第4期34-37,共4页Industrial Microbiology

基　　金：国家"973项目"(2012CB721103);国家"973项目"(2007CB714304)

摘　　要：为了制备特定长度的直链麦芽寡糖,利用PCR方法得到了极端耐热古生菌激烈热球菌Pytococcus furiosus的耐热淀粉酶基因pfta,对其进行序列分析表明,其编码区含有1938 bp,编码含645个氨基酸残基的蛋白质。将该基因系列与表达载体pT7473连接,构建重组质粒pT7473-pfta,以大肠杆菌BL21(DE3)为宿主表达。重组菌株经诱导表达后SDS-PAGE电泳结果显示有明显的分子量约76 kD的特异蛋白条带出现。表达的蛋白经过金属镍离子亲和层析纯化后电泳鉴定出现的条带与预期相符。以环糊精为原料,利用获取的PFTA的单点水解开环反应活性,在90℃、pH5.0、10 min的条件下,能够生产出特定长度的直链麦芽寡糖。An open reading frame that encoded PFTA （Pyrococcus furiosus thermostable amylase） was found out by the genomie analysis of the hyperthermophilic arehaeon Pyrococcusfuriosus. The gene（ 1938 bp） encoded a single polypeptide of 645 amino acids with an estimated molecular mass of 76 kD, was cloned and expressed in Escherichia coll. The recom- binant PFTA was purified with nickel-nitrilotriacetic acid （Ni-NTA） affinity column chromatography and the molecular mass of the recombinant PFTA estimated by SDS-PAGE was the same as the expected. The ring-opening reactions of α-, β-, γ-cyelodextrin were performed at 90℃ and pH 5.0 for 10 rain using PFTA, and the specific-length maltooligosacchar- ides, particularly maltohexaose, maltoheptaose and maltooctaose from cyelodextrin hydrolyzed by PFTA were produced.

关 键 词：PYROCOCCUS furiosus 淀粉酶 环糊精 麦芽寡糖 

分 类 号：Q78[生物学—分子生物学]