麦芽糖转糖基酶在毕赤酵母中的表达及活性分析  

Expression and Activity Assay of Amylomaltase in Pichia pastoris

作　　者：朱国强[1] 王水兴[1] 黄兰[1] 朱石龙[1] 

机构地区：[1]南昌大学食品科学与技术国家重点实验室,江西南昌330047

出　　处：《食品科学》2010年第23期258-261,共4页Food Science

摘　　要：用PCR方法从E.coliBL21(DE3)中获得麦芽糖转糖基酶基因,将该基因插入到酵母表达载体pPIC9K的α分泌信号开放阅读框的下游,对重组质粒pPIC9K-MalQ所含的外源片段进行双向测序。将测序正确的重组质粒用内切酶SalⅠ线性化,电穿孔转化到GS115感受态细胞中,G418梯度筛选高拷贝转化菌及PCR鉴定目的基因的整合,1%甲醇诱导表达。经薄层色谱分析粗酶液处理的麦芽二糖溶液,证实粗酶液具有麦芽糖转糖基酶活性。The gene of MalQ was amplified from genomic DNA of E. coli BL21（DE3） and subcloned intoα secretion signal open reading frame of pPIC9K expression vector to obtain a recombinant plasmid pPIC9K-MalQ. The recombinant plasmid was verified by DNA sequence. The resultant recombinant plasmid bearing MalQ gene was digested by Sal I and transformed into Pichia pastoris strain GS115. The cells with stable expression of amylomaltase were screened in the medium containing G418. The positive clones were induced with methanol to express amylomaltase. The activity of 4-α-glucanotransferase in the crude enzyme was validated through TLC.

关 键 词：麦芽糖转糖基酶 毕赤酵母 基因重组 

分 类 号：Q786[生物学—分子生物学]