海洋α-葡萄糖苷酶的克隆表达及其制备高纯潘糖的研究

Cloning and Expression of Marineα-Glucosidase and Its Preparation of High Purity Panose

潘糖是含α-1,6糖苷键的低聚异麦芽糖,α-葡萄糖苷酶是制备潘糖的关键酶制剂。本研究对深海热液口超嗜热古菌(Thermococcus siculi HJ21)中的α-葡萄糖苷酶序列进行合成,并克隆入载体pET29a,在大肠杆菌BL21中表达,通过His Trap HP层析柱纯化获得纯酶,以SDS-PAGE电泳测定α-葡萄糖苷酶分子量,并研究了酶的性质和其转糖苷作用。结果表明:该基因为729 bp,编码242个氨基酸,分子量约为27.2 kDa。该酶最适催化温度和pH分别为40℃和6.0。在以果糖为受体,与麦芽糖的比例为1:9,反应10 h时,其潘糖的生成量最高可以达到79.1%。研究结果为古细菌α-葡萄糖苷酶制备高纯度潘糖提供了依据。

Panose is iso-malto-oligosaccharide containedα-1,6 glycosidic bonds,andα-glucosidase is the key enzyme to produce panose.In this study,theα-glucosidase gene in deep-sea hydrothermal vents thermophilic archaea Thermococcus siculi HJ21 was synthesized,and cloned into the vector pET29a.Then expressed in Escherichia coli BL21 and purified by His Trap HP column.The molecular weight ofα-glucosidase was determined by SDS-PAGE.The enzymatic properties and transglycosylation were also investigated.The results showed that the gene ofα-glucosidase was 729 bp and encoded 242 amino acids.The molecular weight ofα-glucosidase was about 27.2 kDa.Its optimal temperature and pH were 40℃and 6.0 respectively.When the receptor was fructose,and the ratio of fructose and maltose was 1:9,the highest yield of panose could reached 79.1%after 10 h reaction.The results provide a basis for producing high-purity panose byα-glucosidase from archaea.