Cloning and characterization of glycogen-debranching enzyme from hyperthermophilic archaeon Sulfolobus shibatae

A gene encoding a putative glycogen-debranching enzyme in Sulfolobus shibatae (abbreviated as SSGDE) was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. The recombinant SSGDE was extremely thermostable, with an optimal temperature at 85°C. The enzyme had an optimum pH of 5.5 and was highly stable from pH 4.5 to 6.5. The substrate specificity of SSCIDE suggested that it possesses characteristics of both amylo-1,6-glucosidase and α-1,4-glucanotransferase. SSGDE clearly hydrolyzed pullulan to maltotriose, and 6-O-α-maltosyl-β-cyclodextrin (G2-β-CD) to maltose and β-cyclodextrin. At the same time, SSGDE transferred maltooligosyl residues to the maltooligosaccharides employed, and maltosyl residues to G2-β-CD. The enzyme preferentially hydrolyzed amylopectin, followed in a decreasing order by glycogen, pullulan, and amylose. Therefore, the present results suggest that the glycogen-debranching enzyme from S. shibatae may have industrial application for the efficient debranching and modification of starch to dextrins at a high temperature.