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 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. The recombinant was extremely thermostable, with an optimal temperature at 85 degrees 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 Sduanyu1804E suggested that it possesses characteristics of both amylo-1,6-glucosidase and alpha-1,4-glucanotransferase. Sduanyu1804E clearly hydrolyzed pullulan to maltotrlose, and 6-O-alpha-maltosyl-beta-cyclodextrin (G2-beta-CD) to maltose and beta-cyclodextrin. At the same time, Sduanyu1804E transferred maltooligosyl residues to the maltooligosaccharides employed, and maltosyl residues to G2-beta-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.

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