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 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 SSGDE suggested that it possesses characteristics of both amylo-1,6-glucosidase and alpha-1,4-glucanotransferase. SSGDE clearly hydrolyzed pullulan to maltotrlose, and 6-O-alpha-maltosyl-beta-cyclodextrin (G2-beta-CD) to maltose and beta-cyclodextrin. At the same time, SSGDE 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.
Research Institute of Food and Nutritional Sciences and Department of Food and Nutrition, Brain Korea 21 Project, Yonsei University, Seoul 120-749, Korea.
Trinh Thi Kim
TT
Van
Soo-In
SI
Ryu
Kyung-Ju
KJ
Lee
Eun-Ju
EJ
Kim
Soo-Bok
SB
Lee
D000687:Amylopectin / Q000378:metabolism; D000688:Amylose / Q000378:metabolism; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005936:Glucans / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism; D006004:Glycogen Debranching Enzyme System / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008320:Maltose / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D047392:beta-Cyclodextrins / Q000378:metabolism
D005936:Glucans; D006004:Glycogen Debranching Enzyme System; D009844:Oligosaccharides; D011994:Recombinant Proteins; D047392:beta-Cyclodextrins; C021705:maltooligosaccharides; C075170:6-O-maltosyl-beta-cyclodextrin; D008320:Maltose; C009109:pullulan; D006003:Glycogen; D000688:Amylose; D000687:Amylopectin; C020569:4 alpha-glucanotransferase; C030563:amylo-1,6-glucosidase; C031215:betadex