4-ALPHA-GLUCANOTRANSFERASE FROM THE HYPERTHERMOPHILIC ARCHAEON THERMOCOCCUS-LITORALIS - ENZYME-PURIFICATION AND CHARACTERIZATION, AND GENE CLONING, SEQUENCING AND EXPRESSION IN ESCHERICHIA-COLI

4-alpha-Glucanotransferase was purified from cells of Thermococcus lit oralis, a hyperthermophilic archaeon. The molecular mass of the enzyme was estimated to be approximately 87 kDa by gel filtration. The optim al temperature for its activity was 90 degrees C. The enzyme catalyzed the transglycosylation of maltooligosaccharides, yielding maltooligos accharides of various lengths and glucose. When maltoheptaose was used as the substrate, glucoamylase-resistant and glucoamylase-sensitive s accharides were produced. On incubation of amylose with the T. litoral is enzyme, glucoamylase-resistant but alpha-amylase-sensitive molecule s were produced, but the amount of reducing sugar showed only slight i ncreases. These results indicate that the T. litoralis enzyme catalyze s not only intermolecular transglycosylation to produce linear alpha-1 ,4-glucan, but also intramolecular transglycosylation to produce cycli c alpha-1,4-glucan (cycloamylose), similarly to potato 4-alpha-glucano transferase (called disproportionating enzyme). The gene encoding the T. litoralis 4-alpha-glucanotransferase was cloned, sequenced and expr essed in Escherichia coli. The nucleotide sequence of the gene encoded a 659-amino acid protein with a calculated molecular mass of 77 883 D a. The amino acid sequence of the I litoralis enzyme showed high simil arity with those of alpha-amylases of Pyrococcus furiosus, a hyperther mophilic archaeon, and Dictyoglomus thermophilum, an extremely thermop hilic bacterium, but little similarity with those of other known 4-alp ha-glucanotransferases.