| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
The gene that coded for the subunit of an molecular weight (Mr) 540,000 tightly-subunit-associated homohexameric α-glucosidase II (α-D-glucoside glucohydrolase, EC 3.2.1.20) with both exo-α-1, 4-glucosidase and oligo-1, 6-glucosidase (dextrin 6-α-D-glucanohydrolase, EC 3.2.1.10) activities produced by Bacillus thermoamyloliquefaciens growing at 30 to 66℃ was expressed in Escherichia coli HB101. The resulting homohexameric enzyme had a half-life of 10 min at 80℃. Its purification and characterization showed that the enzyme was identical with the native one except for the latter deleting 7 N-terminal residues found in the former. The primary sequence of the subunit with 787 residues and an Mr of 91,070 deduced from the gene was 32% and 24-34% identical to the corresponding sequences of the subunit (770 residues ; Mr 88,620) of an Mr 530,000 subunit-dissociable/reassociable α-glucosidase III specific for maltosaccharides from the same microorganism and of 15 α-glucosidases in the glycosyl hydrolase family 31 from 14 eukaryotic origins and the archaeon Sulfolobus solfataricus 98/2, respectively.
From the sequence analysis by the neural network method of Rost and Sander[Rost, B.and Sander, C., Proteins : Struct. Funct. Genet.. 19, 55-72 (1994)], we inferred that α-glucosidase II like α-glucosidase III might make each subunit of 3 secondary structural regions, i.e., one N-terminal β region, one central α/β or α+β region with two catalytic residues Asp407 and Asp484 (equivalent to Asp383 and Asp451 in α-glucosidase III), and one C-terminal β region, and that this architecture of a β(α/β)β or β(α+β)β sandwich motif might hold for other α-glucosidases in the family 31.
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