Bacillus thermoamyloliquefaciens KP1071 alpha-glucosidase II is a thermostable M(r) 540,000 homohexameric alpha-glucosidase with both exo-alpha-1,4-glucosidase and oligo-1,6-glucosidase activities.
alpha-Glucosidase II of the facultative thermophile Bacillus thermoamyloliquefaciens KP1071 (FERM-P8477; growth over 30-66 degrees C) was purified to a homogeneous state. Its M(r) was estimated as 90000 by SDS/PAGE. However, the enzyme behaved as an active Mr 540000 protein on gel filtration with each of two gels of different matrices as well as on gel electrophoresis under native conditions. The enzyme was not glycosylated. Its isoelectric point was estimated as 5.7. The N-terminal sequence of 20 residues was determined asAla1-Ile-Gln-Pro-Glu-Gln-Asp-Asp-Lys-Thr-Gln-Glu-Asp-Gly- Tyr-Ile-Asp-Ile-Gly-Asn20. The sequence did not resemble those of procaryotic and eucaryotic proteins hitherto reported including the monomeric exo-alpha-1,4-glucosidase and the monomeric oligo-1,6-glucosidase from the same microorganism. The alpha-glucosidase II had no antigenic group shared with the latter two enzymes. Analysis of substrate specificity showed that the alpha-glucosidase II has dual activity towards oligo-1,6-glucosidases and exo-alpha-1,4-glucosidases, but its preference is for non-reducing terminal alpha-1,4 glucosidic bonds in substrates. Kinetic studies proved that both activities are attributed to the same catalytic site. The enzyme was most active at 81 degrees C and pH 7.0. Its half-life at pH 6.8 was 10 min at 81 degrees C, and 5 h at 55 degrees C in 6.4 M urea, 26% ethanol or 2.5% SDS. We suggest that the alpha-glucosidase II is a thermostable, homohexameric enzyme of origin distinct from the exo-alpha-1,4-glucosidase and the oligo-1,6-glucosidase present in the same strain.
Suzuki
Y
Y
Department of Agricultural Chemistry, Kyoto Prefectural University, Japan. y_suzuki@kpu.ac.jp
Nobiki
M
M
Matsuda
M
M
Sawai
T
T
D000596:Amino Acids / Q000032:analysis; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D004187:Disaccharides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005936:Glucans / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007534:Isomaltose / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D008970:Molecular Weight; D009834:Oligo-1,6-Glucosidase / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D013696:Temperature; D014312:Trisaccharides / Q000378:metabolism; D000520:alpha-Glucosidases / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D004187:Disaccharides; D005936:Glucans; D009844:Oligosaccharides; D014312:Trisaccharides; C021705:maltooligosaccharides; C014637:laminaribiose; C008317:maltotriose; D007534:Isomaltose; D008320:Maltose; C029454:turanose; C040088:1,4-glucan; C008237:kojibiose; C034251:4-nitrophenyl-alpha-glucosidase; D009834:Oligo-1,6-Glucosidase; D000520:alpha-Glucosidases; C008189:isomaltulose