Purification and characterization of an intracellular cycloalternan-degrading enzyme from Bacillus sp. NRRL B-21195.
A novel intracellular cycloalternan-degrading enzyme (CADE) was purified to homogeneity from the cell pellet of Bacillus sp. NRRL B-21195. The enzyme has a molecular mass of 125 kDa on SDS-PAGE. The pH optimum was 7.0, and the enzyme was stable from pH 6.0 to 9.2. The temperature optimum was 35 degrees C and the enzyme exhibited stability up to 50 degrees C. The enzyme hydrolyzed cycloalternan [CA; cyclo(-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(-->3)-alpha-d-Glcp-(1-->)] as the best substrate, to produce only isomaltose via an intermediate, alpha-isomaltosyl-(1-->3)-isomaltose. This enzyme also hydrolyzed isomaltosyl substrates, such as panose, alpha-isomaltosyl-(1-->4)-maltooligosaccharides, alpha-isomaltosyl-(1-->3)-glucose, and alpha-isomaltosyl-(1-->3)-isomaltose to liberate isomaltose. Neither maltooligosaccharides nor isomaltooligosaccharides were hydrolyzed by the enzyme, indicating that CADE requires alpha-isomaltosyl residues connected with (1-->4)- or (1-->3)-linkages. The K(m) value of cycloalternan (1.68 mM) was 20% of that of panose (8.23 mM). The k(cat) value on panose (14.4s(-1)) was not significantly different from that of cycloalternan (10.8 s(-1)). Judging from its specificity, the systematic name of the enzyme should be cycloalternan isomaltosylhydrolase. This intracellular enzyme is apparently involved in the metabolism of starch via cycloalternan in Bacillus sp. NRRL B-21195, its role being to hydrolyze cycloalternan inside the cells.
Kim
Yeon-Kye
YK
Enzyme Laboratory, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
Kitaoka
Motomitsu
M
Hayashi
Kiyoshi
K
Kim
Cheorl-Ho
CH
Côté
Gregory L
GL
D001407:Bacillus / Q000201:enzymology*; D002236:Carbohydrate Conformation; D004591:Electrophoresis, Polyacrylamide Gel; D005959:Glucosidases / Q000737:chemistry* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D009844:Oligosaccharides; C488014:cycloalternan; D005959:Glucosidases; C493352:cycloalternan-degrading enzyme, Bacillus