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United States Department of Agriculture

Agricultural Research Service

Title: Purification and Characterization of An Intracellular Cycloalternan-Degrading Enzyme from Bacillus Sp. Nrrl B-21195

Authors
item Kim, Yeon-Kye - IBARAKI, JAPAN
item Kitaoka, Motomitsu - IBARAKI, JAPAN
item Hayashi, Kiyoshi - IBARAKI, JAPAN
item Kim, Cheorl-Ho - DONGGUK UNIVERSITY
item Cote, Gregory

Submitted to: Carbohydrate Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 1, 2004
Publication Date: April 1, 2004
Citation: Kim, Y., Kitaoka, M., Hayashi, K., Kim, C., Cote, G.L. 2004. Purification and characterization of an intracellular cycloalternan-degrading enzyme from Bacillus sp. NRRL B-21195. Carbohydrate Research. 339:1179-1184.

Interpretive Summary: We found that bacteria previously discovered at NCAUR use an unusual multi-step process to convert starch to sugars that it can then use for energy without having to share its food source with other microbes. The first step involves an enzyme that alters the structure of starch molecules, and the second step then converts this altered starch to a unique sugar with a circular or cyclic structure. This cyclic sugar is then taken up by the bacteria and converted to energy, but cannot be used by other microbes. In this most recent study, we have isolated and characterized the enzyme responsible for breaking open the cyclic sugar structure once it is inside the cell. The multi-step process can also be used by chemists to convert starch into the cyclic sugar, which USDA has patented for a number of potential applications in foods and drugs.

Technical Abstract: 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 degree C and the enzyme exhibited stability up to 50 degree 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-(l-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 Km value of cycloalternan (1.68 mM) was 20% of that of panose (8.23 mM). The kcat value on panose (14.4 s**-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-2l195, its role being to hydrolyze cycloalternan inside the cells.

Last Modified: 9/2/2014
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