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Title: A COLLABORATIVE REACTION MECHANISM OF CYCLO-ALTERNAN-FORMING ENZYME (CAFE) AND GLUCOSYLTRANSFERASE (DE) FOR THE PRODUCTION OF CYCLO-ALTERNAN (CA) IN BACILLUS SP NRRL B-21195

Author
item KIM, YEON-KYE - NATL FOOD RES INST
item KITAOKA, MOTOMITSU - NATL FOOD RES INST
item HAYASHI, KIYOSHI - NATL FOOD RES INST
item KIM, CHEORL-HO - DONGGUK UNIV
item Cote, Gregory

Submitted to: Carbohydrate Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/18/2003
Publication Date: 9/18/2003
Citation: Kim, Y., Kitaoka, M., Hayashi, K., Kim, C., Cote, G.L. 2003. A synergistic reaction mechanism of a cycloalternan-forming enzyme and a D-glucosyltransferase for the production of cycloalternan in Bacillus sp. NRRL B-21195. Carbohydrate Research. 338:2213-2220.

Interpretive Summary: We found that bacteria previously discovered at the National Center for Agricultural Utilization Research use an unusual two-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. The two-step process can also be used by chemists to convert starch into the cyclic sugar, which the U.S. Department of Agriculture has patented for a number of potential applications in foods and drugs.

Technical Abstract: Cyclo-alternan-forming enzyme (CAFE) was partially purified by using isomaltose agarose affinity chromatography. The partially purified CAFE included 140 kDa and 117 kDa proteins which produced CA [cyclo{>6)-alpha-D-Glcp-(1>3)-alpha-D-Glcp-(1>6)-alpha-D-Glcp-(>3)-alpha-D-Glcp-(1>}] from the reaction with maltooligosaccharides (G2-G7) that were not produced by the purified CAFE alone. N-Terminal sequences of the 140 kDa and 117 kDa proteins were FAAALGNI and LGGIWHDPYG, respectively, and no homologous sequences were found in protein data banks, showing that these enzymes were novel proteins. The 140 kDa protein is a transglycosylase, or disproportionating enzyme (DE), which catalyzes alpha-1,6-glucosyl transfer of a glucosyl residue from the non-reducing end of one maltooligosaccharide to the non-reducing end of another, forming an isomaltosyl residue at the non-reducing ends of maltooligosaccharides. The 117 kDa protein, CAFE, initially transferred the isomaltosyl residues to the non-reducing ends of isomaltosyl maltooligosaccharides to form isomaltosyl-alpha-(1>3)-isomaltosyl-alpha-(1>4)-maltooligosaccharides (IMIMGn), and then the enzyme catalyzed the cyclization to produce cyclo-alternan (CA). A collaborative reaction mechanism by DE and CAFE for the production of CA from maltodextrin or starch is proposed as follows: Firstly, DE forms an alpha-(1>6) linkage at non-reducing ends of maltooligosaccharides or starch to produce isomaltosyl maltooligosaccharides (IMGn). In the next stage, CAFE reacts with the IMGn and transfers the isomatosyl residue to the non-reducing ends of another IMGn to produce isomaltosyl-alpha-(1>3)-isomaltosyl-alpha-(1>4)-maltooligosaccharides (IMIMGn). Then CAFE catalyzes intramolecular transglycosylation, or cyclization, of IMIMGn to produce CA. It indicates that the CAFE initially reported to be an enzyme that directly produces CA from alternan, indeed, was an enzyme involved in the effective production of CA from starch or maltodextrin in collaboration with DE.