Purification and characterization of glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide in Bacillus globisporus C11

Glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide (CTS; cyclo {-->6}-alpha-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl(1-->6)-U-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl- (1-->) from alpha-1,4-glucan were purified from Bacillus globisporus C11 The former was a 1,6-alpha-glucosyltransferase (6GT) catalyzing the alpha-1,6-transglucosylation of one glucosyl residue to the nonreducing end of maltooligosaccharides (MOS) to produce alpha-isomaltosyl-MOS from MOS. The latter was an isomaltosyl transferase (IMT) catalyzing alpha-1,3-, alpha-1,4-, and alpha,beta-1,1-intermolecular transglycosylation of isomaltosyl residues. When IMT catalyzed alpha-1,3-transglycosylation, alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS was produced from alpha-isomaltosyl-MOS. In addition, IMT catalyzed cyclization, and produced CTS from alpha-isomaltosyl(1-->3)-alpha-isomaltosyl-MOS by intramolecular transglycosylation. Therefore, the mechanism of CTS synthesis from MOS by the two enzymes seemed to follow three steps: 1) MOS-->alpha-isomaltosyl-MOS (by 6GT), 2) alpha-isomaltosyl-MOS-->alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS (by IMT), and 3) alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS-->CTS + MOS (by IMT). The molecular mass of 6GT was estimated to be 137 kDa by SDS-PAGE. The optimum pH and temperature for 6GT were pH 6.0 and 45degreesC, respectively. This enzyme was stable at from pH 5.5 to 10 and on being heated to 40degreesC for 60 min. 6GT was strongly activated and stabilized by various divalent cations. The molecular mass of IMT was estimated to be 102 kDa by SDS-PAGE. The optimum pH and temperature for IMT were pH 6.0 and 50degreesC, respectively. This enzyme was stable at from pH 4.5 to 9.0 and on being heated to 40degreesC for 60 min. Divalent cations had no effect on the stability or activity of this enzyme.