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)-alpha-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 a-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 45 degrees C, respectively. This enzyme was stable at from pH 5.5 to 10 and on being heated to 40 degrees C 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 50 degrees C, respectively. This enzyme was stable at from pH 4.5 to 9.0 and on being heated to 40 degrees C for 60 min. Divalent cations had no effect on the stability or activity of this enzyme.
Nishimoto
Tomoyuki
T
Hayashibara Biochemical Laboratories, Inc, Okayama, Japan. amaseken@hayashibara.co.jp
Aga
Hajime
H
Mukai
Kazuhisa
K
Hashimoto
Takaharu
T
Watanabe
Hikaru
H
Kubota
Michio
M
Fukuda
Shigeharu
S
Kurimoto
Masashi
M
Tsujisaka
Yoshio
Y
D001407:Bacillus / Q000201:enzymology* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004795:Enzyme Stability; D005936:Glucans / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008320:Maltose / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
D003470:Culture Media; D005936:Glucans; D009844:Oligosaccharides; C474813:cyclic tetrasaccharide, Bacillus globisporus; D008320:Maltose; C040088:1,4-glucan; D005964:Glucosyltransferases