Journal of Applied Glycoscience
Online ISSN : 1880-7291
Print ISSN : 1344-7882
ISSN-L : 1344-7882
Studies on the Development of Functional Oligosaccharides Using Amylases and Related Enzymes
Sumio Kitahata
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2000 Volume 47 Issue 1 Pages 87-97


This paper is composed of the following four research topics, with research carried out at the Osaka Municipal Technical Research Institute. Cyclodextrin glycosyltransferases (CGTases) from Bacillus megaterium, B. circulans, B. macerans and B. stearothermophilus were purified and their catalytic properties were studied. CGTase catalyzed the conversion of α-1, 4-glucans such as starch and glycogen to cyclodextrin (CD) by intramolecular transglycosylation. In the presence of a suitable acceptor such as glucose, CGTase catalyzed the intermolecular transglycosylation, in which the non-reducing end glycosyl residues produced by splitting an α-1, 4-glucan were transferred to the acceptor. In the intramolecular transglycosylation, the enzymes from B. megaterium, B. circulans, B. macerans and B. stearothermophilus produced α-, β- and γ-CDs in ratios of 1.0: 6.3: 1.3, 1.0: 6.4: 1.4, 5.7: 1.0: 0.4 and 1.7: 1.0: 0.3, respectively, on 1 % soluble starch at the initial reaction. B. stearothermophilus CGTase showed the strongest activity in the intermolecular transglycosylation. The effective acceptors of CGTases in the intermolecular transglycosylation were D-glucose, D-xylose, 6-deoxy-D-glucose and L-sorbose, which had a pyranose structure with free equatorial hydroxyl groups at C2, C3 and C4. CGTases transferred glycosyl residues preferentially to the C4-hydroxyl group of D-glucose, D-xylose, and 6-deoxy-D-glucose with the exception of L-sorbose, where the preferred group was the C3-hydroxyl group. The enzyme also catalyzed the hydrolysis of α-1, 4-glucans and CDs. The ratios of hydrolysis to total catalysis were 1.9, 2.0, 2.0 and 8.3 for the CGTases from B. megaterium, B. circulans, B. macerans and B. stearothermophilus, respectively. Using the intermolecular transglycosylation of CGTase, maltooligosyl-sucrose (“coupling sugar, ” commercial name) is produced from the mixture of starch hydrolyzates and sucrose. The cariogenicity of the coupling sugar was studied by a group of the Department of Dental Research, Japanese National Institute of Health, and other universities, and the coupling sugar was proved to be an anticariogenic sweetener. It was the first example of a so-called “functional oligosaccharide” which had a physiological property apart from the conventional functions of sweeteners. Arthrobacter sp. K-1 β-fructofuranosidase (β-FFase), isolated from soil, had very strong transfer activity and broad acceptor specificity. When the β-FFase was incubated with sucrose in the presence of xylose, isomaltose and lactose, the enzyme transferred the fructosyl residue only to the C 1 hydroxyl group of the acceptors and efficiently produced fructosylxyloside (XF), isomaltosylfructoside (IMF) and lactosylfructoside (LacF), respectively. XF competitively inhibited the degradation activity of sucrose by glucosyltransferase (GTase) from Streptococcus mutans as an analogue to sucrose, and IMF acted as an alternative acceptor for the glucosyl transfer reaction of GTase to lessen the formation of insoluble glucan. These saccharides had anticariogenic properties. LacF was nondigestive, but selectively utilized by bifidobacteria in the human intestinal bacteria flora, followed by the improvement of constipation and blood lipid levels of hyperlipemia patients and suppression of putrefactive metabolites such as ammonia, phenol and indole. Stevioside, a sweet steviol glycoside isolated from the leaves of Stevia rebaudiana Bertoni, is about 140-fold as sweet as sucrose, but has a slightly bitter taste and aftertaste. To improve the quality of taste, various stevioside derivatives such as glycosyl-stevioside (G-Ste), fructosyl-stevioside (F-Ste) and galactosyl-stevioside were synthesized with the transfer reaction of CGTase, β-FFase and β-galactosidase.

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