Alpha-Isomaltosylglucosaccharide synthase, process for producing the same and use thereof

#The present invention relates to a novel α-isomaltosylglucosaccharide-forming enzyme, process and uses of the same, more particularly, to a novel α-isomaltosylglucosaccharide-forming enzyme, process for producing the enzyme, α-glucosyl-transferring method using the enzyme, process for producing a cyclotetrasaccharide having the structure of cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→} using both the enzyme and an α-isomaltosyl-transferring enzyme, and compositions comprising these saccharides.

#There have been known saccharides composed of glucose molecules as constituent saccharides, for example, partial starch hydrolysates, produced from starches as materials, including amylose, amylodextrin, maltodextrin, maltooligosaccharide, and isomaltooligosaccharide. Also, these saccharides are known to have usually reducing and non-reducing groups at. their molecular ends and exhibit reducibility. In general, partial starch hydrolysates can be expressed with an index of dextrose equivalent (DE), a scale of reducing power based on the dry solid. Those with a relatively high DE value are usually known to have properties of a relatively low molecular weight, relatively low viscosity, strong sweetness and reactivity, easy reactivity with amino group-containing substances such as amino acids and proteins that may induce browning and unpleasant smell, and easily cause deterioration. To improve these defects, there has long been desired a method for lowering or eliminating the reducing power without altering glucose molecules as constituent saccharides of partial starch hydrolysates. For example, as disclosed in Journal of American Chemical Society, Vol. 71, pp. 353-358 (1949), it was reported that a method for forming α-, β-, and γ-cyclodextrins that are composed of 6-8 glucose molecules linked together via the α-1,4 glucosidic linkage by contacting amylases, derived from microorganisms of the species Bacillus macerans, with starches. Nowadays, these cyclodextrins are produced on an industrial scale and used in diversified fields using their inherent properties such as non-reducibility, tasteless, and enclosing ability. As disclosed, for example, in Japanese Patent Kokai Nos. 143,876/95 and 213,283/95 applied for by the same applicant as the present invention, it is known a method for producing trehalose, composed of two glucose molecules linked together via the α,α-linkage, by contacting a non-reducing saccharide-forming enzyme and a trehalose-releasing enzyme with partial starch hydrolysates such as maltooligosaccharide. At present, trehalose has been industrially produced from starches and used in different fields by using its advantageous non-reducibility, mild- and high quality-sweetness. As described above, trehalose having a glucose polymerization degree (DP) of two, and α-, β-, and γ-cyclodextrins having a DP of 6-8 are produced on an industrial scale and used in view of their advantageous properties, however, the types of non- or low-reducing saccharides are limited, so that more diversified saccharides other than these saccharides are greatly required.

#Recently, a new type of cyclotetrasaccharide, composed of glucose units, was reported. European Journal of Biochemistry, Vol. 226, pp. 641-648 (1994) shows that a cyclic tetrasaccharide having the structure of cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→} (may be called “cyclotetrasaccharide” throughout the specification) is formed by contacting a hydrolyzing enzyme, alternanase, with alternan linked with glucose molecules via the alternating α-1,3 and α-1,6 bonds, followed by crystallization in the presence of methanol as an organic solvent.

#FIG. 1 is an elution pattern of a saccharide, obtained by α-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globiformis C9 strain, when determined on high-performance liquid chromatography.

#FIG. 2 is a nuclear resonance spectrum (1H-NMR) of cyclotetrasaccharide, obtained by the enzymatic reaction using α-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globiformis C9 strain.

#FIG. 3 is a nuclear resonance spectrum (13C-NMR) of cyclotetrasaccharide, obtained by the enzymatic reaction using α-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globiformis C9 strain.

#The following are the identification results of Strain C9, Strain C11, Strain N75, and Strain A19, which produce the novel α-isomaltosylglucosaccharide-forming enzyme of the present invention. The identification tests were conducted in accordance with the methods as described in “Biseibutsu-no-Bunrui-to-Dotei” (Classification and Identification of Microorganisms), edited by Takeji Hasegawa, published by Japan Scientific Societies Press, Tokyo, Japan (1985).

#<Strain C9>

#<A. Morphology>