Patent ID: 7811797

Claim:
A 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→} or a saccharide composition comprising the same, which comprises the steps of: (a) allowing an α-isomaltosylglucosaccharide-forming enzyme to act on a solution containing saccharide having a glucose polymerization degree of at least two and α-1,4 glucosidic linkage as a linkage at the non-reducing end of the saccharide to form α-isomaltosylglucosaccharide having a glucose polymerization degree of at least three, an α-1,6 glucosidic linkage as a linkage at the non-reducing end, and α-1,4 glucosidic linkages at all positions except the linkage at the non-reducing end; whereby said α-isomaltosylglucosaccharide-forming enzyme forms said α-isomaltosylglucosaccharide from said saccharide by catalyzing an α-glucosyl-transferring reaction; (b) allowing an α-isomaltosyl-transferring enzyme to act on the formed α-isomaltosylglucosaccharide to form said cyclotetrasaccharide; whereby said α-isomaltosyl-transferring enzyme forms said cyclotetrasaccharide from said α-isomaltosylglucosaccharide; and (c) collecting the formed cyclotetrasaccharide or a saccharide composition comprising the same from the resulting reaction mixture; wherein said α-isomaltosylglucosaccharide-forming enzyme has the following physicochemical properties: (1) Molecular weight 140,000±20,000 daltons, 137,000±20,000 daltons, 136,000±20,000 daltons, or 94,000±20,000 daltons, when determined on SDS-PAGE: (2) Optimum temperature About 40° C. to 50° C. when incubated at a pH of 6.0 for 60 min, or about 45° C. to 55° C. when incubated in the presence of 1 mM Ca 2+ ; or About 60° C. when incubated at a pH of pH 8.4 for 60 min. or about 65° C. when incubated in the presence of 1 mM Ca 2+ ; 3) Optimum pH About pH 6.0 to 6.5 or pH 8.4 when incubated at 35° C. for 60 min; (4) Thermal stability Stable up to a temperature of about 35° C. to 45° C., or of about 40° C. to about 50° C. in the presence of 1 mM Ca 2+ when incubated at pH 6.0 or 8.0 for 60 min; or Stable up to a temperature of about 60° C. or of about 65° C. in the presence of 1 mM Ca 2+ when incubated at pH 8.0 for 60 min; (5) pH stability Stable at a pH of about 4.5 to about 10.0 when incubated at 4° C. for 24 hours; said α-isomaltosyl-transferring enzyme has the following physicochemical properties: (1) Molecular weight 112,000±20,000 daltons, 102,000±20,000 daltons, or 116,000±20,000 daltons when determined on SDS-PAGE; (2) Optimum temperature About 40° C. to 50° C. when incubated at a pH of 6.0 for 30 min; 4) Optimum pH About 5.5 to 6.5 when incubated at 35° C. for 30 min; (5) Thermal stability Stable up to a temperature of about 40° C. to 45° C. when incubated at pH 6.0 for 60 min; (6) pH stability Stable at a pH of about 3.6 to 10.0 when incubated at 4° C. for 24 hours.