Patent ID: 7537909
Filing Date: 2009-05-26
Classification: C12P

Abstract:
1. A method for producing 4-O-β-D-galactopyranosyl-D-xylose enzymatically in a ratio of at least 68:32 by weight of 4-O-β-D-galactopyranosyl-D-xylose to the combined sum of 2-O-β-D-galactopyranosyl-D-xylose and 3-O-β-D-galactopyranosyl-D-xylose comprising the following steps: (i) preparing a first reaction mixture of 2-20% by weight of D-xylose, 0.5 to 5% by weight of a β-D-galactopyranoside substrate, and 75-97.5% by weight of a reaction medium that comprises buffered water at a pH between 5.0 and 9.0; adding 10 to 1,000 units of a β-D-galactosidase enzyme, per gram of β-D-galactopyranoside, to the first reaction mixture; and obtaining a second reaction mixture; (ii) subjecting the second reaction mixture to a reaction at a temperature comprised between a temperature higher than the freezing point of the second reaction mixture and 45° C., for 2 to 48 hours, in order to form disaccharides in the second reaction mixture; (iii) stopping the reaction when the disaccharides have been formed in the desired amount, by means of a treatment selected from the group consisting of: (iv) separating an aglyconic fragment of the β-D-galactopyranoside substrate used in the first step from the third reaction mixture by extraction or filtration; obtaining a fourth reaction mixture; (v) isolating fractions that contain 4-O-β-D-galactopyranosyl-D-xylose by a method selected from the group consisting of addition of celite to the fourth reaction mixture, followed by solid liquid extraction with a solvent and elution with a first eluent in a column wherein the first eluent is a mixture of water/isopropanol that contains 1 to 10% (v/v) of isopropanol; and directly adding active carbon to the fourth reaction mixture followed by filtration and elution with a second eluent; (vi) crystallizing the fractions that contain 4-O-β-D-galactopyranosyl-D-xylose in a crystallization mixture selected from the group consisting of mixtures of acetone/methanol in a ratio between 5/1 to 20/1 and mixtures of acetone/water in a ratio between 5/1 to 20/1.