Process for preparation of chondroitin fraction
Provided are a method for producing a fraction containing more than 50% of CH represented by the general formula (1), which comprises at least the step of allowing a glucuronic acid donor, an N-acetylgalactosamine donor, a saccharide receptor, a chondroitin polymerase derived from the Escherichia coli K4 strain, and Mn 2+ at a final concentration of 0.02 to 100 mM to coexist, and performing a reaction thereof under conditions of 20 to 40° C. and pH 6 to 8 for 0.5 minutes to 4 hours, and a method for producing a fraction containing more than 50% of CH represented by the general formula (2), which comprises at least the step of performing the reaction under same conditions for 10 hours or longer, which enable industrial scale production of a CH fraction of a controlled even number saccharide and odd number saccharide content ratio by a simple procedure at a low cost. (GlcA-GalNAc) n (1) GalNAc-(GlcA-GalNAc) n (2) (In the formula, GlcA represents a glucuronic acid residue, GalNCAc represents a N-acetylgalactosamine residue, - represents a glycosidic bond, and n represents an arbitrary integer.)
1. A method for producing a fraction comprising more than 50% of chondroitin represented formula (1), comprising the steps:
(a) reacting a glucuronic acid donor, a N-acetylgalactosamine donor, a saccharide acceptor, chondroitin polymerase derived from Escherichia coli K4, and Mn 2+ at a final concentration of 0.02 to 100 mM at 30° C. and pH 7.2 for 5 hours and measuring a chondroitin content ratio, X, in a fraction produced by the reaction, wherein the chondroitin content ratio, X, means a ratio of a number of chondroitin molecules represented by formula (1)/a number of chondroitin molecules represented by formula (2);
(b) determining a reaction time for providing the chondroitin content ratio, X, as determined in step (a) in a fraction produced when the reaction is performed under conditions of arbitrary temperature and pH, other than at 30° C. and pH 7.2, at which the chondroitin polymerase derived from Escherichia coli K4 can act for a reaction time defined to be Y; and
(c) performing the reaction at the same temperature and pH as step (b) for a time shorter than Y,
wherein formulae (1) and (2) are:
(GlcA-GalNAc) n (1)
GalNAc-(GlcA-GalNAc) n (2)
wherein GlcA represents a glucuronic acid residue, GalNac represents an N-acetylgalactosamine residue, - represents a glycosidic bond and n represents an arbitrary integer.
2. A method for producing a fraction comprising more than 50% of chondroitin represented by formula (2), comprising the steps of:
(a) reacting a glucuronic acid donor, a N-acetylgalactosamine donor, a saccharide acceptor, chondroitin polymerase derived from Escherichia coli K4, and Mn 2+ at a final concentration of 5 to 100 mM at 30° C. and pH 7.2 for 8 hours and measuring a chondroitin content ratio, X, in a fraction produced by the reaction, wherein the chondroitin content ratio, X, means a ratio of a number of chondroitin molecules represented by formula (1)/a number of chondroitin molecules represented by formula (2);
(b) determining a reaction time for providing the chondroitin content ratio, X, as determined in step (a) in a fraction produced when the reaction is performed under conditions of arbitrary temperature and pH , other than at 30° C. and pH 7.2, at which the chondroitin polymerase derived from Escherichia coli K4 can act for a reaction time defined to be Y; and
(c) performing the reaction at the same temperature and pH as step (b) for a time longer than Y,
wherein formulae (1) and (2) are:
(GlcA-GalNAc) n (1)
GalNAc-(GlcA-GalNAc) n (2)
wherein, GlcA represents a glucuronic acid residue, GalNAc represents an N-acetylgalactosamine residue, - represents a glycosidic bond, and n represents an arbitrary integer.