Method for producing polysaccharide
The present invention aims to provide a method for producing a polysaccharide with high efficiency using a polysaccharide synthase. The present invention provides a method for producing a polysaccharide, including allowing polysaccharide synthase (B) to act on ribonucleoside diphosphate-monosaccharide (A) to produce a polysaccharide, wherein in 10 to 100% of the duration in which (B) acts on (A), the concentration of ribonucleoside diphosphate in a reaction solution is lower than 100 times an inhibitory concentration IC 50 against polysaccharide synthase (B).
1. A method for producing a hyaluronan,
wherein steps (1) to (3) described below are simultaneously performed:
Step (1): producing a hyaluronan and a uridine diphosphate by allowing a hyaluronan synthase (B-1) to act on a uridine diphosphate-glucuronic acid and a uridine diphosphate-N-acetylglucosamine;
Step (2): producing a uridine triphosphate by allowing a uridine triphosphate synthase (D4-1) to act on the uridine diphosphate and a phosphate-containing compound (F); and
Step (3): producing a uridine diphosphate-glucuronic acid by allowing a uridine triphosphate-monosaccharide-1-phosphate uridylyltransferase (G) to act on the uridine triphosphate and a 1-phospho-glucuronic acid,
wherein initial concentrations of the uridine diphosphate-glucuronic acid and the uridine diphosphate-N-acetylglucosamine are 0.1 mM to 2M, initial concentration of hyaluronan synthase (B-1) is 0.1 to 100,000 U/L,
wherein 1 U of hyaluronan synthase (B-1) represents the amount of enzyme to produce the hyaluronan and the uridine diphosphate from 1 μmol of the uridine diphosphate-glucuronic acid and the uridine diphosphate-N-acetylglucosamine per minute at 30° C. and pH of 7.0,
wherein a duration in which the hyaluronan synthase (B-1) acts on the uridine diphosphate-glucuronic acid and the uridine diphosphate-N-acetylglucosamine is 10 minute or more,
wherein in 10 to 100% of the duration, the concentration of the uridine diphosphate in the reaction solution is lower than 100 times an inhibitory concentration IC 50 against the hyaluronan synthase (B-1), and
wherein the inhibitory concentration IC 50 refers to a concentration of the uridine diphosphate at which an enzyme activity of the hyaluronan synthase (B-1) is reduced by half under a condition where the hyaluronan synthase (B-1) has a concentration at which the hyaluronan synthase (B-1) acts on the uridine diphosphate-glucuronic acid and the uridine diphosphate-N-acetylglucosamine, wherein the uridine diphosphate-glucuronic acid and the uridine diphosphate-N-acetylglucosamine are used as a substrate and the uridine diphosphate is used as an inhibitor, and
wherein the phosphate-containing compound (F) is at least one compound selected from the group consisting of triaminophosphine oxide, ω-phosphono-L-arginine, polyphosphoric acid, phosphoenolpyruvic acid and salts thereof, carbamoyl phosphate, 1,3-Bisphosphoglycerate, phosphocreatine, and nucleoside triphosphate.
2. The method for producing a hyaluronan according to claim 1 ,
wherein an enzyme activity ratio (Y 1 ) calculated from formula (1) is not lower than 0.1,
enzyme activity ratio ( Y 1 )= V max 1 /V max 2 (1),
wherein Vmax 1 is the enzyme activity of the uridine triphosphate synthase (D4-1) on the uridine diphosphate, and Vmax 2 is the enzyme activity of the uridine triphosphate synthase (D4-1) on the uridine diphosphate-glucuronic acid and the uridine diphosphate-N-acetvlglucosamine.
3. The method for producing a hyaluronan according to claim 1 ,
wherein an enzyme activity ratio (Y 2 ) calculated from formula (2) is not lower than 0.1,
enzyme activity ratio ( Y 2 )= V max 1 /V max 3 (2),
where Vmax 1 is the enzyme activity of the uridine triphosphate synthase (D4-1) on the uridine diphosphate, and Vmax 3 is the enzyme activity of the hyaluronan synthase (B-1) on the uridine diphosphate-glucuronic acid and the uridine diphosphate-N-acetylglucosamine.
4. The method for producing a hyaluronan according to claim 1 , wherein a Michaelis constant Km is lower than 100 times the inhibitory concentration IC 50 ,
wherein the Michaelis constant Km refers to a Michaelis constant of reaction to synthesize the uridine triphosphate in the presence of the phosphate-containing compound (F), the using uridine diphosphate as a substrate and the uridine triphosphate synthase as an enzyme, and
wherein the inhibitory concentration IC 50 refers to a concentration of the uridine diphosphate at which an enzyme activity of the hyaluronan synthase (B-1) is reduced by half under a condition where the hyaluronan synthase (B-1) has a concentration at which the hyaluronan synthase (B-1) acts on the uridine diphosphate-glucuronic acid and the uridine diphosphate-N-acetylglucosamine, wherein the uridine diphosphate-glucuronic acid and the uridine diphosphate-N-acetylglucosamine are used as substrates and the uridine diphosphate is used as an inhibitor.