Method for preparing sialic acid derivative
The present invention relates to a method for preparing a sialic acid derivative characterized by performing both of a process for preparing CMP-N-acetylneuraminic acid using N-acetyl-D-glucosamine and a process for preparing the sialic acid (neuraminic acid) derivative that combines a sialic acid with a galactose derivative or a lactose derivative, together, in one reactor. According to the method for preparing a sialic acid derivative of the present invention, expensive cytidine 5′-monophosphate (CMP) is capable of being recycled in a reactor, such that an amount of the CMP introduced into the reactor may be reduced, and the sialic acid derivative is capable of being prepared at a significantly high efficiency by using cheap N-acetyl-D-glucosamine, and pyruvate as substrates.
1. A method for preparing a sialic acid derivative comprising: (a) preparing a sialic acid derivative of a compound including sialyllactose or galactose residues by adding a compound including cytidine 5′-monophosphate (CMP), acetyl phosphate, nucleotide triphosphate (NTP), N-acetyl-D-glucosamine (GlcNAc), Sodium pyruvate, and lactose or galactose residues as substrates, and reacting a reaction solution including cytidine 5′-monophosphate kinase (CMK), acetate kinase (ACK), CMP-N-acetylneuraminic acid synthetase (CMP-NeuAc synthetase: NEU), N-acetylglucosamine-2-epimerase (GlcNAc-2-epimerase: NANE), N-acetylneuraminic acid aldolase (NeuAc aldolase, NAN) and α-2,3-sialyltransferase mutant having an amino acid sequence represented by one selected from the group consisting of SEQ ID NOs: 2-6, or α-2,6-sialyltransferase mutant comprising T substitution from I in 411 th amino acid of α-2,6-sialyltransferase having amino acid sequence of SEQ ID NO:13, or S or T substitution from L in 433 th amino acid of α-2,6-sialyltransferase having amino acid sequence of SEQ ID NO:13, in a single reactor; and (b) obtaining the prepared sialic acid derivative of the compound including sialyllactose or galactose residues prepared according to the step (a).
2. The method according to claim 1 , wherein the α-2,6-sialyltransferase has an amino acid sequence represented by one selected from the group consisting of SEQ ID NOs: 14-18.
3. The method according to claim 1 , wherein the reaction is performed at a temperature of 25° C. to 38° C.
4. The method according to claim 1 , wherein pH of the reaction solution is 7 to 9.
5. The method according to claim 1 , wherein the N-acetylglucosamine-2-epimerase (GlcNAc-2-epimerase: NANE) has an amino acid sequence represented by SEQ ID NO: 25.
6. The method according to claim 1 , wherein the compound including galactose residues is a galactose derivative of a compound selected from the group consisting of monosaccharides, oligosaccharides, linkers, flavonoids, anti-cancer agents, antibiotics, immunosuppressants and antibodies.