Claims
- 1. A process for forming an .alpha.-sialylated galactosyl glycoside that comprises the steps of:
- admixing in a single vessel the following components to form a reaction mixture:
- (i) a catalytic amount of .beta.-galactosidase;
- (ii) a catalytic amount of .alpha.(2,6)- or .alpha.(2,3)sialyltransferase;
- (iii) a .beta.-galactose-containing donor substrate for said .beta.-galactosidase;
- (iv) an acceptor substrate for said .beta.-galactosidase;
- (v) a sialic acid;
- (vi) a CMP-sialic acid regenerating system comprising at least two moles of phosphoenolpyruvate per each mole of said sialic acid, and catalytic amounts of ATP, myokinase, pyruvate kinase, inorganic pyrophosphatase and CMP-sialic acid synthetase; and
- (vii) an aqueous buffer solution containing enzymatically sufficient amounts of metal ion cofactors for said enzymes and having a pH value of about 6 to about 8;
- and maintaining said reaction mixture at a temperature of about zero degrees C. to about 45.degree. C. for a time period sufficient for a .beta.-galactosyl glycoside formed by the .beta.-galactosidase-catalyzed reaction of said galactose-containing donor substrate and said acceptor substrate to be sialylated, said galactosyl glycoside having a K.sub.m /V.sub.max value as a substrate for said sialyltransferase that is less than one-third the K.sub.m /V.sub.max value of said galactose-containing donor substrate for said sialyltransferase.
- 2. The process according to claim 1 including the further step of recovering said formed sialylated galactosyl glycoside.
- 3. The process according to claim 1 wherein said sialylated galactosyl glycoside is Neu5Ac.alpha.2.fwdarw.6Gal.beta.1.fwdarw.4GlcNAc or Neu5Ac.alpha.2-6Gal.beta.1.fwdarw.4GlcNAc-Oallyl.
- 4. The process according to claim 1 wherein said galactose-containing donor substrate (iii) is lactose.
- 5. The process according to claim 1 wherein said acceptor substrate for said .beta.-galactosidase is GlcNAc.
- 6. The process according to claim 1 wherein said .beta.-galactosidase is the .beta.(1,4)galactosidase of Bacillus circulans.
- 7. The process according to claim 1 wherein said sialyltransferase is .alpha.(2,6)sialyltransferase.
- 8. The process according to claim 1 wherein said sialyltransferase is .alpha.(2,3)sialyltransferase.
- 9. The process according to claim 1 wherein said galactosyl glycoside has a K.sub.m /V.sub.max value as a substrate for said sialyltransferase that is less than one-tenth the K.sub.m /V.sub.max value of said galactose-containing donor substrate for said sialyltransferase.
- 10. A process for forming an .beta.-sialylated galactosyl glycoside that comprises the steps of:
- admixing in a single vessel the following components to form a reaction mixture:
- (i) a catalytic amount of Bacillus circulans.beta.(1,4)-galactosidase;
- (ii) a catalytic amount of .alpha.(2,3)sialyltransferase or .alpha.(2,6)sialyltransferase;
- (iii) lactose as a donor substrate for said .beta.(1,4)-galactosidase;
- (iv) N-acetylglucosamine or allyl N-acetylglucosamine glycoside as an acceptor substrate for said .beta.(1,4)-galactosidase;
- (v) sialic acid;
- (vi) a CMP-sialic acid regenerating system comprising at least two moles of phosphoenolpyruvate per each mole of said sialic acid, and catalytic amounts of ATP, myokinase, pyruvate kinase, inorganic pyrophosphatase and CMP-sialic acid synthetase; and
- (vii) an aqueous buffer solution containing enzymatically sufficient amounts of metal ion cofactors for said enzymes and having a pH value of about 6 to about 8;
- and maintaining said reaction mixture at a temperature of about zero degrees C. to about 45.degree. C. for a time period sufficient for N-acetyllactosamine or allyl N-acetyllactosamine glycoside formed by the .beta.-galactosidase-catalyzed reaction of said donor substrate and said acceptor substrate to be sialylated to form an .alpha.-sialylated galactosyl glycoside.
- 11. The process according to claim 10 including the further step of recovering said formed sialylated galactosyl glycoside.
Government Interests
This invention was made with government support under Contract No. GM 44154 awarded by the National Institutes of Health. The government has certain rights in the invention.
US Referenced Citations (4)
Non-Patent Literature Citations (2)
Entry |
Ichikawa et al., J. Am. Chem. Soc., 113(12):4698-4700 (1991). |
David et al., Adv. Carbohyd. Chem. Biochem., 49:175-237 (1991). |