Combined use of .beta.-galactosidase and sialyltransferase coupled with in situ regeneration of CMP-sialic acid for one pot synthesis of oligosaccharides

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.