A NOVEL 3-ENZYME REACTION CYCLE FOR THE SYNTHESIS OF N-ACETYLLACTOSAMINE WITH IN-SITU REGENERATION OF URIDINE 5'-DIPHOSPHATE GLUCOSE AND URIDINE 5'-DIPHOSPHATE GALACTOSE

A new three-enzyme reaction cycle consisting of sucrose synthase, UDP glucose 4'-epimerase, and human beta-1,4-galactosyltransferase was est ablished for the synthesis of N-acetyllactosamine (LacNAc) with in sit u regeneration of UDP galactose. We found that UDP glucose 4'-epimeras e is reductively inactivated in the presence of UMP and acceptor subst rates of beta-1,4-galactosyltransferase. Reactivation of UDP glucose 4 '-epimerase by the transition state analogues dUDP or dTDP 6-deoxy-D-x ylo-4-hexulose in combination with the repetitive batch technique enab led us to use the native enzymes for 11 days in this cycle. With 10 U of sucrose synthase, 5 U of UDP glucose 4'-epimerase, and 1.25 U of be ta-1,4-galactosyltransferase, 594 mg of LacNAc could be synthesized. N -Acetyllactosamine was also subsequently converted to Neu5Ac alpha 2,6 Gal beta 1,4G1cNAc with alpha-2,6-sialyltransferase and CMP-Neu5Ac.