A new three-enzyme reaction cycle consisting of sucrose synthase, UDP glucose 4’-epimerase, and human beta-1,4-galactosyltransferase was established for the synthesis of N-acetyllactosamine (LacNAc) with in situ regeneration of UDP galactose. We found that UDP glucose 4’-epimerase is reductively inactivated in the presence of UMP and acceptor substrates of beta-1,4-galactosyltransferase. Reactivation of UDP glucose 4’-epimerase by the transition state analogues dUDP or dTDP 6-deoxy-D-xylo-4-hexulose in combination with the repetitive batch technique enabled 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 beta-1,4-galactosyltransferase, 594 mg of LacNAc could be synthesized. N-Acetyllactosamine was also subsequently converted to Neu5Ac alpha 2,6Gal beta 1,4G1cNAc with alpha-2,6-sialyltransferase and CMP-Neu5Ac.