Practical preparation of lacto-N-biose I, a candidate for the bifidus factor in human milk

A one-pot enzymatic reaction to produce lacto-N-biose I (LNB), which is supposed to represent the bifidus factor in human milk oligosaccharides, was demonstrated. Approximately 500 mM of LNB was generated in 10-liter of reaction mixture initially containing 660 m,mM of sucrose and 600 mm of GlcNAc by the concurrent actions of four enzymes, sucrose phosphorylase, UDP-glucose-hexose-1-phospate uridylyltransferase, UDPglucose 4-epimerase, and lacto-N-biose phosphorylase, in the presence of UDP-Glc and phosphate, indicating a reaction yield of 83%. LNB was isolated from the mixture by crystallization after yeast treatment. Finally, 1.4 kg of LNB of 99.6% purity was recovered after recrystallization.