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 mm of sucrose and 600 mM of GlcNAc by the concurrent actions of four enzymes, sucrose phosphorylase, UDP-glucose—hexose-1-phospate uridylyltransferase, ...