Enzymatic synthesis and characterization of galactosyl trehalose trisaccharides

α, α-Trehalose was efficiently modified by a transgalactosylation reaction of Escherichia coli β-galactosidase using lactose as a donor to yield two galactosyl trehalose trisaccharides. The reaction products of trehalose by the enzyme were observed by thin layer chromatography (TLC) and high performance anion exchange chromatography (HPAEC) and were purified by BioGel P2 gel permeation chromatography and recycling preparative HPLC. Liquid chromatography-mass spectrometry (LC-MS) and ¹³C nuclear magnetic resonance (NMR) analyses revealed that the structures of the main products were 6²-β-D-galactosyl trehalose (1) and -4²-β-D-galactosyl trehalose (2). A reaction of 30%(w/v) trehalose and 15%(w/v) lactose at pH 7.5 and 45°C resulted in a total yield of approximately 27-30% based on the amount of trehalose used. The galactosyl trehalose products were not hydrolyzed by trehalase. In addition, the mixture of transfer products (9:1 ratio of 1 to 2) showed higher thermal stability than glucose, lactose, and maltose, but less than trehalose, against heat treatment over 100 °C at pH 4 and 7. It also exhibited better thermal stability than sucrose at pH 4 alone.