Fig. 6: R699 has kojibiose synthase activity.

a Schematic representation of the proposed kojibiose synthesis reaction catalyzed by R699. b Enzymatic activity assay of R699 using various substrates, including galactosylhydroxylysine (Gal-Hyl), glucose, galactose, glucose-6-phosphate, and glucose-1-phosphate. Enzymatic activity was measured by detecting UDP production with a luciferase assay. Data are presented as mean values (± S.D.) from triplicate biological samples (n = 3). p values were calculated using two-tailed Student’s t-tests. c–e Gas chromatograms of standards (in c) and cell lysates from untransformed E. Coli strain MEC143 (in d) versus MEC143 transformed with R699 (in e). Chromatograms were generated by extracting m/z 361, a characteristic ion of methyloximated, 8-trimethylsilyl derivatized disaccharides. The positions of maltose and kojibiose major peaks are marked in e. Quantitative analysis using the major peaks indicates that kojibiose yield is approximately 15% of maltose, the most abundant disaccharide in E. coli. Note there are two isomers of derivatized disaccharides.