Fig. 4: Characterization of compounds released by recombinant GlgE of Estrella lausannensis.
a Both histidine-tagged recombinant GlgE-EL and GlgE-WC proteins were purified and incubated in presence of glycogen and inorganic phosphate. The overnight reaction products from crude extract (CE), third washing step (W3) purified enzymes (E1) were subjected to thin-layer chromatography analysis. Orcinol-sulfuric spray reveals a notable production of M1P with recombinant GlgE-EL, which is less visible with recombinant GlgE-WC. b Part of 1D-1H-NMR spectrum of maltoside-1-phosphate. α-anomer configuration of both glucosyl residues was characterized by their typical homonuclear vicinal coupling constants (3JH1A,H2A and 3JH1B,H2B) with values of 3.5 and 3.8 Hz, respectively. A supplementary coupling constant was observed for α-anomeric proton of residue A as shown the presence of the characteristic doublet at 5.47 ppm. This supplementary coupling constant is due to the heteronuclear vicinal correlation (3JH1A,P) between anomeric proton of residue A and phosphorus atom of a phosphate group, indicating that phosphate group was undoubtedly O-linked on the first carbon of the terminal reducing glucosyl unit A. The value of this 3JH1A,P was measured to 7.1 Hz (Table 1). c MS-MS sequencing profile of M1P. The molecular ion [M + 2Na]+ at m/z 466.7 corresponding to M1P + 2 sodium was fractionated in different ions. Peak assignments were determined according to panel incrusted in (c).
