Fig. 7: De novo synthesis of branched polysaccharides.
a Recombinant GlgE activity of Estrella lausannensis from crude extract of E. coli (CE) and purified on nickel affinity column (E1) were loaded on nondenaturing polyacrylamide gel. After migration, slices of native-PAGE were incubated in TRIS/acetate buffer containing 2 mM of maltose-1-phosphate (M1P) over 16 h at 25 °C. The synthesis of de novo glucan chains is visualized as dark-blue bands due to the formation of glucan–iodine complexes. Similar in vitro experiments were conducted by adding GlgB activity of W. chondrophila to a TRIS/acetate buffer containing GlgE activity and 2 mM of M1P. After overnight incubation, reaction mixture was subjected to 1H-NMR analysis. b Part of 1H-NMR spectra of maltose-1-phosphate (M1P), non-purified reaction mixture, and glycogen (gly) from bovine liver in D2O. Peak #1 (5.45–5.3 ppm) and peak #2 (4.98 ppm) represent the signals of protons involved, respectively, in α-1,4 and α-1,6 linkages while peak #3 (5.47 ppm) represents the characteristic doublet of doublet signals of α-anomeric proton located on C1 of maltose-1-phosphate. The appearances of peak #2 and peak #3 in incubation product indicate the formation of branched polysaccharides composed of α-1,4 and α-1,6 linkages. The presence of peak #3 suggests that M1P was not completely polymerized by GlgE activity of EL. α-Polysaccharides were then purified (see “Methods” for details) and incubated with a commercial isoamylase type debranching enzyme. After overnight incubation, the linear glucan chains released from α-polysaccharides (c) and glycogen from bovine liver used as reference (e) were separated according to the degree of polymerization by capillary electrophoresis coupled with a fluorescent labeling of reduced-ends. As control, α-polysaccharide (d) and glycogen (f) samples were directly labeled and analyzed by capillary electrophoresis in order to estimate the content of free-linear glucan chains.
