Figure 3. ¹³C-NMR monitored AlgE6 epimerization of polyMG.

A stacked plot of the anomeric region of ¹³C-NMR spectra (100 MHz) recorded during AlgE6 epimerization of ¹³C-1-enriched polyMG (F_G=0.47 and F_G=0.0) at 40 °C and p²H 7 of buffer solution (Mops) in ²H₂O. The enzymatic reaction was carried out inside the NMR spectrometer. The final concentrations in 500 μl total volume in the NMR tube were 20 mg/ml polyMG-alginate, 5 mg/ml AlgE6 (as total protein) and 5 mM Ca²⁺. Molar ratio of enzyme/mannuronic acid residues susceptible to epimerization (F_M=0.53) was 1:999. Spectra recorded after 19.9 min (F_G=0.51) to 17.1 h reaction time (F_G=0.77) with a time interval of 15.8 min between successive recordings are shown. Front spectrum (dotted line) is recorded before addition of epimerase. Inset: molar fractions of chain trisaccharide combinations versus degree of conversion (F_G) for polyMG epimerized with the mannuronan C-5 epimerases AlgE1 (○) and AlgE6 (◆). Molar fractions were determined from the NMR spectra after 0, 4, 8 and 23 h reaction time for AlgE1, and after 0, 2, 4, 8 and 25 h reaction time for AlgE6. The epimerization reactions were then terminated. F_GGM represents the molar fraction of G-block termination, i.e. the increase in number of G-blocks in the polymer chain during the epimerization reaction.