Characterization of Oligocellulose Synthesized by Reverse Phosphorolysis Using Different Cellodextrin Phosphorylases

Much progress was made in the straightforward and eco-friendly enzymatic synthesis of shorter cellulose chains (oligocellulose). Here, we report the determination of a molar mass distribution of the oligocellulose synthesized from cellobiose (CB) and α-glucose 1-phosphate by reverse phosphorolysis, using enzymes cellodextrin phosphorylase from Clostridium stercorarium or Clostridium thermocellum as catalyst. The oligocellulose molar mass distribution was analyzed using three different methods: ¹H NMR spectroscopy, matrix assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) and size exclusion chromatography (SEC). The molar mass distribution of the synthesized oligocellulose was only dependent on the concentration of cellobiose used in the reaction. Data obtained from MALDI-ToF MS and SEC were almost identical and showed that oligocellulose synthesized using 10 mM CB has an average degree of polymerization (DP_n) of ∼7, while a DP_n of ∼14 was achieved when 0.2 mM CB was used in the reaction. Because of solvent limitation in SEC analysis, MALDI-ToF MS was shown to be the technique of choice for accurate, easy and fast oligocellulose molar mass distribution determination.