Characterization of Oligocellulose Synthesized by Reverse Phosphorolysis Using Different Cellodextrin Phosphorylases

Abstract

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: (1)H 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 (DPn) of ∼7, while a DPn 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.

Water-soluble cellulose oligomer production by chemical and enzymatic synthesis: a mini-review

“…A cellobiose concentration of 10, 5, 1 or 0.2 mmol L −1 gave an average DP of 7, 9, 11 or 13, respectively, with a dispersity below 1.06 in each case. The α ‐ d ‐glucose 1‐phosphate concentration was 20 times higher than the cellobiose concentration and no cellobiose was found in the oligomers obtained, underlying a complete reaction …”

“…The -D-glucose 1-phosphate concentration was 20 times higher than the cellobiose concentration and no cellobiose was found in the oligomers obtained, underlying a complete reaction. 38 The transglycosylation of cellobiose or cellotriose by cellulolytic enzyme endoglucanase I was also investigated. 39 The reaction took place in acetate buffer (pH = 4.0) at 50 ∘ C for 1 h. The oligomer production was followed by an increase of the turbidity which decreased if the reaction lasted more than 1 h. Cellobiose gave a DP of up to 7 (yield not specified) while cellotriose gave oligomers with DP from 4 to 16 with a 30% yield.…”

The structure of a GH149 β‐(1 → 3) glucan phosphorylase reveals a new surface oligosaccharide binding site and additional domains that are absent in the disaccharide‐specific GH94 glucose‐β‐(1 → 3)‐glucose (laminaribiose) phosphorylase

“…Enzymatic synthesis is an attractive approach to such materials due to its regiospecificity and stereospecificity. Emulating what has been achieved with α‐1,4‐glucans and β‐1,4‐glucans, glycoside phosphorylases (GPs) with specificity for β‐(1 → 3)‐glycosidic bonds are potential candidate biocatalysts for such syntheses. GPs acting on β‐ d ‐glucopyranosyl‐(1 → 3)‐ d ‐glucopyranose (laminaribiose) have previously been described in bacteria Paenibacillus sp.…”

Three‐Enzyme Phosphorylase Cascade Immobilized on Solid Support for Biocatalytic Synthesis of Cello−oligosaccharides

“…To avoid cello−oligosaccharide precipitation during reaction in solution, tight control of DP in product is additionally required . Without such control, the CdP would elongate the growing oligosaccharide chains until they become largely insoluble . Besides reaction time, the activity ratio of the three phosphorylases is another critical process parameter.…”

“…[29,31] Without such control, the CdP would elongate the growing oligosaccharide chains until they become largely insoluble. [32] Besides reaction time, the activity ratio of the three phosphorylases is another critical process parameter. The consequent requirement to fine-tune the individual enzyme activities on the support poses a fundamentally difficult challenge for the enzyme co-immobilization to establish.…”