Enzymatic production of cellobiose from starch and its reduction to cellobiitol

Cellobiose (528-50-7) was synthesized from starch using two phosphorylases. α-Glucan phosphorylase (EC 2.4.1.1) converted 39.6% of glucose residues in the starch molecule into glucose 1-phosphate in the presence of 1 M inorganic phosphate. Inorganic phosphate was selectively dialyzed out from the resultant reaction mixture by electrodialysis equipped with an ion exchange membrane having a molecular weight cut-off of 100. Thus glucose1-phosphate solution free from inorganic phosphate was obtained with 82.1% yield. Cellobiose phosphorylase (EC 2.4.1.20) converted 89.2% of added glucose 1-phosphate into cellobiose when the above G1P solution was incubated with a roughly equimolar amount of glucose in the presence of magnesium acetate under alkaline conditions (pH 8.0). As a result of three successive steps using two phosphorylases, cellobiose was produced from starch with a 29.0% yield. Cellobiose was reduced under a sponge nickel catalyst (Raney nickel) and the obtained cellobiitol was crystallized with an 86.7% yield. The 1H- and 13C-NMR spectra of this compound agreed well with those expected from 4-O-β-glucosyl-D-glucitol (cellobiitol, 535-94-4). The X-ray crystallographic analysis of the obtained cellobiitol crystal revealed it to be a monoclinic space group P21 (4), with the following unit-cell parameters: a = 1.0007, b = 0.8683, c = 2.3137 nm, β = 127.27°. From a thermo gravimetric-differential scanning calorimetric analysis, the cellobiitol crystal was found to have a melting point of 103.6°C and to be in the form of a monohydrate. Cellobiitol showed a relative sweetness of around 20, being less sweet than other sugar alcohols such as glucitol (sorbitol) and maltitol. Cellobiitol was found to be much less hygroscopic than sorbitol and maltitol. The median lethal dose of cellobiitol in rats was determined to be more than 5000 mg/kg body weight. All these properties of cellobiitol indicate it to be a promising compound as a food ingredient.