Cellotriose Synthesis Using D-Glucose as a Starting Material by Two Step Reactions of Immobilized β-Glucosidases

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We tried to polymerize D-glucose to cellotriose, the smallest substrate for β-1, 4-glucan synthesis by the β-transglycosylase of Trichoderma longibrachiatum, without participation of high energy compounds such as nucleotide sugars. A commercial β-glucosidase (sweet almond) showed a typical condensation reaction of D-glucose, producing cellobiose when it was entrapped in a visking tube and incubated in 30% D-glucose solution. The reaction was done with immobilized enzyme covalently bound to polyacrylamide beads, and entrapped enzyme. Cellobiose (21.0mg) was obtained from 30 g of D-glucose in a 3-day reaction, where 0.29 unit of the entrapped enzyme preparation was incubated with 100ml of 30% D-glucose at pH 6.0 and 41°C. Gentiobiose was also produced in the mixture as a minor product. The immobilized β-glucosidase (Sumizyme C) preparation covalently bound to polyacrylamide beads could catalyze a transglucosylation reaction to produce cellotriose from cellobiose in a good yield without production of gentiobiose. The transfer reaction was optimal at pH 4.8 and 30°C. Cellotriose (11.2mg) was produced from the reaction mixture containing 68 mg of cellobiose and the enzyme preparation (0.1 unit) after 24-hr of incubation at the optimal conditions. Both immobilized β-glucosidases, sweet almond and Sumizyme C, may be used repeatedly without any loss of the initial activity.
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