The in vitro synthesis of cellulose via a nonbiosynthetic path has been achieved for the first time by condensation of β‐D‐cellobiosyl fluoride as substrate for cellulase in a mixed solvent of acetonitrile/acetate buffer (pH 5,5:1) (enzymatic polymerization). Under reaction condition of a higher substrate concentration or higher acetonitrile concentration, cellooligosaccharides were produced predominantly. The present method of using a glycosyl fluoride as substrate for a hydrolytic enzyme in an organic/water mixed solvent has successfully been applied to synthesis of maltooligosaccharides and unnatural oligosaccharides.
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