Chemoenzymatic synthesis of β-D-glucosides using cellobiose phosphorylase from Clostridium thermocellum

Abstract

Over the past decade, disaccharide phosphorylases have been successfully applied for the synthesis of numerous a-glucosides. In contrast, much less research has been done with respect to the production of beta-glucosides. Although cellobiose phosphorylase was already successfully used for the synthesis of various disaccharides and branched trisaccharides, its glycosylation potential towards small organic compounds has not been explored to date. Unfortunately, disaccharide phosphorylases typically have a very low affinity for non-carbohydrate acceptors, which urges the addition of solvents. The ionic liquid AMMOENG (TM) 101 and ethyl acetate were identified as the most promising solvents, allowing the synthesis of various beta-glucosides. Next to hexyl, heptyl, octyl, nonyl, decyl and undecyl beta-D-glucopyranosides, also the formation of vanillyl 4-O-beta-D-glucopyranoside, 2-phenylethyl beta-D-glucopyranoside, beta-citronellyl beta-D-glucopyranoside and 1-O-beta-D-glucopyranosyl hydroquinone was confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. Moreover, the stability of cellobiose phosphorylase could be drastically improved by creating cross-linked enzyme aggregates, while the efficiency of the biocatalyst for the synthesis of octyl beta-D-glucopyranoside was doubled by imprinting with octanol. The usefulness of the latter system was illustrated by performing three consecutive batch conversions with octanol imprinted cross-linked enzyme aggregates, yielding roughly 2 g of octyl beta-D-glucopyranoside