Enzymatic Synthesis of Oligosaccharides, Alkyl and Terpenyl Glucosides, by Recombinant Escherichia coll-Expressed Pichia etchellsii β-Glucosidase II

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Abstract

The biosynthetic activity of yeast Pichia etchellsii β-glucosidase II (Bglll) expressed in recombinant Escherichia coli was utilized for synthesis of cellooligosaccharides, alkyl and terpene glucosides. Cellooligosaccharides with a degree of polymerization of 3 and greater were resolved by thin-layer chromatography (TLC) using an ethyl acetate:l-propanol:2-propanol:water (8:5:1:1) solvent system followed by visualization with 0.2% naphtho-resorcinol reagent. Using 2M cellobiose and 15IU of partially purified Bglll, 57 mmol/L of oligosaccharides (comprising mostly cellotriose and cello-pentaose) was synthesized in 16 h. Similarly, alkyl glucosides with chain lengths from 6 to 10 carbons were synthesized and products extracted to near purity by ethyl acetate extraction. The same extraction method was employed to separate, to near purity, various monoterpenyl (nerol, geraniol, citronellol) glucosides. A reliable and simple method for separation of cellooligosaccharides using a combination of Bio-Gel P-2 gel filtration and charcoal celite adsorption chromatography wasdeveloped. The cellooligosaccharides were separated to purity as confirmed by TLC. The enzyme was among the very few that could synthesize a wide variety of glycoconjugates.

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