Abstract
The potential of the hyperthermophilic β-glycosidase from Pyrococcus woesei (DSM 3773) for the synthesis of glycosides under microwave irradiation (MWI) at low temperatures was investigated. Transgalactosylation reactions with β-N-acetyl-d-glucosamine as acceptor substrate (GlcNAc-linker-tBoc) under thermal heating (TH, 85 °C) and under MWI at 100 and 300 W resulted in the formation of (Galβ(1,4)GlcNAc-linker-tBoc) as the main product in all reactions. Most importantly, MWI at temperatures far below the temperature optimum of the hyperthermophilic glycosidase led to higher product yields with only minor amounts of side products β(1,6-linked disaccharide and trisaccharides). At high acceptor concentrations (50 mM), transgalactosylation reactions under MWI at 300 W gave similar product yields when compared to TH at 85 °C. In summary, we demonstrate that MWI is useful as a novel experimental set-up for the synthesis of defined galacto-oligosaccharides. In conclusion, glycosylation reactions under MWI at low temperatures have the potential as a general strategy for regioselective glycosylation reactions of hyperthermophilic glycosidases using heat-labile acceptor or donor substrates.
Highlights
Glycosidases are widely used among the well-established enzymatic synthesis strategies of glycoconjugates [1]
We recently demonstrated for two mesophilic β-galactosidases that product hydrolysis is avoided in transglycosylation reactions due to controlled enzyme inactivation by microwave irradiation (MWI) [20,21]
Dabrowski et al reported dissimilarity of two triplets in β-galactoside hydrolase genes between the hyperthermophilic strains Pyrococcus woesei and Pyrococcus furiosus, whereby only one nucleotide variation resulted in an amino acid replacement from isoleucine (P. furiosus) to threonine (P. woesei) at site 436 [25]
Summary
Glycosidases are widely used among the well-established enzymatic synthesis strategies of glycoconjugates [1]. Reaction engineering strategies to lower product hydrolysis includes reaction conditions at high substrate concentrations with addition of organic solvents or ionic liquids, as well as synthesis in frozen solutions [2,3]. In terms of enzyme stability, numerous glycosidases from thermophilic microorganisms were favorably utilized [4]. Their optimum activity at temperatures between 80 and 110 ̋C may be of disadvantage for optimizing transglycosylation reactions in aqueous buffer solutions using heat-labile substrates or products. Performance of synthesis reactions at ambient temperature may not be effective with glycosidases from extreme thermophiles and affords sophisticated reaction engineering
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