Microwave-Assisted Synthesis of Glycoconjugates by Transgalactosylation with Recombinant Thermostable β-Glycosidase from Pyrococcus

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.

Enzymatic Synthesis of 2-(β-Galactosyl)-ethyl Methacrylate by β-Galactosidase fromPyrococcus woeseiand Application for Glycopolymer Synthesis and Lectin Studies

“…Hydrolytic activity was measured following our previously published protocols. , In brief, 100 μL of an appropriately diluted enzyme solution was added to 900 μL of 4.4 mM para -nitrophenyl-β- d -galactopyranoside ( p NP-Gal, 1 ) in citrate-Na 2 HPO 4 buffer (pH 5.5) and incubated for 5 min at 85 °C. The reaction was stopped at specific time intervals by the addition of 200 μL of Na 2 CO 3 (0.2 M) to 100 μL of the sample solution.…”

“…Production of recombinant PwGly was previously described by our group. 71 After harvesting, 3 g of cells was resuspended in 9 mL of Na 2 HPO 4 -citrate buffer (pH 5.5) and disrupted via sonification (four cycles on ice, each cycle 30 s sonification with a 30 s break between cycles). After centrifugation (15 000 rpm, 4 °C, 30 min), the supernatant was heated up to 95 °C for 45 min and centrifuged again.…”

“…coli − In our previous study, we characterized and utilized recombinant Pw Gly for the synthesis of defined galacto-oligosaccharides using microwave irradiation . We herein report on the synthesis of Gal-EMA on gram scale by recombinant Pw Gly and utilization of Gal-EMA in glycopolymer synthesis for lectin-binding studies (Scheme ).…”

Microwave-Assisted Synthesis of 5′-O-methacryloylcytidine Using the Immobilized Lipase Novozym 435

“…Due to the steady heat distribution, the yield in organic syntheses can be increased in a reduced reaction time compared to conventional heating with an oil bath. Combining microwave irradiation with enzyme catalysis is a common method for the synthesis of compounds [26][27][28]. The application of a biocatalyst enables derivatization of carbohydrate moieties without the use of protecting groups, while microwave irradiation significantly increases reaction kinetics, as reflected in reaction time and/or reaction yield.…”

Microwave-assisted cross-linking of milk proteins induced by microbial transglutaminase

“…Microwaves are part of the electromagnetic wave spectrum and have wavelengths ranging from 1 mm to 1 m. MI has been shown to enhance the yield and reaction rate in certain enzymatic reactions12. MI treatment has also been used to accelerate enzyme-catalyzed reactions in food products, thereby enhancing the activity and selectivity of glycosidases and decarboxylases13. In addition, Da Rós, Freitas, Perez and de Castro14 reported the application of lipase/MI treatment to accelerate the enzymatic synthesis of biodiesel, and high catalytic efficiency ( K cat / K m ) values have also been achieved in the microwave-assisted digestion of β-LG by pronase15.…”