Glycosyl benzoates as novel substrates for glycosynthases.

The development of a procedure for the one-pot synthesis of glycosyl benzoates directly from unprotected sugars in aqueous media using 2-chloro-1,3-dimethylimidazolium chloride (DMC), thiobenzoic acid, and triethylamine is reported. These glycosyl donors are excellent substrates for wild-type and mutant glycosidases. ß-Glucosyl benzoate was hydrolysed by the GH1 ß-glucosidase derived from Halothermothrix orenii (HorGH1). Subsequent use of this substrate in thioligase-mediated glycosylation of p-nitrothiophenol demonstrated their superiority as donors compared to their p-nitrophenol counterparts with excellent conversions. Using a series of arene nucleophiles, we also demonstrate good to excellent conversions (up to 94%) of ß-glucosyl benzoate to the corresponding p-nitrophenyl- and thioglycosides.

Novel triple mutant of an extremophilic glycosyl hydrolase enables the rapid synthesis of thioglycosides.

In order to expand the toolbox of enzymes available for thioglycoside synthesis, we describe here the first example of an extremophilic glycosyl hydrolase from Halothermothrix orenii (HorGH1) engineered towards thioglycosynthase activity with a novel combination of mutations. Using the triple mutant, HorGH1 M299R/E166A/E354G, a range of thioglycosides from glycosyl fluoride donors and aromatic thiols could be synthesised with exquisite stereoselectivity and good to excellent conversions (61-93%).

Copper-induced Production of Laccases for Lignin Depolymerisation and Micropollutant Degradation by Laccase-mediator Systems.

Contaminants deriving from human activities represent a constantly growing threat to our environment and have a direct impact on plant and animal health. To alleviate this ecological imbalance, biocatalysis offers a green and sustainable alternative to conventional chemical processes. Due to their broad specificity, laccases are enzymes possessing excellent potential for synthetic biotransformations in various fields as well as for the degradation of organic contaminants. Herein, we produced laccases in submerged cultures of P. ostreatus and T. versicolor in three different media. The fungi/medium combination leading to the highest enzymatic activity was malt extract (2%) + yeast extract (3%) + glucose (0.8%). Laccase production was further increased by supplementing this medium with different concentrations of Cu2+, which also provided a better understanding of the induction effect. Additionally, we disclose preliminary results on the interaction of laccases with mediators (ABTS and violuric acid - VA) for two main applications: lignin depolymerisation with guaiacylglycerol-ß-guaiacyl ether (GBG) as lignin model and micropollutant degradation with Remazol Brilliant Blue (RBB) as enzymatic bioremediation model. Promising results were achieved using VA to increase depolymerization of GBG dimer and to enhance RBB decolorisation.