The two Rasamsonia emersonii α-glucuronidases, ReGH67 and ReGH115, show a different mode-of-action towards glucuronoxylan and glucuronoxylo-oligosaccharides

Background: The production of biofuels and biochemicals from grass-type plant biomass requires a complete utilisation of the plant cellulose and hemicellulosic xylan via enzymatic degradation to their constituent monosaccharides. Generally, physical and/or thermochemical pretreatments are performed to enable access for the subsequent added carbohydrate-degrading enzymes. Nevertheless, partly substituted xylan structures remain after pretreatment, in particular the ones substituted with (4-O-methyl-) glucuronic acids (UA(me)). Hence, alpha-glucuronidases play an important role in the degradation of UA(me)xylan structures facilitating the complete utilisation of plant biomass. The characterisation of alpha-glucuronidases is a necessity to find the right enzymes to improve degradation of recalcitrant UA(me)xylan structures. Results: The mode-of-action of two alpha-glucuronidases was demonstrated, both obtained from the fungus Rasamsonia emersonii; one belonging to the glycoside hydrolase (GH) family 67 (ReGH67) and the other to GH115 (ReGH115). Both enzymes functioned optimal at around pH 4 and 70 degrees C. ReGH67 was able to release UA(me) from UA(me)-substituted xylo-oligosaccharides (UA(me)XOS), but only the UA(me) linked to the non-reducing end xylosyl residue was cleaved. In particular, in a mixture of oligosaccharides, UA(me)XOS having a degree of polymerisation (DP) of two were hydrolysed to a further extent than longer UA(me)XOS (DP 3-4). On the contrary, ReGH115 was able to release UA(me) from both polymeric UA(me)xylan and UA(me)XOS. ReGH115 cleaved UA(me) from both internal and non-reducing end xylosyl residues, with the exception of UA(me) attached to the non-reducing end of a xylotriose oligosaccharide. Conclusion: In this research, and for the first time, we define the mode-of-action of two alpha-glucuronidases from two different GH families both from the ascomycete R. emersonii. To date, only four alpha-glucuronidases classified in GH115 are characterised. ReGH67 showed limited substrate specificity towards only UA(me)XOS, cleaving UA(me) only when attached to the non-reducing end xylosyl residue. ReGH115 was much less substrate specific compared to ReGH67, because UA(me) was released from both polymeric UA(me)xylan and UA(me)XOS, from both internal and non-reducing end xylosyl residues. The characterisation of the mode-of-action of these two alpha-glucuronidases helps understand how R. emersonii attacks UA(me)xylan in plant biomass and the knowledge presented is valuable to improve enzyme cocktails for biorefinery applications.