A new synergistic relationship between xylan-active LPMO and xylobiohydrolase to tackle recalcitrant xylan

Hemicellulose accounts for a significant part of plant biomass, and still poses a barrier to the efficient saccharification of lignocellulose. The recalcitrant part of hemicellulose is a serious impediment to the action of cellulases, despite the use of xylanases in the cellulolytic cocktail mixtures. However, the complexity and variety of hemicelluloses in different plant materials require the use of highly specific enzymes for a complete breakdown. Over the last few years, new fungal enzymes with novel activities on hemicelluloses have emerged. In the present study, we explored the synergistic relationships of the xylan-active AA14 lytic polysaccharide monooxygenase (LPMO), PcAA14B, with the recently discovered glucuronoxylan-specific xylanase TtXyn30A, of the (sub)family GH30_7, displaying xylobiohydrolase activity, and with commercial cellobiohydrolases, on pretreated natural lignocellulosic substrates.

PcAA14B and TtXyn30A showed a strong synergistic interaction on the degradation of the recalcitrant part of xylan. PcAA14B was able to increase the release of xylobiose from TtXyn30A, showing a degree of synergism (DS) of 3.8 on birchwood cellulosic fibers, and up to 5.7 on pretreated beechwood substrates. The increase in activity was dose- and time- dependent. A screening study on beechwood materials pretreated with different methods showed that the effect of the PcAA14B-TtXyn30A synergism was more prominent on substrates with low hemicellulose content, indicating that PcAA14B is mainly active on the recalcitrant part of xylan, which is in close proximity to the underlying cellulose fibers. Simultaneous addition of both enzymes resulted in higher DS than sequential addition. Moreover, PcAA14B was found to enhance cellobiose release from cellobiohydrolases during hydrolysis of pretreated lignocellulosic substrates, as well as microcrystalline cellulose.

The results of the present study revealed a new synergistic relationship not only among two recently discovered xylan-active enzymes, the LPMO PcAA14B, and the GH30_7 glucuronoxylan-active xylobiohydrolase TtXyn30A, but also among PcAA14B and cellobiohydrolases. We hypothesize that PcAA14B creates free ends in the xylan polymer, which can be used as targets for the action of TtXyn30A. The results are of special importance for the design of next-generation enzymatic cocktails, able to efficiently remove hemicelluloses, allowing complete saccharification of cellulose in plant biomass.