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.

中文翻译：

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木聚糖活性 LPMO 和木糖二糖水解酶之间新的协同关系以解决顽固的木聚糖。

半纤维素占植物生物质的重要部分，并且仍然对木质纤维素的有效糖化构成障碍。尽管在纤维素分解混合物混合物中使用了木聚糖酶，但半纤维素的顽固部分是纤维素酶作用的严重障碍。然而，不同植物材料中半纤维素的复杂性和多样性需要使用高度特异性的酶才能完全分解。在过去几年中，出现了对半纤维素具有新活性的新型真菌酶。在本研究中，我们探索了木聚糖活性 AA14 裂解多糖单加氧酶 (LPMO) PcAA14B 与最近发现的（亚）家族 GH30_7 的葡糖醛酸木聚糖特异性木聚糖酶 TtXyn30A 的协同关系，显示木二糖水解酶活性，和商业纤维二糖水解酶，在预处理的天然木质纤维素基质上。PcAA14B 和 TtXyn30A 对木聚糖顽固部分的降解表现出强烈的协同作用。PcAA14B 能够增加 TtXyn30A 中木二糖的释放，在桦木纤维素纤维上的协同作用程度 (DS) 为 3.8，在预处理的山毛榉基材上高达 5.7。活性的增加是剂量和时间依赖性的。对用不同方法预处理的榉木材料进行的筛选研究表明，PcAA14B-TtXyn30A 协同作用在低半纤维素含量的底物上更显着，表明 PcAA14B 主要作用于木聚糖的顽固部分，该部分与木聚糖非常接近。底层的纤维素纤维。同时添加两种酶导致比顺序添加更高的DS。此外，发现 PcAA14B 在预处理的木质纤维素底物以及微晶纤维素的水解过程中增强了纤维二糖水解酶的纤维二糖释放。本研究的结果揭示了一种新的协同关系，不仅在最近发现的两种木聚糖活性酶 LPMO PcAA14B 和 GH30_7 葡糖醛木聚糖活性木二糖水解酶 TtXyn30A 之间，而且在 PcAA14B 和纤维二糖水解酶之间。我们假设 PcAA14B 在木聚糖聚合物中产生自由末端，可用作 TtXyn30A 作用的目标。该结果对于下一代酶混合物的设计特别重要，能够有效去除半纤维素，使植物生物质中的纤维素完全糖化。