Støpamo Fredrik G, Sulaeva Irina, Budischowsky David, Rahikainen Jenni, Marjamaa Kaisa, Kruus Kristiina, Potthast Antje, Eijsink Vincent G H, Várnai Anikó
Norwegian University of Life Sciences (NMBU), Ås, Norway.
University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.
Biotechnol Biofuels Bioprod. 2024 Aug 24;17(1):118. doi: 10.1186/s13068-024-02564-8.
In recent years, lytic polysaccharide monooxygenases (LPMOs) that oxidatively cleave cellulose have gained increasing attention in cellulose fiber modification. LPMOs are relatively small copper-dependent redox enzymes that occur as single domain proteins but may also contain an appended carbohydrate-binding module (CBM). Previous studies have indicated that the CBM "immobilizes" the LPMO on the substrate and thus leads to more localized oxidation of the fiber surface. Still, our understanding of how LPMOs and their CBMs modify cellulose fibers remains limited.
Here, we studied the impact of the CBM on the fiber-modifying properties of NcAA9C, a two-domain family AA9 LPMO from Neurospora crassa, using both biochemical methods as well as newly developed multistep fiber dissolution methods that allow mapping LPMO action across the fiber, from the fiber surface to the fiber core. The presence of the CBM in NcAA9C improved binding towards amorphous (PASC), natural (Cell I), and alkali-treated (Cell II) cellulose, and the CBM was essential for significant binding of the non-reduced LPMO to Cell I and Cell II. Substrate binding of the catalytic domain was promoted by reduction, allowing the truncated CBM-free NcAA9C to degrade Cell I and Cell II, albeit less efficiently and with more autocatalytic enzyme degradation compared to the full-length enzyme. The sequential dissolution analyses showed that cuts by the CBM-free enzyme are more evenly spread through the fiber compared to the CBM-containing full-length enzyme and showed that the truncated enzyme can penetrate deeper into the fiber, thus giving relatively more oxidation and cleavage in the fiber core.
These results demonstrate the capability of LPMOs to modify cellulose fibers from surface to core and reveal how variation in enzyme modularity can be used to generate varying cellulose-based materials. While the implications of these findings for LPMO-based cellulose fiber engineering remain to be explored, it is clear that the presence of a CBM is an important determinant of the three-dimensional distribution of oxidation sites in the fiber.
近年来，可氧化裂解纤维素的裂解多糖单加氧酶（LPMO）在纤维素纤维改性方面受到越来越多的关注。LPMO是相对较小的依赖铜的氧化还原酶，以单结构域蛋白形式存在，但也可能含有一个附加的碳水化合物结合模块（CBM）。先前的研究表明，CBM将LPMO“固定”在底物上，从而导致纤维表面更局部的氧化。然而，我们对LPMO及其CBM如何改性纤维素纤维的理解仍然有限。
在这里，我们使用生化方法以及新开发的多步纤维溶解方法，研究了CBM对来自粗糙脉孢菌的双结构域AA9家族LPMO NcAA9C的纤维改性特性的影响，该方法可以绘制LPMO从纤维表面到纤维核心在整个纤维上的作用。NcAA9C中CBM的存在改善了对无定形（PASC）、天然（纤维素I）和碱处理（纤维素II）纤维素的结合，并且CBM对于未还原的LPMO与纤维素I和纤维素II的显著结合至关重要。催化结构域的底物结合通过还原得到促进，使得截短的无CBM的NcAA9C能够降解纤维素I和纤维素II，尽管与全长酶相比效率较低且存在更多的自催化酶降解。顺序溶解分析表明，与含CBM的全长酶相比，无CBM的酶切割在纤维中分布更均匀，并且表明截短的酶可以更深入地渗透到纤维中，从而在纤维核心中产生相对更多的氧化和裂解。
这些结果证明了LPMO从表面到核心改性纤维素纤维的能力，并揭示了酶模块性的变化如何用于生成不同的纤维素基材料。虽然这些发现对基于LPMO的纤维素纤维工程的影响仍有待探索，但很明显，CBM的存在是纤维中氧化位点三维分布的重要决定因素。
