Department of Bioproducts and Biosystems, Aalto University, Espoo, Finland.
Department of Food and Nutrition, University of Helsinki, Helsinki, Finland.
Appl Environ Microbiol. 2023 Jan 31;89(1):e0186322. doi: 10.1128/aem.01863-22. Epub 2023 Jan 16.
Microbial expansin-related proteins are ubiquitous across bacterial and fungal organisms and reportedly play a role in the modification and deconstruction of cell wall polysaccharides, including lignocellulose. So far, very few microbial expansin-related proteins, including loosenins and loosenin-like (LOOL) proteins, have been functionally characterized. Herein, four LOOLs encoded by Phanerochaete carnosa and belonging to different subfamilies (i.e., PcaLOOL7 and PcaLOOL9 from subfamily A and PcaLOOL2 and PcaLOOL12 from subfamily B) were recombinantly produced and the purified proteins were characterized using diverse cellulose and chitin substrates. The purified PcaLOOLs weakened cellulose filter paper and cellulose nanofibril networks (CNF); however, none significantly boosted cellulase activity on the selected cellulose substrates (Avicel and Whatman paper). Although fusing the family 63 carbohydrate-binding module (CBM63) of BsEXLX1 encoded by Bacillus subtilis to PcaLOOLs increased their binding to cellulose, the CBM63 fusion appeared to reduce the cellulose filter paper weakening observed using wild-type proteins. Binding of PcaLOOLs to alpha-chitin was considerably higher than that to cellulose (Avicel) and was pH dependent, with the highest binding at pH 5.0. Amendment of certain PcaLOOLs in fungal liquid cultivations also impacted the density of the cultivated mycelia. The present study reveals the potential of fungal expansin-related proteins to impact both cellulose and chitin networks and points to a possible biological role in fungal cell wall processing. The present study deepens investigations of microbial expansin-related proteins and their applied significance by (i) reporting a detailed comparison of diverse loosenins encoded by the same organism, (ii) considering both cellulosic and chitin-containing materials as targeted substrates, and (iii) investigating the impact of the C-terminal carbohydrate binding module (CBM) present in other expansin-related proteins on loosenin function. By revealing the potential of fungal loosenins to impact both cellulose and chitin-containing networks, our study reveals a possible biological and applied role of loosenins in fungal cell wall processing.
微生物扩展蛋白相关蛋白在细菌和真菌生物中普遍存在，据报道，它们在细胞壁多糖（包括木质纤维素）的修饰和解构中发挥作用。到目前为止，只有很少的微生物扩展蛋白相关蛋白，包括松弛素和松弛素样（LOOL）蛋白，被功能表征。本文中，从 Phanerochaete carnosa 中编码的四个属于不同亚科的 LOOL（即 A 亚科的 PcaLOOL7 和 PcaLOOL9 以及 B 亚科的 PcaLOOL2 和 PcaLOOL12）被重组产生，并使用各种纤维素和几丁质底物对纯化的蛋白质进行了表征。纯化的 PcaLOOL 削弱了纤维素滤纸和纤维素纳米纤维网络（CNF）；然而，在所选纤维素底物（Avicel 和 Whatman 纸）上，它们都没有显著促进纤维素酶的活性。尽管将枯草芽孢杆菌编码的家族 63 碳水化合物结合模块（CBM63）融合到 PcaLOOL 中增加了它们与纤维素的结合，但 CBM63 融合似乎减少了野生型蛋白观察到的纤维素滤纸弱化。PcaLOOL 与 alpha-几丁质的结合明显高于与纤维素（Avicel）的结合，并且依赖于 pH 值，在 pH5.0 时结合最高。在真菌液体培养中添加某些 PcaLOOL 也会影响培养的菌丝体密度。本研究揭示了真菌扩展蛋白相关蛋白对纤维素和几丁质网络的潜在影响，并指出了其在真菌细胞壁处理中的可能生物学作用。本研究通过（i）报告同一生物体编码的不同松弛素的详细比较，（ii）考虑纤维素和含几丁质的材料作为靶向底物，以及（iii）研究其他扩展蛋白相关蛋白中存在的 C 端碳水化合物结合模块（CBM）对松弛素功能的影响，加深了对微生物扩展蛋白相关蛋白及其应用意义的研究。通过揭示真菌松弛素对纤维素和含几丁质网络的潜在影响，我们的研究揭示了松弛素在真菌细胞壁处理中的可能生物学和应用作用。
