Fungal loosenin-like proteins boost the cellulolytic enzyme conversion of pretreated wood fiber and cellulosic pulps.

Microbial expansin-related proteins, including loosenins, can disrupt cellulose networks and increase enzyme accessibility to cellulosic substrates. Herein, four loosenins from Phanerochaete carnosa (PcaLOOLs), and a PcaLOOL fused to a family 63 carbohydrate-binding module, were compared for ability to boost the cellulolytic deconstruction of steam pretreated softwood (SSW) and kraft pulps from softwood (ND-BSKP) and hardwood (ND-BHKP). Amending the Cellic® CTec-2 cellulase cocktail with PcaLOOLs increased reducing products from SSW by up to 40 %, corresponding to 28 % higher glucose yield. Amending Cellic® CTec-2 with PcaLOOLs also increased the release of glucose from ND-BSKP and ND-BHKP by 82 % and 28 %, respectively. Xylose release from ND-BSKP and ND-BHKP increased by 47 % and 57 %, respectively, highlighting the potential of PcaLOOLs to enhance hemicellulose recovery. Scanning electron microscopy and fiber image analysis revealed fibrillation and curlation of ND-BSKP after PcaLOOL treatment, consistent with increasing enzyme accessibility to targeted substrates.

Loosenin-Like Proteins from Phanerochaete carnosa Impact Both Cellulose and Chitin Fiber Networks.

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. IMPORTANCE 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.

PACER: a novel 3D plant cell wall model for the analysis of non-catalytic and enzymatic responses.

BACKGROUND: Substrate accessibility remains a key limitation to the efficient enzymatic deconstruction of lignocellulosic biomass. Limited substrate accessibility is often addressed by increasing enzyme loading, which increases process and product costs. Alternatively, considerable efforts are underway world-wide to identify amorphogenesis-inducing proteins and protein domains that increase the accessibility of carbohydrate-active enzymes to targeted lignocellulose components. RESULTS: We established a three-dimensional assay, PACER (plant cell wall model for the analysis of non-catalytic and enzymatic responses), that enables analysis of enzyme migration through defined lignocellulose composites. A cellulose/azo-xylan composite was made to demonstrate the PACER concept and then used to test the migration and activity of multiple xylanolytic enzymes. In addition to non-catalytic domains of xylanases, the potential of loosenin-like proteins to boost xylanase migration through cellulose/azo-xylan composites was observed. CONCLUSIONS: The PACER assay is inexpensive and parallelizable, suitable for screening proteins for ability to increase enzyme accessibility to lignocellulose substrates. Using the PACER assay, we visualized the impact of xylan-binding modules and loosenin-like proteins on xylanase mobility and access to targeted substrates. Given the flexibility to use different composite materials, the PACER assay presents a versatile platform to study impacts of lignocellulose components on enzyme access to targeted substrates.