SANS investigation of fungal loosenins reveal substrate dependent impacts of protein action on inter-fibril distance and packing order of cellulosic substrates.

Background: Microbial expansin-related proteins include fungal loosenins, which have been previously shown to disrupt cellulose networks and enhance the enzymatic conversion of cellulosic substrates. Despite showing beneficial impacts to cellulose processing, detailed characterization of cellulosic materials after loosenin treatment is lacking. In this study, small-angle neutron scattering (SANS) was used to investigate the effects of three recombinantly produced loosenins that originate from Phanerochaete carnosa, PcaLOOL7, PcaLOOL9, and PcaLOOL12, on the organization of holocellulose preparations from Eucalyptus and Spruce wood samples. Results: Whereas the SANS analysis of Spruce holocellulose revealed an increase in interfibril spacing of neighboring cellulose microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL7, the analysis of Eucalyptus holocellulose revealed a reduction in packing number following treatment with PcaLOOL12 and to a lesser extent PcaLOOL9. Parallel SEC-SAXS characterization of PcaLOOL7, PcaLOOL9, and PcaLOOL12 indicated the proteins likely function as monomers; moreover, all appear to retain a flexible disordered N-terminus and folded C-terminal region. The comparatively high impact of PcaLOOL12 motivated its NMR structural characterization, revealing a double-psi ß-barrel (DPBB) domain surrounded by three α-helices - the largest nestled against the DPBB core and the other two part of loops extending from the core. Conclusions: The SANS analysis of PcaLOOL action on holocellulose samples confirms their ability to disrupt cellulose fiber networks and suggests a progression from reducing microfibril packing to increasing interfibril distance. The most impactful PcaLOOL, PcaLOOL12, was previously observed to be the most highly expressed loosenin in P. carnosa. Its structural characterization herein reveals its stabilization through two disulfide linkages, and an extended N-terminal region distal to a negatively charged and surface accessible polysaccharide binding groove.

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.