Publications

Abstract

Loosenins are non-hydrolytic proteins that enhance cellulose accessibility by disrupting hydrogen bonding. This study investigates the effect of a novel fungal loosenin-like protein, PcaLOOL12, on never-dried hardwood kraft pulp fibers pre-treated by mechanical ball milling. Ball milling at varying energy levels (50–150 kWh/t) was used to open the fiber structure and improve protein accessibility. A treatment energy of 50 kWh/t was selected for subsequent experiments. Fiber morphology, water retention, and dissolution behavior were analyzed as a function of PcaLOOL12 dosage and incubation time. Mechanical treatment increased fiber width, fibrillation, and water retention, with minimal impact on fiber length or nanoscale porosity. PcaLOOL12 further enhanced fibrillation and water retention, particularly at higher dosages (5–10 wt%) and longer treatment times (24–72 h), suggesting surface-level delamination. No significant increase in nanoscale porosity was observed, indicating surface-specific action. CED-solubility decreased following protein treatment, possibly due to fibril aggregation or altered fiber–solvent interactions. These results demonstrate that combining mechanical and protein treatments can modulate fiber morphology and accessibility, supporting their use in bio-based product development.

https://doi.org/10.1016/j.indcrop.2025.122177

Abstract

Expansins loosen plant cell wall networks through disrupting non-covalent bonds between cellulose microfibrils and matrix polysaccharides. Whereas expansins were first discovered in plants, expansin-related proteins have since been identified in bacteria and fungi. The biological function of microbial expansins remains unclear; however, several studies have shown distinct binding preferences toward different structural polysaccharides. Earlier studies of bacterial expansin-related proteins uncovered sequence and structural features that correlate to substrate binding. Herein, 20 fungal expansin-related sequences were recombinantly produced in Komagataella phaffii, and the purified proteins were compared in terms of substrate binding to cellulosic and chitinous substrates. The impact of pH on the zeta potential of prioritized substrates was also measured, and Principal Component Analysis was performed to uncover correlations between protein characteristics (e.g., pI, hydrophobicity, surface charge distribution) and measured substrate binding preferences. Whereas acidic proteins with a predicted pI less than 5.0 preferentially bound to chitin, basic proteins with pI greater than 8.0 preferentially bound to xylan and xylan-containing fiber. Similar to many cellulases, binding to cellulose was correlated to relatively high aromatic amino acid content in the protein sequence and presence of a carbohydrate binding module (CBM), which in the case of expansins is a C-terminal CBM63. Whereas overall sequence characteristics could be correlated to substrate binding preference, the identity of amino acids occupying conserved positions that impact protein activity was better correlated with loosenin versus expansin classifications.

https://doi.org/10.1002/prot.70029

Abstract

Overcoming lignocellulose recalcitrance to enzymatic or chemical processing is a prerequisite for biorefinery applications. Expansins and loosenins are non-lytic proteins that could assist reducing this recalcitrance by disrupting the intermolecular contacts between plant cell wall components. Here, immunolocalization with fluorescence and transmission electron microscopy (TEM) were used to study the ability of a Bacillus subtilis expansin-like protein (BsEXLX1), a Phanerochaete carnosa loosenin protein (PcaLOOL12) and a fusion protein of PcaLOOL12 with the carbohydrate-binding module 63 (CBM63) of BsEXLX1 (i.e., PcaLOOL12-CBM63) to bind secondary cell walls (SCW) of aspen fibres, including fresh aspen wood, milled wood fibres (MWF) and MWF subjected to subcritical water extraction.

Results The immunofluorescence labelling of fresh wood samples showed a weak signal for PcaLOOL12 and a strong signal for BsEXLX1 and PcaLOOL12-CBM63, suggesting the importance of CBM63 for protein adsorption to SCW components. TEM analysis after immunogold labelling revealed the presence of BsEXLX1 and PcaLOOL12-CBM63 in all secondary cell wall layers. Pretreatment of wood samples with the proteins reduced the binding of glucomannan- and glucuronoxylan-specific monoclonal antibodies. Similarly, protein adsorption to MWF was higher before subcritical water extraction. Together, these results suggest the adsorption of BsEXLX1 and PcaLOOL12-CBM63 to SCWs was mediated at least in part by their interaction with hemicelluloses.

Conclusion Our study demonstrates that microbial expansin-related proteins can bind to the secondary walls of aspen wood through potential interaction of CBM63 with hemicelluloses.

https://doi.org/10.1186/s13068-025-02674-x

Abstract

The fabrication of cellulose-based barrier paper by coating base paper with regenerated cellulose is presented. Various concentrations of cellulose solutions and coating weights, akin to conventional polymeric coatings, are employed. The coated papers' properties, including morphological changes, mechanical properties, and barrier properties, are evaluated and compared to the reference base paper. It is observed that cellulose concentrations of 6 and 9 wt.% in the coating solution provide good paper coverage, evidenced by a reduction in porosity. The oxygen barrier properties surpass those typically achieved with laminated/extruded polyolefins or cellulose esters. Additionally, enhanced grease barrier properties against hot oil are attained. The tensile indices of the coated papers decrease, likely due to the wetting and redrying process during coating. However, the strain at break shows a slight improvement, and no significant difference is noted in the tear index compared to the reference.

https://doi.org/10.1002/mame.202500184

Abstract

Expansins and expansin-like proteins are found in plants and microbes, and can disrupt the cellulosic microfibril network of plant cell walls. While plant expansins play a role in cell wall formation, microbial expansin-like proteins reportedly enhance the activity of lignocellulolytic enzymes. Herein, two novel fungal expansin-like proteins, AmaEXLX1 from Allomyces macrogynus and ApuEXLX1 from Aureobasidium pullulans, were recombinantly produced in Pichia pastoris. While both AmaEXLX1 and ApuEXLX1 retain typical expansin structure, they share low sequence identity (22.5 %) and different predicted pI values (5.8 and 8.8, respectively), which was expected to impact their action on cellulosic substrates. Accordingly, adsorption of the proteins on cellulose nanofibrils (CNF) and the impact of the expansin-like proteins on the hydrolysis of CNF by an endoglucanase (Cel7B) was investigated using quartz crystal microbalance with dissipation (QCM-D). AmaEXLX1 showed higher affinity towards cellulose compared to ApuEXLX1, which was correlated to missing key aromatic residues in the polysaccharide binding surface of ApuEXLX1. The pretreatment of a CNF film with AmaEXLX1 and ApuEXLX1 increased the initial rate of Cel7B activity. This study underscores similarities between the impacts that bacterial and fungal expansin-like proteins can have on the enzymatic deconstruction of cellulose, and sequence properties that could impact expansin performance.

https://doi.org/10.1016/j.crbiot.2025.100296

Abstract

Whereas the enzymatic deconstruction of lignocellulosic materials is well established, comparatively few studies investigate the application of enzymes in bio-based material manufacturing. In this study, we demonstrate the potential of an ancestral endoglucanase from Bacillus subtilis (LFCA_EG) together with a loosenin from the white-rot fungus Phanerochaete carnosa (PcaLOOL12) to produce cellulose nanocrystals (CNC) with smaller size and higher yield than CNCs prepared using LFCA_EG alone. Moreover, CNCs prepared using both LFCA_EG and PcaLOOL12 retained the chemical purity, crystallinity, and thermal stability of previously described enzymatically prepared CNCs, and could be used to exfoliate graphite to generate low resistance, graphene-based conductive inks. Accordingly, this study highlights the potential of loosenins such as PcaLOOL12 to not only enhance the enzymatic deconstruction of lignocellulose but also the preparation of value-added cellulosic materials.

https://doi.org/10.1016/j.carbpol.2025.123469

Abstract

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 inter-microfibril spacing of neighboring cellulose microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL7, the analysis of Eucalyptus holocellulose revealed a reduction in the ordered arrangement of microfibrils 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 β-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.

Conclusion The SANS analysis of PcaLOOL action on holocellulose samples confirms their ability to disrupt cellulose fiber networks and suggests a progression from reducing regular order in the microfibril arrangement to increasing inter-microfibril spacing. 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.

https://doi.org/10.1186/s13068-025-02618-5

Abstract

The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs from hydrobacteria to reconstruct a common ancestor, ReChb, characterized by near-PAMless targeting and the recognition of diverse nucleic acid activators and collateral substrates. ReChb shares 53% sequence identity with the closest Cas12a ortholog but no longer requires a T-rich PAM and can achieve genome editing in human cells at sites inaccessible to the natural FnCas12a or the engineered and PAM-flexible enAsCas12a. Furthermore, ReChb can be triggered not only by double-stranded DNA but also by single-stranded RNA and DNA targets, leading to non-specific collateral cleavage of all three nucleic acid substrates with similar efficiencies. Finally, tertiary and quaternary structures of ReChb obtained by cryogenic electron microscopy reveal the molecular details underlying its expanded biophysical activities. Overall, ReChb expands the application space of Cas12a nucleases and underscores the potential of ASR for enhancing CRISPR technologies.

https://doi.org/10.1038/s41587-024-02461-3

Abstract

Microbial expansins (EXLXs) are non-lytic proteins homologous to plant expansins involved in plant cell wall formation. Due to their non-lytic cell wall loosening properties and potential to disaggregate cellulosic structures, there is considerable interest in exploring the ability of microbial expansins (EXLX) to assist the processing of cellulosic biomass for broader biotechnological applications. Herein, EXLXs with different modular structure and from diverse phylogenetic origin were compared in terms of ability to bind cellulosic, xylosic, and chitinous substrates, to structurally modify cellulosic fibrils, and to boost enzymatic deconstruction of hardwood pulp.

Results Five heterogeneously produced EXLXs (Clavibacter michiganensis; CmiEXLX2, Dickeya aquatica; DaqEXLX1, Xanthomonas sacchari; XsaEXLX1, Nothophytophthora sp.; NspEXLX1 and Phytophthora cactorum; PcaEXLX1) were shown to bind xylan and hardwood pulp at pH 5.5 and CmiEXLX2 (harboring a family-2 carbohydrate-binding module) also bound well to crystalline cellulose. Small-angle X-ray scattering revealed a 20–25% increase in interfibrillar distance between neighboring cellulose microfibrils following treatment with CmiEXLX2, DaqEXLX1, or NspEXLX1. Correspondingly, combining xylanase with CmiEXLX2 and DaqEXLX1 increased product yield from hardwood pulp by ~ 25%, while supplementing the TrAA9A LPMO from Trichoderma reesei with CmiEXLX2, DaqEXLX1, and NspEXLX1 increased total product yield by over 35%.

Conclusion This direct comparison of diverse EXLXs revealed consistent impacts on interfibrillar spacing of cellulose microfibers and performance of carbohydrate-active enzymes predicted to act on fiber surfaces. These findings uncover new possibilities to employ EXLXs in the creation of value-added materials from cellulosic biomass.

https://doi.org/10.1186/s13068-024-02500-w

Abstract

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.

https://doi.org/10.1016/j.biortech.2023.130188

Abstract

Cellulose activation is a necessary step in many industrial processes including production of cellulose derivatives, regenerated cellulose, biofuels and biochemicals. Expansins and expansin-related proteins have been shown to disrupt the fibrillar aggregation and loosen the structure of lignocellulosic materials but typically lack lytic activity. Therefore, they offer a new but rather unexploited possibility for biomass to obtain better accessibility and reactivity. From an applied perspective, expansin-related proteins have been investigated for their potential to promote enzymatic hydrolysis of cellulosic substrates for the purpose of producing biofuels. The aim of this review is to compare conventional and emerging technologies relevant to cellulose activation, and critically evaluate the potential of expansin-related proteins for this purpose. As part of this assessment, methods to evaluate the action of expansin-related proteins on cellulosic substrates are summarized, and reported impacts are discussed in relation to source of the cellulosic substrate and treatment conditions. An outlook on prospective applications of expansin-related proteins is presented.

https://doi.org/10.1007/s10570-023-05637-3

Abstract

Fast growing hardwoods are one of the major renewable resources available to produce bio-based materials, platform chemicals and biofuels. However, the industrial processing of lignocellulosic biomass is hindered by the complex molecular structure of the cell wall components and their supramolecular organization. This highlights the necessity of improving green processing strategies to enhance biomass conversion to valuable products from industrial wood production species. In the present study, we implemented a hydrothermal step by sequential subcritical water (SW) in aspen wood prior to saccharifi- cation and validated the process for trees grown in greenhouse and field conditions. Subcritical water enables extraction of non-cellulosic cell wall polysaccharides in native polymeric form. A major part of the pectic fraction was easily extracted within the first 10 min, while acetylated xylan was enriched in the subsequent extracts after 20- and 30-min rounds. Prolonged extraction (above 60 min) resulted in partial deacetylation and a reduction of the molar mass of xylan. The analysis of the residues enriched with cell- ulose and lignin showed several micromorphological changes caused by subcritical water treatment, such as an increased porosity, a loosening of the fibre matrix and a decrease in the macrofibrillar dimensions. These morphological and molecular changes in the organization of cell wall polymers after SW treatment significantly enhanced saccharification yields compared to those of non-treated aspen wood chips from both field and greenhouse conditions. Our study demonstrates that SW can be implemented as pretreat- ment prior to saccharification reducing the requirements for chemical acid pretreatments. This process enables the extraction of native non-cellulosic cell wall polymers for potential material applications and promotes the subsequent biochemical conversion of the residual biomass into fermentable sugars and platform chemicals in future biorefineries.

https://doi.org/10.1039/D3GC01020A

Abstract

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.

https://doi.org/10.1128/aem.01863-22

Abstract

Ultrafast folding proteins have become an important paradigm in the study of protein folding dynamics. Due to their low energetic barriers and fast kinetics, they are amenable for study by both experiment and simulation. However, single molecule force spectroscopy experiments on these systems are challenging as these proteins do not provide the mechanical fingerprints characteristic of more mechanically stable proteins, which makes it difficult to extract information about the folding dynamics of the molecule. Here, we investigate the unfolding of the ultrafast protein Engrailed Homeodomain (EnHD) by single-molecule atomic force microscopy experiments. Constant speed experiments on EnHD result in featureless transitions typical of compliant proteins. However, in the force-ramp mode we recover sigmoidal curves that we interpret as a very compliant protein that folds and unfolds many times over a marginal barrier. This is supported by a simple theoretical model and coarse-grained molecular simulations. Our results show the ability of force to modulate the unfolding dynamics of ultrafast folding proteins.

https://doi.org/10.1038/s42005-022-01125-5

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR–Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.

https://doi.org/10.1038/s41564-022-01265-y

Abstract

Numerous enzymes have the potential to upgrade biomass, converting it into high-tech materials for new applications. However, the features of natural enzymes often limit their use beyond chemical conversion of the substrate. The development of strategies for the enzymatic conversion of biomass into high-value materials may broaden the range of applications of enzymes and enzyme design techniques. A relevant case is lytic polysaccharide monooxygenase (LPMO), a class of enzymes that catalyzes the oxidative cleavage of glycosidic bonds. Here, we show that an ancestral LPMO can generate chitin nanocrystals. Physicochemical characterization of the chitin nanocrystals demonstrates modifications that make it superior compared to chitin obtained by chemical treatments. We show that the nanocrystals are suitable for controlled 2D and 3D cell cultures, as well as for engineering a biomatrix that combines with graphene oxide, forming a hybrid conductive bioink.

https://doi.org/10.1038/s43246-022-00277-9