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

by 82 % and 28 %, respectively. Xylose release from ND-BSKP and ND-BHKP increased by 47 % and 57 %, respectively, highlighting the potential of Pca LOOLs to enhance hemicellulose recovery. Scanning electron microscopy and fiber image analysis revealed fibrillation and curlation of ND-BSKP after Pca LOOL treatment, consistent with increasing enzyme accessibility to targeted substrates.


Introduction
Plant fiber (i.e., lignocellulose) is largely comprised of cellulose, hemicellulose and lignin, and is a major feedstock for the production of renewable fuels and chemicals that reduce societal reliance on fossil resources.The evolved functional resilience of lignocellulose, however, presents a challenge to cost-effective enzymatic conversion (Himmel et al., 2007).In nature, lignocellulose deconstruction is achieved through the concerted action of multiple carbohydrate-active enzymes secreted by diverse microorganisms.These enzymes are classified in the carbohydrate-active enzyme (CAZy) database and include glycoside hydrolases as well as auxiliary activities that display cellulolytic and hemicellulolytic activities (Drula et al., 2022).Despite major improvements to corresponding enzyme cocktails (Lopes et al., 2018) and lignocellulose pretreatment (Mankar et al., 2021), enzyme cost remains a barrier to the economic feasibility of lignocellulose conversion to fuels and commodity chemicals (Ferreira et al., 2021).Accordingly, there is considerable interest to identify additional protein families that enhance enzyme access to targeted lignocellulosic substrates (Kim et al., 2014).One such class of proteins with reported amorphogenesis activity on cellulosic substrates are the expansins and expansin-related proteins (Arantes and Saddler, 2010;Cosgrove, 2000;Georgelis et al., 2015;Sampedro and Cosgrove, 2005).
The potential of microbial expansin-related proteins to boost enzymatic deconstruction of lignocellulosic substrates was predicted based on their observed impacts to cellulosic fiber.For example, swollen areas were observed by light microscopy in mercerized cotton fiber after sonication and treatment with SWO1 (Saloheimo et al., 2002) and the loosenin LOOS1 from Bjerkandera adusta (Quiroz-Castañeda et al., 2011).Moreover, light microscopy images of microcrystalline cellulose (Avicel) treated with the AfSWO swollenin from Aspergillus fumigatus show a reduction in cellulose particle size (Chen et al., 2010).Cellulose filter paper weakening has also been reported for several microbial expansin-related proteins, including recombinant SWO1 (Jäger et al., 2011), BsEXLX1 from Bacillus subtilis (Kim et al., 2009), and loosenins from Phanerochaete carnosa (Monschein et al., 2023).In practice, the impact of expansin-related proteins on enzymatic deconstruction of lignocellulose varies depending on several factors, including substrate, reaction time, expansin dose, the enzyme dosage and enzyme system used (Baker et al., 2000;Kim et al., 2014;Liu et al., 2015).For example, SWO1 enhances cellulase treatment of native grass substrates but not microcrystalline cellulose (Avicel) (Eibinger et al., 2016) and enhances xylanase treatment of steam pretreated corn stover over cellulase treatment of the same substrate (Gourlay et al., 2013).Moreover, HcEXLX2 from Hahella chejuensis and BsEXLX1 enhance the enzymatic release of soluble sugars from cellulose filter paper at low cellulase loadings (e.g., 0.06 FPU/ g cellulose), but the gains are considerably reduced at higher cellulase loadings (Kim et al., 2009;Lee et al., 2010).In the case of SWO2 from Trichoderma pseudokoningii and ScExlx1 from Schizophylum commune, pretreatment of the cellulose substrate with the expansin-related protein prior to the addition of cellulase maximized cellulose hydrolysis (Tovar-Herrera et al., 2015;Zhou et al., 2011).
Four expansin-related proteins from Phanerochaete carnosa known as loosenin-like proteins (PcaLOOLs) were previously recombinantly expressed and functionally characterized (Monschein et al., 2023).The PcaLOOLs were shown to weaken cellulose filter paper and reduce the yield strain of cellulose nanofibers; however, none of the four PcaLOOLs appeared to boost cellulase hydrolysis of microcrystalline cellulose (Avicel) or filter paper.Herein, the four PcaLOOLs along with a Pca-LOOL-CBM63 fusion are evaluated for their potential to enhance lytic enzyme action on lignocellulosic substrates, including steam pretreated wood fiber and pulps that lack lignin but retain hemicelluloses.The results underscore the importance of including complex lignocellulosic substrates to reveal synergistic impacts between expansin-related proteins and carbohydrate-active enzymes.
The PcaLOOL12-CBM63 fusion protein comprised a PcaLOOL12 at the N-terminus and CBM63 from BsEXLX1 at the C-terminus of the protein; the two domains were connected by the linker sequence taken from BsEXLX1 including two last amino acids from BsEXLX1 domain 1 (Monschein et al., 2023).PcaLOOL12-CBM63 was recombinantly produced in P. pastoris as previously reported (Monschein et al., 2023).
CBM63 from BsEXLX1 was produced in Escherichia coli strain BL21 (DE3) from the codon optimized gene subcloned to pET21a(+) plasmid.The amino acid sequence and the production and purification of CBM63 is described in Monschein et al. (2023).
In all cases, protein purity was verified by SDS-PAGE before flash freezing and storing the protein at −80 • C. Protein concentration was determined by measuring absorption at 280 nm (Thermofischer Scientific NanoDrop™ Lite Spectrophotometer).

Bioreactor production of PcaLOOLs
In an effort to increase protein yield, PcaLOOL7, PcaLOOL9 and PcaLOOL12 were also produced in a 7 L Sterilizable-In-Place (SIP) Fermenter (BIOSTAT® Cplus from Sartorius).The reactor was fitted with pH (Article no.BB-34090812), oxygen (Article no.BB-8848663) and turbidity HAMILTON probes from Sartorius, and all probes were calibrated using manufacturer's instructions prior to initiating the cultivation.
The preculture for the bioreactor was prepared by transferring fresh colonies of each transformant into 300 mL BMGY and growing the culture at • C and 180 rpm for 16-24 h until the OD 600nm reached between 20 and 25.The cells were then centrifuged and suspended in 50 mL of sterile minimal basal salts media (BSM) under the laminar hood.
The production process in the bioreactor was started with Glycerol Batch Phase (GBP) containing 4 % (w/v) glycerol as carbon source and 0.4 % (v/v) PTM 1 trace salts in 3.5 L BSM.The media in the bioreactor was inoculated with the resuspended cell pellet.Media and trace salt preparations were carried out in accordance with the manufacturer's guidelines for Pichia Fermentation (Invitrogen™, Thermo Fisher Scientific).The bioreactor cultivation was performed at 30 • C and the pH was maintained at pH 6.0 throughout the productions.The air flow rate in the bioreactor was set to 40-60 % to maintain the volume of air per volume of liquid per minute (VVM) between 0.5 and 1.0.The oxygen level was set to 35 % by putting an automatic cascade with stirring and gas flow using a partial oxygen controller that works on the Proportional-Integral-Derivative (PID) controller function in the bioreactor.The maximum stirring rate was 900 rpm with a flat six-blade Rushton turbine type impeller.
The GBP was completed in 4-6 h after which the Glycerol Fed-Batch Phase (GFBP) was initiated where up to 4 % (w/v of media) glycerol was fed to the culture over 5-6 h.This was done to achieve higher cell density.Following the GFBP and a resting phase with no added carbon for 3 h, the remaining glycerol was consumed and the Methanol Fed-Batch Phase (MFBP) was initiated to induce recombinant protein production.The temperature of the MFBP phase was reduced to 20 • C. Methanol was added at up to 6.5 mL/ h per liter of media for up to 110 h.The pH of all the productions were maintained with 14 % NH 4 OH.An antifoaming agent, Struktol J 647, was used as needed to prevent the formation of foam during fermentation.The pH of the harvested culture was adjusted to pH 7.8 using 4 M NaOH, centrifuged to remove cells, and the recovered supernatant was filtered through a 0.45 µm PES membrane before the recombinant protein was purified using Ni-NTA resin as previously described (Monschein et al., 2023).

Circular dichroism (CD)
The purified PcaLOOL proteins were adjusted to 0.3 mg/mL using 20 mM sodium acetate (pH 6.0) and 200 µL was added to a 1 mm high transparency rectangular quartz cell under constant nitrogen purge.The absorbance spectra were obtained using a J-1500-150ST (JASCO) at wavelengths between 170 and 290 nm.This was repeated for protein samples incubated at 40 • C and 50 • C and 1000 rpm for 24 h in a thermoshaker.Three scans were obtained for each sample and baseline measurements were performed using the 20 mM sodium acetate buffer alone using default parameters at 25 • C. The JASCO Spectra Manager software was used for data analysis.

Impact of PcaLOOLs and appended CBM63 on the enzymatic hydrolysis of cellulosic substrates
A master stock of SSW substrate was prepared by directly suspending the pretreated material in 200 mM potassium phosphate (K-P) buffer (pH 5.4) to 14 % w/v.To evaluate practical benefits of reactions amended with PcaLOOLs, the SSW substrate not washed or otherwise further processed before being used in the experiments.The substrate suspension was mixed using a magnetic stirrer for 2 h and the dry content of the resulting substrate stock was measured after overnight incubation at 105 ℃.Enzymatic treatments were performed in 2 mL Eppendorf® Protein LoBind tubes from Sigma Aldrich in a total reaction volume of 1 mL.Triplicate reactions comprised 7.5 % w/v SSW in 100 mM K-P buffer (final concentration) and were initiated by adding Cel-lic® CTec-2 (2 % w/w substrate) alone and in addition to each PcaLOOL (1 % w/w substrate).Reactions were incubated up to 24 h at 40 ℃ using a thermo-shaker set to 1000 rpm.
Enzymatic treatments of ND-BSKP and ND-BHKP substrates were similarly performed in 2 mL Eppendorf® Protein LoBind tubes in a total reaction volume of 1 mL.Triplicate reactions comprised 2.0 % w/v pulp in 50 mM sodium acetate (pH 5.3) and were initiated by adding Cellic® CTec-2 (0.5 % w/w substrate) alone and in the presence of each Pca-LOOL (0.5 % w/w substrate), as well as PcaLOOL12-CBM63 or CBM63 (8.8 μM final concentrations, equivalent to 0.5 % of the wild-type Pca-LOOL12).Reactions were incubated for up to 24 h at 40 ℃ using a thermo-shaker set to 1000 rpm.Notably, the pulp consistency was retained at 2.0 % w/v because higher consistencies were too viscous to permit efficient shaking.
In all cases, the Cellic® CTec-2 loading was adjusted so as not to achieve maximum conversion over the course of the reaction.Aliquots (100 µL) were recovered from each reaction at 0 h, 4 h, 8 h and 24 h, and centrifuged before measuring sugars released to the supernatant using the 4-hydroxybenzoic acid hydrazide (PAHBAH) assay (Lever, 1972).The 96-well microtiter plate containing the samples and assay reagent was incubated at 70 • C for 30 min after which it was cooled to room temperature prior to the analysis.The reaction between the PAHBAH reagent and total reducing sugars was then measured at 410 nm using an Eon Microtiter plate reader from BioTek.
For monosaccharide analysis, high-performance anion-exchange chromatography equipped with pulsed amperometric detection (HPAEC-PAD) was used with a PA20 column.Ion Chromatograph was operated at a flow rate of 0.37 mL/min.The supernatants were diluted to 1:500 with MilliQ water and then heated to 95 • C for 15 min to denature the proteins present in the supernatant; once cool, the samples were passed through 0.45 µm filters.Arabinose, rhamnose, galactose, glucose, xylose and mannose were used as standards at concentrations between 1 and 50 mg/L.Triplicate samples were used for the standard curve preparation and further quantification of dissolved monosaccharides in the samples.

Scanning electron microscopy (SEM) of kraft pulps
Approximately 22 mg (dry mass) of ND-BSKP and ND-BHKP D. Dahiya et al. substrates were suspended in 1 mL 50 mM sodium acetate (pH 5.3) (2.0 % w/v final pulp fiber consistency) and separately treated with 0.5 % (w/w substrate) of each PcaLOOL for 24 h at 40 • C. The treated fibers were rinsed with MilliQ water at least five times to neutralize the pH and remove bound protein.A Zeiss Sigma VP SEM equipped with a Schottky FEG emitter was used to collect the images.The acceleration voltage was 2 kV with a current of 60 pA.The wet samples were held in aluminium SEM sample stubs with double sided carbon tape and air-dried in the fumehood overnight.The samples were sputter coated using EM ACE200, Leica, Germany with 3 nm of Au/Pd prior to SEM imaging.

Fiber morphology using a fiber image Analyser (FIA)
The impact of PcaLOOL treatment on ND-BSKP and ND-BHKP on fiber length, width, extent of fibrillation and percent fines were measured using a Valmet Fiber Image Analyser FS5.Reactions (1 mL for ND-BHKP and 2 mL for ND-BSKP) were performed in triplicates in 15 mL round bottom Falcon tubes and comprised 50 mM sodium acetate (pH 5.3) amended with 40 mg ND-BSKP or 20 mg ND-BHKP, and 0.5 % w/w or 5.0 % w/w PcaLOOL12.Different dry mass values were chosen based on recommendations from the device manufacturer; in both cases, the pulp fiber consistency was 2 % w/v.Reactions were incubated for 24 h at 40 • C with shaking at 1000 rpm and then washed five times with MilliQ water.The fiber consistency was adjusted to 13-18 mg/L using MilliQ water before the fiber measurements were collected in triplicate.

Protein production and thermal stability
All four PcaLOOLs were initially produced in 2 L non-baffled Erlenmeyer flasks at pH 6.0, leading to approximately 70 mg/ L, 13 mg/ L, 12 mg/ L and 16 mg/ L protein yield for PcaLOOL2, PcaLOOL7, PcaLOOL9 and PcaLOOL12 respectively.In an effort to increase the yield of Pca-LOOL7, PcaLOOL9 and PcaLOOL12, these recombinant proteins were also produced using a 7 L bioreactor.In all cases, the protein yields increased in comparison to shake flask productions, generating 110 mg/ L PcaLOOL7, 479 mg/ L PcaLOOL9 and 386 mg/ L PcaLOOL12; accordingly, the corresponding proteins were used for functional characterizations.
The stability of each PcaLOOL at 40 • C and 50 • C was evaluated by circular dichroism; these temperatures were chosen based on the preferred temperature range of the Cellic® CTec-2 enzyme cocktail used in subsequent synergism studies.At 40 • C, all four PcaLOOLs retained their expected DPBB folding pattern with β-sheet-rich structures (Fig. 1) (Monschein et al., 2023).By contrast, a change in ellipticity values was observed indicative of protein unfolding at 50 • C for 24 h (Fig. 1).Accordingly, all subsequent reactions with lignocellulosic substrates were performed at 40 • C.

Impact of PcaLOOLs on the enzymatic deconstruction of lignocellulosic substrates
All four PcaLOOLs boosted the enzymatic conversion of steamexploded softwood (SSW) by Cellic® CTec-2, albeit to different extents (Fig. 2).After 24 h, the highest yield of total reducing products released by Cellic® CTec-2 were reached in reactions comprising PcaLOOL12, where the increase relative to Cellic® CTec-2 alone was approximately 40 % (Fig. 2a).More specifically, compositional analyses of the enzymatic products confirmed higher glucose content in Cellic® CTec-2 reactions amended with PcaLOOLs, especially PcaLOOL12 where glucose content increased by 28 % after 24 h (Fig. 2b).Besides glucose, reactions amended with PcaLOOLs had up to 21 % higher mannose content; no significant change in product formation was observed for other monosaccharides.
Whereas reducing end measurements (Fig. 2a) and monosaccharide analyses (Fig. 2b) consistently showed higher product release from SSW by Cellic® CTec-2 amended with PcaLOOL9 and PcaLOOL12, the results were less consistent in Cellic® CTec-2 reactions amended with Pca-LOOL2 and PcaLOOL7.It is conceivable the early increase in reducing products from enzyme reactions amended PcaLOOL2 compared to Cel-lic® CTec-2 alone is explained by the release of oligosaccharides not quantified in the monosaccharide analyses.On the other hand, the consistent benefit of PcaLOOL7 amendment on monosaccharide release by Cellic® CTec-2 was not reflected in the corresponding reducing end measurements, likely due to the lower sensitivity of the reducing end assay.Although the molecular basis for differences in PcaLOOL performance is still to be resolved, the consistent performance of PcaLOOL12 observed herein is congruent with previous studies that show higher filter paper weakening by PcaLOOL12 compared to PcaLOOL2 and PcaLOOL7 (Monschein et al., 2023).
Although PcaLOOLs boosted the cellulolytic conversion of SSW albeit to different extents, similar results were not observed when previously using purified cellulose substrates (Monschein et al., 2023).The beneficial contribution of PcaLOOLs to the enzymatic conversion of comparatively complex lignocellulosic substrates is consistent with earlier studies that investigate the impact of substrate composition on synergism between microbial expansin-related proteins and lytic enzymes.For example, Lin et al. (2013) report a positive correlation between hemicellulose content and the synergistic activity of BsEXLX1 on cellulase deconstruction of diverse lignocellulosic substrates.Lin et al. (2013) also report a negative correlation between lignin content of the feedstock and the potential of BsEXLX1 to boost cellulolytic conversion of the substrate.The potential importance of hemicellulose content on the synergistic activity of microbial expansin-related proteins and lytic enzymes is further evidenced by the higher potential of SWO1 to boost xylanase over cellulase conversion of steam-pretreated corn stover (Gourlay et al., 2013).Specifically, xylose release from steam-pretreated corn stover increased more than 300 % when amending a xylanase with swollenin (SWO1) from T. reesei (Gourlay et al., 2013); SWO1 was also shown to increase xylanase hydrolysis of xylan in bleached eucalyptus kraft pulp by nearly 10 % (Cebreiros et al., 2021).Certainly, hydrolase dose also influences measured synergisms between microbial expansinrelated proteins and lytic enzymes.For example, BsEXLX1 and HcEXLX2 enhance the release of soluble sugars from cellulose filter paper at low cellulase loadings (e.g., 0.06 FPU/ g cellulose), but at higher cellulase loadings the measured synergy is considerably reduced (Kim et al., 2009;Lee et al., 2010).Still, despite impacts of reaction time, expansin dose, and lytic enzyme on the synergistic action of expansin-related proteins (Baker et al., 2000), comparatively high hemicellulose and low lignin contents in the substrate frequently correlate with synergism between expansin-related proteins and cellulolytic enzymes (Liu et al., 2015).
The differing impacts of PcaLOOLs on Cellic® CTec-2 performance omits explanations based on non-specific affects, such as reducing nonproductive binding of the hydrolytic enzymes to lignin present in the SSW substrate (Huang et al., 2022).Still, to evaluate whether PcaLOOLs boost Cellic® CTec-2 performance on cellulosic substrates lacking lignin, the enzyme treatments were repeated using never-dried bleached softwood kraft pulp (ND-BSKP) and hardwood kraft pulp (ND-BHKP).Besides lacking lignin, these substrates comprise different hemicellulose contents, where ND-BSKP is enriched in galactoglucomannan and to a lesser extent arabinoglucuronoxylan, and ND-BHKP is enriched in glucuronoxylan (see supplementary material).Amending Cellic® CTec-2 treatments of ND-BSKP and ND-BHKP with PcaLOOLs increased glucose yields after 24 h by up to 82 % and 28 %, respectively, indicating the PcaLOOLs benefit Cellic® CTec-2 activity beyond reducing enzyme binding to lignin (Fig. 3).The Cellic® CTec-2 enzyme cocktail comprises cellulolytic and xylanase activities (Sun et al., 2015), and differences in glucose yields measured after treating ND-BSKP and ND-BHKP with Fig. 2. Total reducing sugars (a) and monosaccharide release (b) from 7.5 % w/v steam pretreated softwood (SSW) by Cellic® CTec-2 (2 % w/w substrate) alone and in the presence of PcaLOOLs (1 % w/w/ substrate).Monosaccharide compositions were measured by HPAEC-PAD after 4 h, 8 h and 24 h at 40 • C. For each time point, treatments with PcaLOOL that show statistically significant difference in total monosaccharide amount from the treatment with Cellic® CTec-2 alone are indicated by one asterisk (p-value is less than 0.05), two asterisks (p-value is less than 0.001) or three asterisks (p-value is less than 0.0001).n = 3. Cellic® CTec-2 alone were not statistically significant.This suggests the cellulolytic enzymes in Cellic® CTec-2 similarly access the cellulosic fraction of the pulp substrates.In this case, the higher impact of Pca-LOOLs on enzymatic release of glucose from ND-BSKP could be explained by enhanced access of endoglucanases to both cellulose and galactoglucomannan components of ND-BSKP.Evidence that PcaLOOLs can enhance enzymatic access to hemicellulose components in cellulosic substrates is furthered by the higher xylose content in Cellic® CTec-2 reactions amended with PcaLOOLs, where the enzymatic release of xylose from ND-BSKP and ND-BHKP increased by up to 57 % and 47 %, respectively (Fig. 3).The potential of PcaLOOLs to increase the enzymatic hydrolysis of both cellulose and hemicelluloses is congruent with the earlier studies highlighted above that investigate the impact of microbial expansins and swollenins on the enzymatic conversion of lignocellulosic substrates.
Loosenins, including PcaLOOLs, lack the C-terminal CBM63 domain present in swollenins and other expansin-related proteins.The construction and testing of a PcaLOOL12 fusion to the CBM63 from BsEXLX1 was previously reported and showed that the addition of CBM63 enhanced PcaLOOL12 binding to cellulosic substrates but reduced the potential of PcaLOOL12 to loosen cellulose filter paper networks (Monschein et al., 2023).Herein, the PcaLOOL12-CBM63 fusion was evaluated for potential to boost Cellic® CTec-2 activity on ND-BSKP and ND-BHKP over the wild-type PcaLOOL12.Remarkably, fusing the CBM63 to PcaLOOL12 eliminated the beneficial impact of PcaLOOL12 on Cellic® CTec-2 hydrolysis of ND-BSKP (Fig. 3a), possibly through reducing the accessibility of PcaLOOL12 to cellulose fibers within the ND-BSKP substrate.Similar effects of the CBM63 fusion were observed after 4 h and 8 h of ND-BHKP treatment; however, the negative impacts of the CBM63 fusion were alleviated after 24 h (Fig. 3b).The benefical impact at 24 h of amending Cellic® CTec-2 with CBM63 alone was unexpected; however, family 1 cellulose binding modules (CBM1) have been reported to boost cellulase activities through disrupting cellulosic substrates.For example, ThCBM1 from Trichoderma harzianum was shown to boost cellulase action on filter paper in a dose dependent manner, which was explained by ThCBM1-induced disruption of the cellulose fiber network (Bernardes et al., 2019).Since the CBM63 from BsEXLX1 used herein preferentially binds xylans over cellulose (Georgelis et al., 2011;Wang et al., 2016), it is conceivable the presense of the CBM63 increases xylanase accessibility to the xylan component that is enriched in ND-BHKP compared to ND-BSKP.Notably, the beneficial impact at 24 h of treating ND-BHKP with Cellic® CTec-2 plus Pca-LOOL12-CBM63 was less than the combined benefit of amending Cel-lic® CTec-2 with PcaLOOL12 or CBM63 separately.This result underscores the lack of cooperativity between PcaLOOL12 and fused CBM63, similarly observed using the ND-BSKP as well as earlier studies that evaluated impacts of filter paper weakening (Monschein et al., 2023).

Impact of PcaLOOL12 on cellulose fiber morphology
The ability of wild-type PcaLOOLs to enhance the enzymatic hydrolysis ND-BSKP and to a lesser extent ND-BHKP could be explained by their ability to disrupt cellulosic networks and thereby increase enzyme access to targeted substrates.To investigate this possibility, fiber morphology was imaged by SEM following the treatment of ND-BSKP and ND-BHKP with each PcaLOOL (see supplementary material).SEM images of ND-BSKP after treatment with PcaLOOL12 revealed clear evidence of fibrillation; this was less apparent for ND-BHKP.Morphological impacts of PcaLOOL12 to the pulp samples were then measured with a fiber image analyzer (FIA,Valmet FS5).Whereas the pulp preparations for SEM imaging used 0.5 % w/w PcaLOOL12, statistically significant impacts on fiber morphology could only be quantified by FIA when using 5.0 % w/w PcaLOOL12 (see supplementary material).
Treating ND-BSKP with 5 % w/w PcaLOOL12 increased the percent fibrillation and the percent fines of the pulp by approximately 40 % and 10 %, respectively.Whereas PcaLOOL12 treatments did not impact fiber length and only slightly reduced the width of ND-BSKP fiber, the Pca-LOOL12 treatment increased the percent curl by approximately 10 % while reducing fiber kinks (Table 1).Similarly, the ND-BHKP treatment with 5 % w/w PcaLOOL12 increased the percent fibrillation and curlation of the pulp by 21 % and 11 % respectively, without substantially by Cellic® CTec-2 (0.5 % w/w substrate) alone and in the presence of PcaLOOLs (0.5 % w/w substrate), PcaLOOL12-CBM63 (8.8 µM, equivalent to 0.5 % Pca-LOOL12) or CBM63 (8.8 µM).Monosaccharide compositions were measured by HPAEC-PAD after 4 h, 8 h and 24 h at 40 • C. For each time point, treatments with non-lytic proteins that show statistically significant difference in total monosaccharide amount from the treatment with Cellic® CTec-2 alone are indicated by one asterisk (p-value is less than 0.05), two asterisks (p-value is less than 0.001) or three asterisks (p-value is less than 0.0001).n = 3. impacting fiber length or width (Table 1).Notably, treating ND-BHKP with 5 % w/w PcaLOOL12 also increased fiber kinks by approximately 10 %.Fiber curls and kinks are examples of cellulose fiber deformations that can occur naturally and be induced through mechanical processing at high fiber consistency (Page et al., 1985).While curl describes the extent to which a fiber deviates from being straight, kink describes exceptionally sharp bends in the fiber structure (Hirn and Bauer, 2006;Page et al., 1985) and can include breaks in associated glucan chains (Ciesielski et al., 2019).Chandra et al (2019) show that inducing fiber curlation substantially increases cellulase hydrolysis of unbleached softwood kraft pulp (Chandra et al., 2019).Moreover, the impact of curlation on cellulose hydrolysis is retained after fiber straightening, suggesting lasting deformations in the fiber structure that increase enzyme accessibility (Chandra et al., 2019).The observation herein that PcaLOOL12 induces fiber curl and increases enzymatic hydrolysis of the pulp samples is thus consistent with earlier studies that induce fiber curl through mechanical treatments.A compelling explanation for the corresponding decrease in fiber kinks in ND-BSKP following PcaLOOL12 treatment is action of PcaLOOL12 at comparatively severe dislocation sites, potentially relaxing stresses in the hemicellulose-cellulose matrix.Studies that focus on localizing PcaLOOL12 and other expansin-related proteins in different cellulosic materials, including pretreated pulps, will be important to testing this possibility.
Observing different impacts of the same expansin-related protein on the morphology of different cellulosic substrates was similarly reported for SWO1 from T. reesei.For example, swelling of mercerized cotton fiber was observed by light microscopy following the cotton treatment with SWO1 from T. reesei (Saloheimo et al., 2002b).On the other hand, SEM analyses of filter paper (Jäger et al., 2011) and mercerized cotton (Gourlay et al., 2012) did not reveal fiber swelling after SWO1 treatment; instead, the SWO1 treatment reduced the particle size of filter paper cellulose (Jäger et al., 2011) and increased the surface smoothness of mercerized cotton fiber (Gourlay et al., 2012).The reported impact of substrate source and preparation on the performance of expanin-related proteins points to there being different and unknown substrate specificities within this protein family.

Conclusions
Four recombinantly produced loosenins from Phanerocheate carnosa were shown to boost the enzymatic conversion of lignocellulosic substrates.This result contrasts with earlier studies showing PcaLOOLs did not enhance enzymatic deconstruction of cellulosic substrates (Monschein et al., 2023) and reinforces the importance of both cellulose and hemicellulose in targeted substrates.Fusing a CBM63 to PcaLOOL12 reduced the potential of PcaLOOL12 to augment the enzymatic deconstruction of the pulps, and SEM and fiber image analysis of never-dried bleached kraft pulps revealed fibrillation and curlation of fibers following PcaLOOL12 treatment.These physical changes to the fiber morphology could explain their improved enzymatic processability.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 1 .
Fig. 1.Circular Dichroism (CD) spectra of the affinity purified PcaLOOLs [0.3 mg/ mL] at 40 • C and 50 • C. CD data were collected between 190 and 280 nm using a 0.1 cm path-length quartz cuvette.Raw data were averaged, smoothed and the buffer baseline was subtracted using JASCO spectra manager software.