# Probing the colloidal behavior of a cell wall polysaccharides-degrading enzyme in a highly constrained model system

Hugo Voisin, Estelle Bonnin, Mélanie Marquis, Camille Alvarado, Suzanne Lafon, Teresa Lopez-Leon, Frédéric Jamme, Isabelle Capron

► To cite this version:

Hugo Voisin, Estelle Bonnin, Mélanie Marquis, Camille Alvarado, Suzanne Lafon, et al. Probing the colloidal behavior of a cell wall polysaccharides-degrading enzyme in a highly constrained model system. Journal of Colloid and Interface Science, 2025, 694, pp.137685. 10.1016/j.jcis.2025.137685. hal-05048803

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Regular Article

# Probing the colloidal behavior of a cell wall polysaccharides-degrading enzyme in a highly constrained model system

Hugo Voisin<sup>a, *</sup>, Estelle Bonnin<sup>a</sup>, Mélanie Marquis<sup>b, c</sup>, Camille Alvarado<sup>a</sup>, Suzanne Lafon<sup>c, d</sup>, Teresa Lopez-Leon<sup>c</sup>, Frédéric Jamme<sup>d</sup>, Isabelle Capron<sup>a</sup>

<figure><img src="image_2.png" /></figure>

HIGHLIGHTS

* Short XG chains adsorb flatly on cellu-
lose and are resistant to enzyme action.

* Covering of CNC hydrophobic plane with short XG prevent enzyme adsorption.

* This surface modification influences enzyme diffusion in concentrated media.

* These results in model confined condi-
tions may explain enzyme diffusion in
plants.

GRAPHICAL ABSTRACT

<figure><img src="image_3.png" /></figure>

ARTICLE INFO

Keywords:

Enzyme diffusion

Quartz crystal microbalance (QCM-D)

Polysaccharides

Confined conditions

Surface functionalization

Deep-UV fluorescence microscopy

A B S T R A C T

Understanding the specific interaction of cell wall polysaccharides degrading-enzymes with their substrates is of fundamental and practical interest for the fine grasp of their activity. Such interactions are difficult to unveil in real-life conditions due to the structural complexity of the plant cell wall. In this work, we present complex and highly confined model systems of cholesteric cellulose nanocrystal suspensions reaching dimensions close to those found in the plant cell wall. Cellulose-xyloglucan (XG) assemblies of various surface chemistries were studied, allowing to probe the interactions and diffusion behavior of a glucanase both in diluted dynamic conditions and in an organized and concentrated (110-140 g/L) cholesteric environment. QCM-D and synchrotron source-deep UV analyses showed that XG adsorbed in a flat and extended conformation changed the glucanase interaction with the polysaccharides from attractive to repulsive, making it prone to spontaneously migrate to regions with lower solid content. Furthermore, it strongly increased the enzyme diffusion kinetics. Our results confirm the preferential interaction of both enzyme and XG on the cellulose hydrophobic crystalline plane. This work provides new insights on the influence of the interaction of a protein with a substrate on its transport in a constrained environment.

<footnote><sup>a</sup> INRAE, UR BIA, 44316 Nantes, France</footnote>

<footnote><sup>b</sup> INRAE, PANTHER, 44307 Nantes, France</footnote>

<footnote><sup>c</sup> Laboratoire Gulliver, UMR CNRS 7083, ESPCI Paris, Université PSL, 10 rue Vauquelin, 75005 Paris, France</footnote>

<footnote><sup>d</sup> SOLEIL Synchrotron, DISCO Beamline, 91192 Gif-sur-Yvette, France</footnote>

<footnote>* Corresponding author.
E-mail address: hugo voisin@inrae.fr (H. Voisin).</footnote>
# 1. Introduction

Enzymes such as glycosyl-hydrolases naturally interact with polysaccharides of the plant cell wall in natural biological processes, but also during biomass treatment employed e.g. for the production of biofuel or platform molecules, or for new food resources [1]. The valorization of the biomass using enzymatic processes is environmentally more favorable than using chemical- or energy-consuming ones, but in order to employ them properly the enzyme action in plant cell walls needs to be clearly identified.

Cell wall is a complex concentrated system that can be approached as a hydrated composite material with a fiber-reinforced matrix structure [2,3]. These fibers are cellulose nanofibrils, tethered together or covered by biopolymers such as hemicelluloses and pectin that bear most of load thanks to their high mechanical properties. Cellulose and hemicelluloses (mainly xyloglucan, XG) are present in the primary cell walls within a matrix of pectin [4]. XG chains consist of a linear backbone of β-1,4-linked glucose residues, most of which are substituted with 1-6 linked xylose residue, often followed by a galactose residue. These side chains sterically prevent XG chains from coalescing into a tightly packed microfibril and yet still allow binding to cellulose, with stronger binding of segments lacking side chain [5]. XG is present in plants primary cell wall and is only a minor constituent of their secondary cell wall [6]; however, it still plays an important role in the cell wall development, influencing cell wall thickening [7] or tuning the interaction between cellulose microfibrils and pectin [8]. The interaction of XG with cellulose has been extensively studied both for its role in tethering the cellulose fibrils in a load-bearing network in the plant cell walls [9] and for the production of biobased materials [10–14]. The microstructures formed between XG and cellulose fibrils have been the subject of much debate in the plant science community [15–17], and strongly influence both the cell wall mechanical properties and enzyme accessibility. In particular, XG can adsorb on cellulose surface in flat conformation or as dangling loops and tails. These polymer loops and tails, in larger number with increasing XG chain length, have been widely studied since they can be accessed and degraded by enzymes and because of their influence on the assembly of cellulose nanoparticles. However, the influence of flatly adsorbed XG on cellulose fibers properties and enzyme behavior are less characterized due to their apparent recalcitrance. Their role should nevertheless not be underestimated, as in planta XG covers an appreciable fraction of cellulose microfibrils in an extended conformation [15]. Furthermore, its role in cellulose-XG interactions sites is now considered potentially crucial for cell wall properties as these sites are notably described as “biomechanical hot spots” [16].

The structure and composition of cell wall polysaccharides are expected to influence not only the colloidal and mechanical properties of their assemblies, but also their interaction with enzymes naturally present in the plant. Enzymes can interact with polysaccharides through carbohydrates binding modules (CBM), which present specific structure leading to specific affinity. For example, type A CBMs that include CBM1 and CBM3 present a flat hydrophobic surface made by three aromatic amino acids [18], which favors their binding with the cellulose hydrophobic (200) crystalline face. While the characterization of such CBM-mediated interaction with plant material is highly relevant, it can be difficult to investigate due to the high complexity of the plant cell wall, requiring the design of more or less complex model systems.

Several model systems of the plant cell wall have been developed to investigate the action mechanism of enzymes or other biological phenomena [19–23]. However, they generally do not combine high concentration of biopolymers (>10 wt%) and high complexity of composition and organization. Cellulose nanocrystals (CNCs) are crystalline form of nanocellulose presenting high purity. Those obtained through the acidic hydrolysis from wood are nanorods of approx. 6 nm wide for 100–200 nm in length. They are known to self-assemble in a chiral nematic liquid crystalline phase called cholesteric liquid crystal line (CLC) phase above a critical concentration. Such cholesteric

organization approaches the helicoidal organization observed in plant cell walls [3,24], in which the cellulose microfibrils separated by a distance of few tens of nm (20–50 nm) present a rotating orientation. We recently developed a model system in which concentrated CNCs organized in cholesteric suspensions that displayed similar rotating organization and inter CNC distance [25]. Such system thus presented features approaching the plant cell wall structures and composition, especially when further combined with hemicelluloses. These suspensions could be emulsified as droplet-type reactors that offered several advantages for the study of enzymes: medium solid content (7 wt%), low volume (in the pico- to nanoliter range), which is of high interest for the study of rare and expensive enzymes, and a clearly defined onion-like cholesteric organization due to CNC surface anchoring. Their size (100–250 µm) is also compatible with the UV synchrotron microscopic setup, which can be used to monitor native enzymes concentration without labeling with fluorescent probes that could potentially modify their activity [26]. Finally, we recently developed very low molar masses XG and demonstrated that they adsorbed exclusively in train conformation [12], presumably on the hydrophobic face of CNCs [27] and were compatible with CLC phase formation [28]. They could thus be incorporated with CNC suspensions in droplet-based micro-reactors making them good candidates for studying enzymes in cell wall models.

In this work, we investigated the interaction at the nano and macroscale of an endo-glucanase, an enzyme hydrolyzing the β(1-4)-glucan backbone in cellulose and hemicelluloses, with cellulose surface. The effect of the surface functionalization of model cellulose with short and flatly adsorbed XG on the glucanase diffusion properties was investigated. The behavior of the glucanase in a complex colloidal environment in a multiscale study was characterized by testing its interaction with CNCs and XG-coated CNCs from dilute to highly concentrated conditions. Highly organized and concentrated model systems were prepared by adapting previously developed micro-reactors to a solid content up to 14 wt% with the addition of hemicelluloses. The results showed that XG adsorbed in train conformation on cellulose surface had a very significant influence on the glucanase by preventing its adsorption on cellulose surface. It ultimately changed the nature of the interaction between the glucanase and the XG-covered CNC surface from attractive to repulsive, which resulted in its over-concentration in low solid content regions. This further allowed its transport in this crowded environment, influencing its flow and accelerating its diffusion in plant cell wall-like conditions.

# 2. Experimental

## 2.1. Reagents

Wood sulfated CNCs in their sodium form were purchased from Celluforce (Montreal, QC, Canada). They presented a typical length of 165 ± 82 nm, cross section of 6 ± 2 nm (estimated from STEM observation of over 100 particles), and sulfur content of approximately 0.6 wt % (obtained from elementary analysis). They were used as received without dialysis. Native XG from tamarind seed was purchased from DSP Gokyo Food & Chemical Co., Ltd. Osaka, Japan (Mw ≈ 850,000 g/mol). The GH7 endo-β-1,4-glucanase from Trichoderma longibrachiatum was purchased from Megazyme (E- CELTR, Lot 111102b). Hexadecane and sulfuric acid were purchased from Sigma-Aldrich.

## 2.2. Preparation of CNCs

CNCs used in the synchrotron experiments on droplets were prepared from commercial CNCs through acidic hydrolysis. This was achieved by first heating at 45 °C a volume of 370 mL of 64 wt% sulfuric acid (d = 1.54). Twenty g of commercial CNCs were then progressively incorporated under strong stirring, then the suspension was left under agitation for 45 min, before being quenched in 350 mL of cold water. The CNCs were centrifugated 3 times at 15000 g for 10 min and redispersed in
deionized water, before being extensively dialyzed against milli-Q water until the conductivity of the dialysis bath remained constant. The suspension was then concentrated to the desired concentration using rotary evaporator and ambient condition evaporation. The resulting CNCs were approx. 120 nm long on average with 1.17 % sulfur content.

## 2.3. CLC structure characterization

A suspension of a CNC sample was prepared at 110 g/L into milli-Q water, free from added salt, using magnetic stirring followed by ultrasonication using Q700 sonicator (20 kHz, 700 W maximum output, Qsonica LLC, Newtown, CT). The dispersion was performed using a power of at least 40 W and 160 J/mL CNC suspension, considering the sonicator nominal power. After 5–10 min they presented a cholesteric structure characterized by fingerprint structure that was observed using an upright Olympus microscope (Model BX51) equipped with a x10 objective and cross polarizers [10].

## 2.4. XG preparation

Low-M<sub>w</sub> XG was prepared by enzymatic degradation of native XG (XG<sub>850</sub>) to reach a M<sub>w</sub> of 20,000 g/mol (XG<sub>20</sub>) following a previously developed protocol [12]. Solutions of 10 g/L XG<sub>850</sub> were prepared by dissolving the powder in Milli-Q water under magnetic stirring at 50 °C for 2 h, then at room temperature overnight. The typical volume for enzymatic degradation was 10 mL. The required volume of the enzyme solution was then added to the XG solution to obtain 1 unit of glucanase per gram of XG. The reaction medium was incubated at 40 °C for 4 h on a stirring wheel at 20 rpm. It was then heated at 100 °C for 10 min to inactivate the enzyme. The low M<sub>w</sub> XGs were then isolated by precipitation in water:ethanol mixture (ratio = 40:60) by stirring the mixture for 10 min at 4 °C and resting overnight. It was then centrifuged at 17,500g for 30 min. The precipitate was redispersed in water, and the ethanol was evaporated using rotary evaporator. The M<sub>w</sub> distribution was measured using HPSEC-RALS (Fig. S1). The resulting solutions were then frozen and freeze-dried.

## 2.5. XG characterization High-Performance Size-Exclusion Chromatography (HPSEC)

XG samples were dissolved at 5 g/L in 50 mM NaNO<sub>3</sub> (99 % Sigma Ultra S8170-250G) and filtered at 0.45 µm on a PVDF membrane before analysis. The eluent consisted of 50 mM NaNO<sub>3</sub> and was filtrated through a 0.1 µm pore membrane. The samples were eluted at 0.7 mL/min on a Shodex OHpak SB-805 HQ column (8 mm × 300 mm). The molar mass, refractive index, and intrinsic viscosity were measured on an online multidetector system OMNISEC Malvern consisting of a right-angle laser light scattering (RALLS) detector, a differential refractometer, and a differential viscometer. Molar masses were determined by using OMNISEC 11.4 software (Malvern, UK). The concentrations of eluted XG were calculated using a refractive index increment of dn/dc = 0.147 mL/g. This method allows an accurate determination of peak concentrations and thus an absolute distribution of molar mass.

## 2.6. Zeta potential

Determination of the CNC and the CNC-XG zeta potential was achieved using a Zetasizer Nano ZS (Malvern Instruments, UK). CNC concentration was fixed at 5 g/L, XG concentration at 1 g/L and enzyme concentration at 0.02 g/L. They were placed in DTS1070 cuvettes. The measurements were performed at 5 mM NaCl, each measurement was composed of 15 runs of at least 20 s, and every data point presented is the average of three measurements.

## 2.7. Emulsion preparation

After hydrolysis, the concentration of the CNC suspension was adjusted at 110 g/L. CNC-XG mixtures were prepared by adding a mass of freeze-dried XG as a powder in the CNC suspension in order to reach 30 g/L XG. Sonication was carried out following the same protocol than above, and before any addition of the enzyme to prevent its alteration. When enzyme was present, its concentration (0.4 g/L) was chosen in order to insure enough signal under fluorescence microscopy without disrupting the organization of the CNCs. Hexadecane was purified from any amphiphilic compound through several passes in a neutral alumina column (Alumina N - Super I, MP) until its surface tension against water was stable at 55 mN/m [29]. The emulsion was prepared by mixing 0.1 mL suspension with 0.9 mL hexadecane. To stabilize the system, surfactants are generally added. However, it has been shown that micelles form at the interface interfering with the experiment due to several mechanisms. First, they drain water from the main droplets, which increases the solid content inside the droplet and strongly change the experimental conditions. Then, the swelling micelles can hamper the microscopic observation through light scattering, and finally the water diffusion can pump the proteins into the micelles. We prevented such issues by preparing the emulsion by hand shaking without any surfactant and quickly depositing it on a quartz slide to prevent the droplet coalescence. The sample chamber was immediately closed by a silicone joint and another quartz slide.

## 2.8. Synchrotron based Deep-UV fluorescence microscopy

Imaging was performed at the DISCO beamline of the SOLEIL synchrotron radiation facility (Gif-sur-Yvette, France) [30]. For synchrotron deep UV (DUV) fluorescence acquisitions, the widefield microscope, so-called TELEMOS, was used [31,32] with both × 40 and x100 Ultrafluar objectives (Zeiss, Germany). The excitation wavelength was set at 275 nm and reflected towards the sample with a DM-300 dichroic mirror (Semrock, USA). Fluorescence emission was collected using a 2D detector (CCD camera, Pixis BUV, Princeton Instruments, USA). The samples were placed between quartz fused silica (R52–5000, ESCO Optics) and acquisition time was set at 10 s. Images were dark subtracted and processed to compensate for illumination inhomogeneity. Fluorescence emission was evaluated based on the gray scale intensity of the pixels. Photobleaching experiments were performed to ensure that no significant bleaching was happening in the chosen timescale. Polarized optical microscopy (POM) was simultaneously performed by observing the samples in-between crossed polarizers.

## 2.9. QCM-D functionalization of sensors

CNC-covered QCM-D sensors (QSX 301, Qsense) were prepared following a previously described protocol [33]. Briefly, a poly(allylamine hydrochloride) (PAH) layer was adsorbed and dried on the gold by depositing a 4 g/L PAH solution at 25 mM NaCl on a gold-covered sensor using a spin-coater (Spin 150, APT Gmbh). The sensors were then rinsed and dried before the deposition of a 5 g/L CNC suspension at 25 mM NaCl. They were then finally rinsed and dried, and indefinitely stored under ambient conditions before use. The covering of the sensors' surface by the CNCs was assessed with Atomic Force Microscopy (AFM) using an Innova AFM (Bruker) in the tapping mode (see Fig. S2).

## 2.10. QCM-D measurements

QCM-D experiments were carried out on a Q-Sense E4 instrument (AB, Gothenburg, Sweden). Frequency (Δf<sub>n/n</sub>) and dissipation changes (Δn<sub>n</sub>) were simultaneously registered at fundamental resonance frequency, and its various overtones as a function of time. Spin-coated CNC surfaces were placed in the QCM-D cells at 20 °C and rinsed with water until the resonance response was stable. “While not corresponding to the
temperature of maximum activity of the glucanase, we verified that hydrolysis occurred at 20 °C with the liquefaction of a gel made from CNC and XG850 (results not shown here); this temperature was thus chosen for further ease of experimentation. Then, frequency and dissipation signals were offset to zero just before the measurement. Xylo-glucan solutions at 5 g/L and enzyme solutions at 0.02 g/L were injected at 50 µL min⁻¹ and allowed to adsorb. Rinsing step consisted in the injection of water in order to remove any loosely bound material. The experiments were repeated at least twice. All data shown correspond to the fifth overtone of the sensor fundamental resonance frequency.

## 2.11. *Diffusion experiments setup*

The setup was inspired from previous synchrotron-based diffusion experiments [34,35] but adapted to prevent evaporation during the time necessary for the CNC suspensions to equilibrate and form CLC. A sheet of polydimethylsiloxane (PDMS) at 10 wt% crosslinking agent content (RTV 615, Momentive Performance Materials Gmbh) at a constant thickness of 0.75 mm was prepared by casting in between glass slide with a controlled gap and heated at 70 °C for 30 min. PDMS disks of ca. 25 mm diameter were cut out of the sheet, and a rectangular chamber of 4 mm x 15 mm was cut out in every disk (see Fig. 1.a). The PDMS disks were then set in between two quartz slides to seal the chamber. 55 µL of CNC or CNC-XG₂₀ suspension were then injected in the chamber, which was clogged by a drop of hexadecane and left to equilibrate for 2 h. The observation was achieved by placing the chamber on the DUV microscopy setup, pumping out the hexadecane drop with paper and injecting 3 µL of a 0.4 g/L glucanase solution at the chamber entrance with a syringe. Its diffusion along the chamber was followed by acquiring fluorescence images at 6 positions each separated by 500 µm (Fig. 1.c). Every position was imaged every 15 min until 120 min after the enzyme injection.

# 3. Results and discussion

## 3.1. *Glucanase interaction with cellulose surface with and without flatly adsorbed XG*

In order to mimic the chemical composition and the structure of cellulose-hemicellulose assemblies in plant cell wall, the cellulose surface of the CNCs was covered with XG chains. We focused on flatly adsorbed XG to study the influence of recalcitrant hemicelluloses on

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Top (a) and side (b) views of the experimental setup used to monitor enzyme diffusion. The PDMS chamber (gray), squeezed in-between quartz slides (light gray) was filled with the suspensions (dark blue) before addition of the enzyme (light green) and plugging with hexadecane (gold). The diffusion was monitored with the chamber mapped as described in (c), with the injection of the enzyme around Pos0. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

glucanase behavior. To do so, very low molar mass XG (XG₂₀, Mw ≈ 20,000 g/mol) (see Fig. S1-HPSEC) were obtained from the native tamarind seed XG (XG₈₅₀, Mw ≈ 850,000 g/mol) by enzyme hydrolysis. It was shown that at such low Mw the XG chains (33 nm, calculated from the molar mass using 0.5 nm as the dimension of a glucose ring in the backbone, see Table 1.) are long enough to adsorb irreversibly onto the cellulose surface but too short and rigid to present tails or loops [12] due to the low number of persistence lengths in the chain, with the XG persistence length estimated at 8 nm from neutron scattering measurement [36].

No or very limited change of the CNCs bearing the XG₂₀ layer was detected in term of suspension viscosity or the CNC hydrodynamic radius [10,12]. Here, the CNC apparent size was unchanged in shape and thickness as observed using electronic and atomic force microscopes (Fig. S3 TEM, AFM). The typical dimensions and indication of the XG chains flexibility are summarized in the Table 1, with the length calculated using 1206 g/mol as the molar mass of an average XG oligomer containing four anhydroglucose, three xyloses, and one galactose minus four water molecules, i.e., 301.5 g/mol per anhydroglucose backbone.)

QCM-D measurement was used to follow i) the adsorption of XG on the CNCs (Fig. 2), and after that ii) the behavior of glucanase on the CNC surface modified by the XG (Fig. 3). Indeed, the adsorption of molecules on a CNC-covered piezoelectric quartz sensor translates into a shift of its resonance frequency (Δf). A qualitative estimation of the visco-elasticity of the adsorbed layer can simultaneously be obtained from the dissipation factor (ΔD) related to its tendency to attenuate the quartz oscillation. This technique had already demonstrated its potential for the study of the interaction of either specific CBM with polysaccharides surface [37,38], or the enzymatic degradation of cellulose [39] or cellulose-XG assemblies [40]. After flushing the chamber with the solution of XG₂₀, a clear decrease of the frequency down to Δf = -40 Hz was observed that demonstrated the adsorption of XG₂₀ (Fig. 2.a). The increase of ΔD demonstrated the increasingly viscoelastic nature of the adsorbed layer. After rinsing, a slight release of the less strongly adsorbed chains was observed with a Δf increase of 6 Hz and a significant reduction of the dissipation due to the alignment of the small chains at the cellulose surface and the elimination of transient XG aggregates. Interestingly, when comparing the short chains XG (XG₂₀) with native long XG (Fig. 2.b), the frequency changes after surface rinsing were very similar (-35 Hz) despite the large difference of chain dimension (see Table 1). Conversely, the dissipation value (ΔD) due to the adsorption of the XG₂₀ was of 2 × 10⁻⁶ after rinsing, compared to 6 × 10⁻⁶ for the native XG, confirming the denser nature of the low Mw adsorbed layer. This highlighted the more efficient covering of the cellulose surface with short rigid XG. The observed adsorption of the XG can be reasonably considered to be exclusively with CNC surface as AFM observation of the functionalized sensors revealed a homogeneous CNC layer (Fig. S2). Furthermore, the injection of XG on PEI covered sensors, without the CNC layer, showed a significantly decreased adsorption (Δf ≈ -10 Hz and -12 Hz for XG₂₀ and XG₈₅₀, respectively, Fig. S4.a and b).

In the second step, the interaction of the glucanase with the bare, and XG₂₀- and XG₈₅₀-covered CNC surface was analyzed through monitoring of the mass variation when glucanase was injected (Fig. 3). The profiles of interaction of the enzyme on the three surfaces were very different. On bare CNCs (Fig. 3.a), the sharp frequency drop indicated a quick increase of weight revealing an adsorption of the glucanase in relatively

<table><caption>**Table 1**
Dimensions of the XG described in this study.</caption><thead><tr><th></th><th>Native XG (XG<sub>850</sub>)</th><th>XG<sub>20</sub></th></tr></thead><tbody><tr><td>M<sub>w</sub> (g/mol)</td><td>≈850,000</td><td>≈20,000</td></tr><tr><td>Number of glucose in the backbone</td><td>2819</td><td>66</td></tr><tr><td>Backbone length (nm)</td><td>1403</td><td>33</td></tr><tr><td>Number of persistence length</td><td>170</td><td>4</td></tr></tbody></table>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** QCM-D analysis of the adsorption of XG20 (a) and XG850 (b) on CNC covered sensors. In green are the frequency shifts and in orange the change of dissipation of the fifth overtone. The dotted correspond to the start of XG injection (black) and the end of injection/the rinsing step (light blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** QCM-D analysis of the interaction of the glucanase with CNC covered sensors (a), CNC coated with XG<sub>20</sub> (b), CNC coated with XG<sub>850</sub> (c). In green are the changes of frequency and in orange the change of dissipation of the fifth overtone. The black dotted lines correspond to the start of glucanase injection and the light blue dotted lines to the end of injection and the rinsing step. Right side are the cartoons illustrating the behavior of the glucanase on the cellulose covered sensors. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

large quantities, with $\Delta f = -30$ Hz. This adsorption was mainly irreversible as the rinsing led to a 5 Hz frequency increase to a final $\Delta f$ of -25 Hz. The $\Delta D$ value of $6 \times 10^{-6}$ of the enzyme layer on the bare CNCs indicated that the enzyme deposited on the surface constituted a rather visco-elastic layer. On XG<sub>20</sub>-covered surface (Fig. 3.b), the enzyme adsorption revealed to be slow and very weak, with a final $\Delta f = -4.1$ Hz and a $\Delta D$ value of $2 \times 10^{-6}$. QCM-D thus revealed that the prior adsorption of XG<sub>20</sub> on CNC surface prevented further interaction with the glucanase. It confirmed that while glucanase hydrolyze unadsorbed XG, it does not interact with XG aligned along CNC. Furthermore, in a previous study, we hypothesized that glucanase interacted *in vitro* with the CNC surface through its CBM in a mostly irreversible mode and with the enzyme hanging from the CBM by the linker, forming a soft visco-elastic corona around CNCs [25]. The QCM-D experiment described in Fig. 3.a confirmed this hypothesis for bare CNC, and that in contrast the

covering of the CNC surface with flatly adsorbed XG<sub>20</sub> chains prevented the interaction of the glucanase with the cellulose surface. The glucanase interaction with the PEI layer was also checked using QCM-D and showed very little adsorption ($\Delta f \approx -1$ Hz), demonstrating that the studied interaction was between the glucanase and the CNC or the CNC-XG (Fig. S4.c).

On the opposite, the injection of enzyme on CNC covered with long XG<sub>850</sub> chains (Fig. 3.c) quickly led to a clear increase of the frequency and a decrease of the dissipation. The enzyme hydrolyzed the dangling, accessible chains, decreasing the mass of XG on the CNC surface as previously described [33,40]. Following this degradation, a slow decrease of frequency and increase of the dissipation were observed similarly to Fig. 3.b when only short chains fully adsorbed on CNC were remaining on CNC surface starting 12 min after injection. This also had been observed elsewhere but not commented on [33]. It could
reasonably be assigned to the adsorption of the enzyme on the accessible CNC surface as it was not expected to be integrally covered. As both CNCs and the glucanase are charged entities, electrostatic forces may play a role in their interaction. Zetametry was thus carried out to assess the influence of XG on the CNC surface charge; the zeta potential of bare CNCs was −27.1 ± 1.1 mV, for CNC-XG<sub>20</sub> −19.4 ± 1.4 mV and for CNC-XG<sub>850</sub> it was −17.9 ± 2.0 mV. While it showed that the XG adsorption on the CNCs slightly screened their surface charge, the difference was too small to influence their electrostatic interaction with the marginally negatively charged glucanase (ζ = −7.3 ± 1.1 mV).

We thus demonstrated the natural affinity of glucanase for the bare
cellulose surface and accessible XG chains, in contrast with surface
covered with flatly adsorbed XG chains.

## 3.2. Cholesteric shells in droplets: A 3D highly concentrated and organized cellulosic model structure

In order to analyze the behavior of the enzyme in a system approaching the cell wall conditions, a concentrated and ordered medium was prepared, involving solid content above 100 g/L and a supra-particular organization. Usually, CNCs tend to form a gel at high concentration [41]. In order to reach a high enough cellulose content, CNCs of reduced length and higher surface charge density were prepared through a stronger sulfuric acid hydrolysis of commercial CNCs as such process is known to degrade CNCs by the extremities; an average length of 120 nm was reached. The suspension thus retained a liquid behavior up to 140 g/L and organized in a cholesteric structure with the characteristic fingerprint structure that can be observed using polarized optical microscope (POM) (Fig. 4). Droplets of CNC suspensions in hexadecane were prepared and presented diameters varying from 70 to 250 µm. An onion-like structure was clearly identified with the characteristic feature of an anisotropic shell visible as alternate bright and dark circular lines under POM (Fig. 4a and b). The formation of this cholesteric shell was promoted by the oil-water interface that initiated the organization and accelerated the CLC phase formation; the core appeared as a dark area since it remained isotropic, corresponding to a lower CNC concentration [42,43]. This core-shell structure remained stable for several hours. It is known that the isotropic-to-anisotropic volume ratio is directly related to the CNC concentration [44]. In the case of this study, the anisotropic phase appeared for CNC concentration around 50 g/L, and the samples were fully anisotropic at 130 g/L (see Fig. S5). This was higher than for the commercial CNCs (before hydrolysis) for which isotropic-to-fully-anisotropic transition occurs from 40 to 80 g/L (Fig. S5) due to their lower aspect ratio. The CNC

concentration of 110 g/L was chosen in order to obtain a radius of the isotropic core of approximately 50 % of the droplet radius, which was in line with the isotropic phase volume content of 10–20 % measured in tube at this concentration (see Fig. S5) ($VR^3$, $0.5^3$ = 0.125). This enabled the assessment of the respective influence of both phases on further enzyme distribution.

The pitch of the cholesteric phase, corresponding to the period of one full rotation of the CNCs in the helicoidal structure and measured as twice the distance between bright lines in POM pictures, was 2.1 ± 0.3 µm (see Fig. 4.a). From our previous study, a 110 g/L CNC concentration corresponded to an inter-CNC distance of approximatively 17 nm¹⁰. Droplets of CNC-XG₂₀ suspensions were prepared following the same methods. As the CNC surface saturation by XG₂₀ has been previously estimated at a XG-to-CNC weight ratio $g_{XG}/g_{CNC} = 0.30$ [45], the XG concentration was fixed at 30 g/L to extensively cover the CNC surface (corresponding to $g_{XG}/g_{CNC} = 0.27$), while limiting the concentration of free XG in solution. The pitch of the CNC-XG suspension had an average value of 2.2 ± 0.3 µm (Fig. 4.b). The addition of XG₂₀ to the CNC suspension thus did not notably influence the property of the cholesteric phase, with both its pitch and isotropic-to-anisotropic ratio unchanged. However, the microscopic observation of the whole distribution of droplets revealed some disruptions of the circular CLC organization into more elongated or crescent shaped shells, hinting for specific interaction. Droplets of CNC-XG₈₅₀ suspensions were not studied as it was previously shown that long XG chains are incompatible with CLC phase formation due to gel formation [12] and as the focus of this study was to assess the influence of the CLC phase on enzyme behavior.

## 3.3. Macroscopic distribution of the glucanase in the organized system: Analysis of its behavior in the droplet and influence of cellulose surface state

The glucanase was incorporated in the concentrated CNC suspension that was emulsified and the droplets were simultaneously observed using POM and DUV synchrotron-based fluorescence microscopy (Fig. 5).

Fig. 5.e-h show the fluorescence intensity directly related to the concentration of the enzyme in CNC droplets at 110 g/L without and with XG at 30 g/L at two different magnifications. In the absence of XG (Fig. 5.e), the enzyme appeared uniformly distributed over the whole droplet and no specific accumulation could be observed. In contrast, the droplets containing XG₂₀ at 30 g/L (Fig. 5.f) presented some accumulation of enzymes at the isotropic-cholesteric phases' interface.

The synchrotron setup is a widefield microscope for maximum

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** POM pictures of the cholesteric shell of droplets containing 110 g/L of (a) pure CNCs and (b) CNCs 110 g/L with 30 g/L of XG<sub>20</sub>, 120 min after emulsification and observed with a x100 objective. The arrows indicate the water-hexadecane interface, and the dotted line the interface between anisotropic bright shell and the isotropic dark core. Scale bar = 25 µm.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** POM (a, b, c, d) and DUV fluorescence microscopy (e, f, g, h) pictures of droplets containing 110 g/L CNC without (a, c, e, g) and with (b, d, f, h) 30 g/L XG<sub>20</sub>, using x40 (a, b, e, f) and x100 (c, d, g, h) objectives. On the right is displayed the glucanase autofluorescence intensity profiles (i) along the arrows on (g) and (h). The gray dotted lines in (i) highlight the outlines of the droplets as shown on (g) and (h).</figcaption></figure>

fluorescence sensitivity; as such, when studied with a x40 objective (Fig. 5.e and 5.f), the fluorescence of the whole droplet thickness was taken into account, thanks to the large depth of field (see Fig. S6.a). Under these observation conditions, a small variation in enzyme concentration over the droplet diameter would not be visible. A higher magnification (x100) and high numerical aperture (N.A 1.25) together with a shorter depth of field enabled the study of a thinner volume of the droplets (Fig. S6.b and c). In the absence of XG<sub>20</sub>, the flat fluorescence profile of the droplet (Fig. 5.g, 5.i, orange profile) indicated uniform enzyme concentration regardless of isotropic and anisotropic phases, confirming the homogeneous distribution of glucanase along the droplet diameter. However, in the presence of XG, a higher fluorescence intensity in the isotropic core of the droplet indicated enzyme accumulation (Fig. 5.h and 5.i blue profile).

The glucanase thus appeared sensitive to the cellulose organization only when the cellulose was coated with XG<sub>20</sub>. It appeared clearly that the enzyme was visible in both phases, implying that during the CLC formation, the glucanase was not strictly excluded towards the isotropic phase as might have been expected from previous results from Kumacheva *et al.* in similar CNC biphasic systems. These authors reported that particles tended to concentrate into the isotropic core [43]. However, they used latex particles of 184 nm in diameter, which is an order of magnitude larger than the glucanase (11 nm) and the partitioning they observed conjointly involved some disruption of the CLC phase, which was not the case in our system. The accumulation of enzyme in the core was thus probably driven by a random migration into the less concentrated isotropic phase rather than long-range elastic forces arising from cholesteric disruption [46]. This confirmed that the presence of XG on the cellulose surface influenced the glucanase diffusion and its macroscopic distribution by making it sensitive to variation of solid content in concentrated systems, such as found in the plant cell wall.

### 3.4. Diffusion of the enzyme in a cell wall-like organization

The previous hypothesis was tested by quantifying the diffusion dynamics of the glucanase in complex concentrated cellulosic media. This was made using a specifically designed setup that allowed to follow the glucanase diffusion as a function of time in a fully self-organized cholesteric system on a distance of 2838 µm. Chambers were filled with bare or XG<sub>20</sub>-coated CNCs that were left to organize as checked using POM before each experiment. CNC-XG<sub>850</sub> suspensions were not studied as long XG chains not only influences the XG diffusion by modulating its interaction with CNC surface, but also by the activity of the enzyme on the dangling XG chains. No autofluorescence of the CNCs could be observed (visible as the black squares on the Fig. 6). After deposition of the solution of enzyme at Position 0 at the entrance of the chamber, its diffusion was monitored by following its autofluorescence

in DUV microscopy (see Fig. 1 in the Experimental Section). The spot intensity was directly proportional to the amount of enzyme at each position since no bleaching was observed. For both systems and 5 min after deposition, only the region of deposition area displayed a spot characteristic of the enzyme presence (Pos 0 column, Fig. 6.a and b). After 60 min, fluorescence intensity could be observed at Pos 0, Pos 1 and Pos 2 in the CNC suspension (Fig. 6.a, 60 min row) and at Pos 0, Pos 1, Pos 2 and Pos 3 in the CNC-XG<sub>20</sub> suspension (Fig. 6.b, 60 min row), demonstrating the enzyme diffusion in the chamber, as well as a seemingly faster diffusion kinetic in the CNC-XG<sub>20</sub> suspension. The direct comparison of the intensities over the experiment 120 min timescale further revealed a much faster diffusion in the CNC-XG<sub>20</sub> suspension compared to CNC alone (Fig. 6.a and b, 120 min row).

The fluorescence intensity was evaluated based on the gray scale intensity of the pixels and the profiles of the enzyme diffusion in both systems were plotted as a function of time and distance (Fig. 7). In the presence of bare CNC (Fig. 7.a,c), the enzyme progressed with a quick diffusion to Pos 1, showing a spike after 15 min (t1) in Pos1 and one after 45 min (t4) in Pos 2 before stalling and not displaying any significant consequent diffusion. No signal was detectable in Pos3 even after 120 min. In contrast, in the CNC-XG<sub>20</sub> system the diffusion was a lot faster and more constant with the enzyme progressing approximately 500 µm every 15 min until Pos 6 (Fig. 7.b, d).

### 3.5. Consequences for the understanding of enzyme behavior in biological-like conditions

In this study, the QCM-D experiments showed that the glucanase does not interact with cellulose surface when covered by short, extended XG chains, unlike bare CNCs (Fig. 3). Previous studies on layer-by-layer systems focused on long, coiled XG chains (length of 1403 nm), revealing a mix of glucanase adsorption and substrate degradation. They revealed two XG populations, distinguishing accessible and inaccessible fractions [45]. In such systems, endo-glucanase degraded accessible XG segments, causing mass loss due to desorption of the hydrolyzed fragments and complicating the detection of an eventual adsorption of glucanase on cellulose surface [33,40].

Previous studies described the cellulose binding domain CBM1 serving as binding module to cellulose [38,47]. Transmission electron microscopy images provided unequivocal evidence that the fungal family 1 CBMs as well as the family 3 CBMs have defined binding sites on two opposite corners of cellulose crystals [48] and it has been established that the three aromatic rings in their structure led them to preferentially stack at their flat exposed surface onto the (200) hydrophobic crystalline face of cellulose [49]. Driven by entropy, the interaction was observed to be mainly irreversible in the droplet-based systems [25] under in vitro conditions. Similarly, XG adsorption on cellulose is
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Fluorescence images acquired during diffusion experiment of the enzyme through dense organized system 5 min, 60 min and 120 min after enzyme deposition on position 0 in bare CNC (a) and CNC-XG<sub>20</sub> (b) suspensions. Each position is separated by 500 µm from the previous one, and the regions of interest are 332 µm wide and high. For each sample, the intensity scale was weighted by the highest intensity measured.</figcaption></figure>

irreversible, also driven by entropy [50] and preferably on the (200) hydrophobic face [27]. This leads to competition between hemicellulose and CBM adsorption on cellulose's hydrophobic face and are in line with the observed results from the QCM-D experiments. The recalcitrance of extended XG to enzymes could thus be linked to CBM inaccessibility.

The obvious difference of the glucanase distribution between CNC and CNC-XG₂₀ suspensions occurred whereas the two systems exhibited similar liquid crystalline organization with similar pitch and inter-CNC distances [28]. This indicated that enzyme distribution was only influenced by the cellulose surface chemistry. In bare CNC suspensions, glucanase molecules strongly adhered to the CNC surface, spreading evenly in the droplet (Fig. 5.a). On the opposite, in CNC-XG₂₀ suspensions, the enzyme remained mostly free due to lack of interaction with the XG₂₀-covered CNC surface (Fig. 5.b). Considering the size of the enzyme (R<sub>h</sub> = 5.6 nm) compared to inter-CNC distances (15–17 nm, see SI), the enzyme diffusion in the cholesteric CNC suspension can be related to the dynamics of colloid transport involved in a confined system [51]. Here, the repulsive nature of the interaction between the glucanase and the XG₂₀-covered CNC surface initiated an osmosis flow towards the isotropic domain where the interfacial pressure was lower. The glucanase thus concentrated in the isotropic core, which presented a less confined environment.

In plant cell wall conditions, which involve a similarly crowded environment, enzyme diffusion could also be affected by such factors. It

could thus modulate enzyme activity through its selective adsorption on cellulose microfibrils depending on its surface state. The coating of microfibrils by XG could thereby enhance glucanase availability by preventing its adsorption after degradation of polysaccharidic tethers between microfibrils, optimizing the amount of necessary enzyme. Indeed, we showed that obstructing the CBM-mediated interaction of glucanase with cellulose surfaces due to XG adsorption sharply increased the diffusion speed of the enzyme compared to bare cellulose surface on which the enzyme adsorbed until saturation, further preventing its diffusion (see Fig. 8). It demonstrated how the cellulose surface state strongly modulated the enzyme behavior over distance of several millimeters and a timescale of 2 h, potentially allowing it to be distributed more efficiently in plant. More generally, the covering of the hydrophobic cellulose plane by hydrophilic hemicelluloses decreases the tendency of any amphiphilic entities to attach to cellulose. This would ultimately make them more readily and quickly available in the plant tissues, lowering the amount of enzyme required at equal activity, hence the quantity of energy necessary for their synthesis.

4. Conclusion

We described here new insights on the influence of short XG chains adsorbed on cellulose surface in a train like conformation, in diluted conditions as well as in a highly concentrated new model of a plant cell
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Evolution of the autofluorescence intensity of glucanase diffusing in a cholesteric CNC medium as a function of position (a, b) and observation times at 3 positions (c, d) in chamber filled with CNC (a, c), CNC-XG<sub>20</sub> (b, d).</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Diffusion behavior of the enzyme (blue objects) in the concentrated systems. (a) In bare-CNC (gray cuboids) suspension the enzyme adheres to the cellulose surface and fully saturates it before further diffusing, reducing its speed; (b) in XG-covered CNC (gold colored cuboids) suspension the enzyme does not adhere to the CNC surface and can freely diffuse at a higher speed. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
wall. This system proposed an unprecedented complexity of composition and organization. It showed that the coating of the crystalline cellulose surface by short XG chains had little influence on the self-organization state of CNCs, but it prevented endo-glucanase interaction with cellulose in both diluted and concentrated conditions. It revealed the free diffusion of a glucanase towards the less concentrated and organized areas exclusively when XG was present, due to the preferential adsorption of both the enzyme hydrophobic CBM and XG on the same cellulose crystalline plane. It also drastically increased the macroscopic diffusion speed of the enzyme in such medium compared to a pure cellulose system. This work demonstrates that increased complexity of medium (mixture of soluble and insoluble polysaccharides) strongly influences the nature of the interaction of enzymes with solid components. This modulation then changed the enzyme diffusion in concentrated and highly organized conditions. Such in vitro behavior could thus relate to the diffusion and action of enzymes observed in plant tissues. The role of flat-adsorbed XG could be more diversified than simply establishing a mechanical "hot-spot" of interaction between cellulose microfibrils, as XG could also modulate enzyme diffusion. These results open new horizon and further experiments will be designed with a more complex structure, with new enzymes presenting different CBM as well as mechanism of action (exo instead of endo modes of action, for instance) and substrates with various recalcitrance. Studies will also focus on the role of the XG chain sequence, as it was shown to influence both its adsorption on cellulose [5] and enzyme mode of action [52]. Concerning the new substrates, a specific attention will be given to polysaccharides more predominant in plant secondary cell walls that are extensively present in plant biomass, in particular xylans.

## CRediT authorship contribution statement

**Hugo Voisin:** Writing – original draft, Methodology, Investigation, Conceptualization. **Estelle Bonnin:** Writing – review & editing, Methodology, Investigation, Conceptualization. **Mélanie Marquis:** Writing – review & editing, Methodology, Investigation. **Camille Alvarado:** Investigation. **Suzanne Lafon:** Writing – review & editing, Investigation. **Teresa Lopez-Leon:** Methodology, Conceptualization. **Frédéric Jamme:** Writing – review & editing, Methodology, Investigation. **Isabelle Capron:** Writing – review & editing, Project administration, Methodology, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Isabelle Capron reports financial support was provided by French National Research Agency. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

This work was supported by the French Agence Nationale de la Recherche (project Clicteam No ANR-18-CE09-0028). We acknowledge Synchrotron SOLEIL facilities for providing access to synchrotron radiation facilities on the DISCO beamline proposal number 20191953. The authors also thank Nadège Leray (BIA-INRAE, France) for the acquisition of the AFM pictures, as well as Pierre Sartelet (BIA-INRAE, France) and Bruno Novales (BIBS platform, BIA-INRAE, France) for the STEM pictures.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jcis.2025.137685.

## Data availability

Data will be made available on request.

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