# One-point covalent immobilization of a xylanase on elastomer: effects of surface stretching on enzymatic activity

Maxime Artico, Cécile Formosa-Dague, Dimitri Kamak, Frédéric Peruch, Anne-Françoise Mingotaud, Clément Roux, Cédric Y Montanier

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Maxime Artico, Cécile Formosa-Dague, Dimitri Kamak, Frédéric Peruch, Anne-Françoise Mingotaud, et al.. One-point covalent immobilization of a xylanase on elastomer: effects of surface stretching on enzymatic activity. Applied Surface Science, 2025, 706, 10.1016/j.apsusc.2025.163554. hal-05083766

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Full Length Article

# One-point covalent immobilization of a xylanase on elastomer: effects of surface stretching on enzymatic activity

Maxime Artico<sup>a,b,d</sup>, Cécile Formosa-Dague<sup>b,d</sup>, Dimitri Kamak<sup>b</sup>, Frédéric Peruch<sup>c</sup>, Anne-Françoise Mingotaud<sup>a,d</sup>, Clément Roux<sup>a,d,*</sup>, Cédric Y. Montanier<sup>b,d,*</sup>

ARTICLE INFO

Keywords:

Polymer

Elastomer

Enzyme immobilization

AFM

Mechanical stretching

ABSTRACT

Protein immobilization on solid surfaces is a prevalent strategy in biotechnology and industry, offering advantages in terms of stability and reusability. Various methods, including covalent attachment, adsorption, covalent cross-linking, and entrapment, have been developed, with challenges in adapting these techniques to enzymes due to their catalytic pockets. In a previous study, we introduced a method for grafting the glycoside hydrolase Neocallimastix patriciarum endo-β-1,4-xylanase (NpXyn11A) onto paramagnetic beads, preserving its biological activity. Here, we extend this approach to polymeric surfaces, specifically polyisoprene (PI) and polydimethylsiloxane (PDMS), known for their elasticity. The surfaces are plasma-activated and functionalized with maleimide functions to immobilize the engineered protein Jo through a one-point thiol-maleimide click reaction. In a second step, the xylanase fused to the protein In is immobilized on the membrane by a spontaneous and specific covalent bond between Jo and In. A custom device is employed to stretch the elastomer surfaces, altering the distance between immobilized enzymes. The specific activity of the immobilized xylanase, evaluated with a chromogenic substrate, mirrors that of the free enzyme in solution. However, the use of natural polysaccharides reveals activity variations based on the extent of surface stretching, providing insights into the impact of spatial proximity on enzyme dynamics. Our study contributes to overcoming limitations in traditional enzyme immobilization, offering valuable insights into the dynamics of multi-enzymatic complexes.

1. Introduction

The immobilization of proteins on solid surfaces is a widely used strategy in industry and biotechnology [1]. Supports such as silica, agarose or nanomaterials offer distinct advantages in terms of protein stability and reusability in industrial processes [2]. A variety of immobilization methods have been developed over time, including covalent attachment [3], adsorption [4], covalent cross-linking [5] and entrapment [6]. Although multi-point immobilization is suitable for a wide range of proteins, it may not be the best approach for enzymes. Enzymes are a class of proteins whose three-dimensional structure in solution enables them to accommodate a substrate and carry out a chemical reaction in a highly selective way. Modifying this three-dimensional conformation can influence the manner in which the enzyme interacts

with its substrate, thereby affecting its activity [7]. For example, non-specific immobilization may lead to inactivity due to their catalytic pockets that require full accessibility to substrates for catalysis [8]. To address this issue, enzyme immobilization may require single-point grafting to a solid support, utilizing for instance specific chemical functions present in the amino acid sequence of the protein [9] or introduced by genetic modification [10].

In a previous study [11], we proposed an alternative method for grafting the *endo*-β-1,4-xylanase A from *Neocallimastix patriciarum* NpXyn11A, a glycoside hydrolase (GH) belonging to family 11 [12], to porous paramagnetic beads displaying a large specific surface aera. This was achieved using two engineered protein fragments, Jo and In, which have the ability to covalently bind post-translationally [13] very selectively. We demonstrated that the grafting of NpXyn11A fully preserved

<footnote><sup>a</sup> Laboratoire Softmat, Université de Toulouse, CNRS UMR 5623, Université Toulouse III – Paul Sabatier, Toulouse, France</footnote>

<footnote><sup>b</sup> TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France</footnote>

<footnote><sup>c</sup> Univ. Bordeaux, CNRS, Bordeaux INP, LCPO, UMR 5629, F-33600 Pessac, France</footnote>

<footnote><sup>d</sup> FR FERMAT, Université de Toulouse, CNRS, INPT, INSA, UPS, Toulouse, France</footnote>

<footnote>* Corresponding authors at: Laboratoire Softmat, Université de Toulouse, CNRS UMR 5623, Université Toulouse III – Paul Sabatier, Toulouse, France for Clément Roux and TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France.

*E-mail addresses:* clement.roux1@univ-tlse3.fr (C. Roux), cedric.montanier@insa-toulouse.fr (C.Y. Montanier).</footnote>
its enzymatic activity on its natural substrate, namely catalyzing the hydrolysis of the β-1,4 glycosidic bonds between adjacent sugar units in the backbone of xylan, a natural plant cell polymer of xylose residues. This approach allows the immobilization of the xylanase without modifying its intrinsic activity, thus making the surface density (i.e. the average relative distance between enzymes) the only variable parameter to consider. The results indicate that the proximity of the enzyme has a significant effect on both the reaction kinetics and the nature of the released products. However, creating beads with different grafting densities, and so different average relative distance between enzymes, can be difficult to establish.

Protein immobilization on polymeric surfaces, particularly on elastomers, is a challenging task, especially when dealing with enzymes [14]. However, elastic surfaces such as polydimethylsiloxane (PDMS) or silicone offer new possibilities in mechanochemistry, a relatively new field of research that investigates the effect of mechanical forces on chemical modifications in macromolecules [15]. For instance, Rios *et al.* [16] applied mechanical constraints on a covalently immobilized β-galactosidase on elastic silicone to induce enzyme-conformational changes and thus modulated the enzymatic activity by stretching the elastomer. A decrease of 40 % of the enzymatic activity was observed by stretching the elastomer by 100 %, and 87 % of the initial rate was obtained by the release of the stretch.

The significance of spatial proximity between enzymes has been explored by various researchers in recent years [17–20]. Multi-enzyme cascades with spatial arrangements are common in cells, particularly in metabolic pathways [21]. Enzymatic cascades function as organized biochemical orchestras, with different enzymes catalyzing complex metabolic reactions sequentially. This sequential organization ensures maximum efficiency in substrate conversion. The cellulosome is another example of spatial organization that enhances a complex enzymatic process [22]. Cellulosomes, which are expressed by some cellulolytic microorganisms, break down plant cell wall polysaccharides into simple sugars. They present a scaffold protein immobilized to the outer membrane of the microorganisms on which up to 160 GHs are grafted to prevent their dispersion and increase catalytic synergism [23]. Their high dynamics and spatial organization contribute to the overall catalytic efficiency [24].

The aim of our present study is to immobilize *NpXyn11A* on two commercially available elastomers, based on polyisoprene (PI) and polydimethylsiloxane (PDMS) structures, in order to control density of grafting in a similar way as described in a previous study [11] and summarized in Fig. 1. Both elastomers were plasma activated and functionalized with maleimide functions to immobilize Jo protein through a thiol-maleimide click reaction. Following this, the xylanase expressed in fusion with In is covalently grafted to the surface. A dedicated device was developed to stretch the elastomer surface, increasing its surface area and therefore the distance between the enzymes. The specific activity of the immobilized xylanase assessed using a small and easily diffusible chromogenic substrate is comparable to the free enzyme in solution, whereas the use of a long natural xylose-based polysaccharide clearly highlights a variation of the specific activity on the enzyme according to the extent of the stretching of the surface, and thus the distance between the immobilized *NpXyn11A*. Altogether this work addresses the limitations of conventional enzyme immobilization approaches, and proposes an approach that will facilitate understanding of the multifaceted dynamics in multi-enzymatic complexes.

## 2. Materials and methods

### 2.1. Chemicals and instruments

The polyisoprene (PI) surfaces were from Durex® with the reference Natural 5000 3544, the silicone (PDMS) surfaces from GoodFellow with the references SI30-FM-000135 and SI30-FM-000150, with a thickness of respectively 0.175 mm and 0.05 mm. The first reference (PDMSr) was a surface reinforced by a polyester fiber mesh. All surfaces were washed before use with two successive ultrasonic baths in MilliQ water, each lasting 5 min at 30 °C, followed by two additional baths in technical ethanol for 5 min at 30 °C. The surfaces were then dried under an argon flow and were ready for use. In case of non-instantaneous use, they were stored under an inert atmosphere at 4 °C. The procedure for the synthesis of polybutadiene samples (PB) and elastomer formation are provided in supplementary information.

The water used in all experiments was produced by a Flex PF2L260088 system and deionized using a Veolia ELGA ELECT50

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Schematic illustration of the immobilization of In-NpXyn11A on the PI and PDMS elastomer surfaces. $Jo_{cys}$ stands for a cysteine-modified Jo protein and is represented by its 3D structure in red. The In domain of In-NpXyn11A is represented in green, and the NpXyn11A itself in brown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
dispenser. All following compounds were used as received from the supplier. NaCl (Aldrich); yeast extract (Biokar Diagnostics); tryptone (Biokar Diagnostics); glycerol (VWR); monopotassium phosphate (KH₂PO₄, VWR); dipotassium hydrogen phosphate (K₂HPO₄, VWR); agar (bacteriological grade type E, Biokar); SDS-Page gel (TGX, stain-free gels, Mini-Protean® precast gels, BioRad); Cobalt resin (Talon® metal affinity resin from TAKARA, Thermo Scientific); lysozyme (>90 %, Chicken egg white, Sigma); isopropyl β-D-1-thiogalactopyranoside (IPTG, >99 %, Molecular Biology Grade, Euromedex); ampicillin (Cell Culture Grade, Euromedex); kanamycin (Cell Culture Grade, Euromedex); chloramphenicol (Cell Culture Grade, Euromedex); Imidazole (>99 %, Merck); rCutSmartBuffer (rCutSmart®, BioLab); restriction enzyme (rCutSmart®, BioLab); ethidium bromide (BET, 1 %, Euro-medex); β-mercaptoethanol (BioRad); Laemmli Sample Buffer 4X (Bio-Rad); SDS gel (Mini-Protean® Precast Gels, TGX Stainfree gels, BioRad); molecular weight marker (Protein Unstained Standards, BioRad); Coomassie blue (Page Blue Protein Staining Solution, Thermo Scientific). Acetone (>99.8 %, HPLC grade, Sigma); 6-maleimidohexanoic acid (90 %, Merck); triethylamine (>99.5 %, Sigma); ethyl chloroformate (>98 %, Sigma); tris(2-carboxyethyl)phosphine (TCEP, Immobilized TCEP disulfide reducing resin: 50 v/v% in water, ThermoScientific); polyoxyethylene sorbitan monolaurate (TWEEN®20, Sigma-Aldrich); 2-(N-Morpholino)ethanesulfonic acid and 4-morpholineethanesulfonic acid monohydrate (MES, >99 %, Sigma-Aldrich); tris(hydroxymethyl)aminomethane (Tris/HCl, Gen Apex, VWR); bovine serum albumin (BSA, >98 %, Sigma-Aldrich).

Plasma treatment was conducted using a Femto device marketed by Diene Electronic. Incubations were performed in an Eppendorf Thermomixer C. Contact angle measurements were performed on a PAT1M v4 from Sinterface using droplets of 3 µL. For each sample, 10 different droplets were deposited at various locations. All autoclave steps were carried out with a Sanoclav Adolf Wolf, 20 min at 121 °C. The centrifuge used was an Avanti® J-26 XP model from Beckman Coulter. For volumes of 400 mL, a J-Lite Series JLA 10,500 rotor was used. For volumes below 50 mL, a J-Series Instrument, JA 25,50 rotor was used. The sonicator FB705, Fisher Scientific, was used with two cycles of 1 min and then 30 s at 25 % amplitude. Agarose gel electrophoresis was migrated in a Mupid® One from Advance. SDS-PAGE electrophoresis gels were migrated in a Power Pac from BioRad. They were imaged on a Chemidoc MP Imaging System from BioRad.

## 2.2. Protein expression

The xylanase 11A from Neocallimastix patriciarum (NpXyn11A) [12] was cloned in pET28b in fusion with In as previously described [11], displaying a His₆ tag at the N-terminus. As native Jo did not display a cysteine residue in its amino acid sequence, a cysteine residue was introduced by point mutation in the MCS1 of pBMW1 [13], using a QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent technology) and the following primers: 5' CCA TCA TCA CCA CTG CCA GGA TCC GTC TGA CCA G 3' and 5' CTG GTC AGA CGG ATC CTG GCA GTG GTG ATG ATG G 3'. Both In-NpXyn11A and Jo<sub>cys</sub> were expressed and purified as previously described, using strain BL21 (DE3) of *Escherichia coli* harboring plasmids of interest and cultured in Luria-Bertani broth at 37 °C [11]. Proteins of interest were purified by immobilized metal ion affinity chromatography (IMAC) using Talon resin (Clontech, Takara) as previously described [11]. Protein concentrations were determined by measuring absorbance at 280 nm (DS-11 Spectrophotometer, DeNovix). Proteins were stored at 4 °C in 50 mM phosphate buffer pH 7.2.

## 2.3. Surface functionalization

**Plasma activation.** The surfaces to be treated were placed on a glass plate in the plasma generator. It is noteworthy that PDMS and PDMSr surfaces were slightly adhesive to the glass plates, unlike PI surfaces. Therefore, PDMS surfaces were treated only on the front side, while the

PI surface was treated on both sides. The plasma treatment was carried out under the following conditions: 40 kHz, 0.5 mbar of argon for 30 s.

**Maleimide functionalization.** Following the plasma treatment, the surfaces were immersed in approximately 100 mL of MilliQ water with stirring for 10 min. Subsequently, the surfaces were sequentially dipped in three baths of acetone (25 mL) for 30 s and then in a reactive solution prepared in advance. The preparation of this solution is described in the following paragraph.

0.25 g of 6-maleimidohexanoic acid (1.18 mmol) was dissolved in 1.5 mL of acetone and injected into a Schlenk tube at 4 °C with stirring under an inert atmosphere. The solution was cooled to -5 °C by adding NaCl to the ice bath. 1.4 equivalents of triethylamine dried over KOH (1.62 mol, 119 µL) were added while maintaining an inert atmosphere. 0.9 equivalents of ethyl chloroformate (1.09 mmol, 104 µL) were diluted with 1.5 mL of acetone before being added to the solution under an inert atmosphere. The solution was stirred for 10 min at -5 °C, and followed by the addition of 4 mL of acetone.

The prepared surfaces were introduced into the reactive solution. The ratio used for all experiments was 2.67 cm² of surface per 1 mL of reactive solution, leading to 3 surfaces of 2.5 × 2.5 cm for 7 mL of reactive solution. The solution containing the surfaces was incubated for 6 h at 20 °C and 300 rpm under an inert atmosphere. The surfaces were then immersed for 2 min in two baths of acetone (25 mL) and subsequently for 1 min in a 50 mM phosphate buffer bath, pH 7.2 (10 mL).

**Jo<sub>cys</sub> immobilization.** 1 mL of TCEP suspension (TCEP immobilized on agarose beads, beads at 50 v/v% in water) was introduced into a 2 mL Eppendorf tube. The TCEP-agarose beads were recovered by centrifugation for 30 s at 3,000 rpm and washed twice using 500 µL of 50 mM phosphate buffer pH 7.2 (Buffer 1). 1 mL of Jo<sub>cys</sub> at 1.5 mg.mL⁻¹ was added over the TCEP-agarose beads and incubated using a thermomixer at 20 °C for 15 min at 900 rpm. TCEP-agarose beads were pelleted by centrifugation and the supernatant was collected. The Jo<sub>cys</sub> solution was diluted to 0.75 mg.mL⁻¹ and 1.7 mL was incubated overnight in the presence of the maleimide-functionalized surface, approximately 16 h, at 20 °C with stirring at 900 rpm.

After 16 h, the surface grafted with Jo<sub>cys</sub> was thoroughly washed by vortexing 30 s with a series of different buffers (50 mM phosphate, 250 mM NaCl, TWEEN 20 1 %, pH 7; 500 mM MES, 500 mM NaCl, pH 5; 500 mM Tris-HCl, 500 mM NaCl, pH 9) and finally stored in Buffer 1 at 4 °C.

**In-NpXyn11A immobilization.** For PDMS and PDMSr surfaces, the 50 mM phosphate buffer pH 7.2 was supplemented with 1 mg.mL⁻¹ BSA and the enzymatic concentration was set at 2 µM. For PI surfaces, no BSA was added and the enzymatic concentration was set at 1 µM. Both enzymatic solution (1.7 mL) and surface functionalized with Jo<sub>cys</sub> were incubated in a thermomixer for 6 h at 20 °C with stirring at 900 rpm. Surfaces were rinsed, washed, and stored as described for Jo<sub>cys</sub> immobilization.

**Set-up for the elastomer stretching and measurement of elastomer surfaces at different stretching stages and calculation of graft density.**

The procedures are provided in supplementary information.

## 2.4. Enzymatic assays

**Enzymatic kinetic of free enzyme.** The specific activity of the xylanase was determined using 4-nitrophenyl-β-D-xylotrioside (pNP-X₃) as substrate and measuring absorbance at 401 nm of the released 4-nitrophenolate product (ε = 12,578 M⁻¹.cm⁻¹). Typically, 450 µL of 5 mM pNP-X₃ in 50 mM sodium phosphate, 1 mg.mL⁻¹ of BSA, pH 7.2 were preheated at 37 °C for 5 min in quartz cuvettes with a chamber volume of 500 µL (cuvettes Hellma Analytics), using a UV-vis spectrophotometer (Cary, Multi-Cell Peltier, Agilent Crosslab). The reaction was then initiated by the addition of 50 µL of the xylanase.

Dinitrosalicylic acid (DNS) assay [25] was performed to estimate reducing sugar when wheat arabinoxylan WAX (Megazyme) was the substrate. Substrate concentration was set to 1 % w/v, in 50 mM phosphate, 1 mg.mL⁻¹ BSA, pH 6. The enzymatic reactions (1,950 µL),
initiated by adding 50 µL of enzyme (1 to 10 nM final concentration), were performed at 37°C under constant agitation at 1,400 rpm (ThermoMixer®C, Eppendorf) in 2 mL centrifuge tube. At specific time (1, 2, 4, 6, 8, 10, 12 and 15 min), an aliquot of 100 µL was mixed to 100 µL of DNSA and incubated 10 min at 95 °C. After cooling down on ice, 1 mL of deionized water was added. Samples of 300 µL were transferred to a 96-well microplate and absorbance at 540 nm was measured using a microplate spectrophotometer (Infinite 200 Pro, TECAN). Aliquots of Dxylose were prepared from 0 to 2 mg·mL⁻¹ as standard curve. Specific activity was expressed in µmoles of product formed per min per µmole of enzyme (IU/µmole).

**Determination of amount of immobilized enzyme.** To determine the amount of immobilized enzyme on elastomer as accurately as possible, a standard curve using increasing amounts of free In-NpXyn11A was set up. Enzymatic reaction was initiated using 22 µL of enzyme (0, 10, 20, 50, 100 and 200 nM) in 1.5 mL Eppendorf tube containing 198 µL of reaction medium (50 mM sodium phosphate, 1 mg·mL⁻¹ of BSA, pH 7.2 and 5 mM of pNP-X₃). In parallel, the surface sample with grafted xylanase was immersed in 1.7 mL of reaction medium. Enzymatic reactions were incubated at 37 °C under constant agitation (900 rpm, ThermoMixer®C, Eppendorf) for 30 min whereupon 200 µL of each sample were mixed with 100 µL of Na₂CO₃ 1 M in a 96-well microplate to stop reactions. Final absorbance was then measured at 401 nm in a microplate reader (Infinite 200 Pro, TECAN). Based on the specific activity of the free In-NpXyn11A, the amount of enzyme immobilized to the surface sample was determined according to the standard curve. In the specific instance of PI, with both surfaces treated, once the membrane was affixed to the apparatus, the quantity of enzyme exposed to the reaction medium was reduced by half.

**Measurement of elastomer surfaces at different stretching stages and calculation of graft density.** Details are provided in Supplementary Information

**Enzymatic kinetic with immobilized enzyme.** The specific activity of grafted In-NpXyn11A on WAX was determined by DNS assay in a similar way as above, using the dedicated stretching device described above. The reactor (8 mL) containing 1.7 mL of reaction buffer (WAX 1 % w/v, in 50 mM phosphate, 1 mg/mL BSA, pH 6) was incubated at 37 °C under magnetic stirring. The grafted surface mounted on the stretching device was immersed inside the reactor, while aliquots of 50 µL were collected at regular time (5, 10, 15, 20, 30 and 45 min), mixed to 50 µL of DNS and incubated 10 min at 95 °C. After addition of 90 µL of deionized water, samples were displayed on a 96 wells microplate and absorbance at 540 nm was measured using a microplate spectrophotometer (Infinite 200 Pro, TECAN).

## 2.5. Atomic force microscopy (AFM) experiments

**Surface characterization.** High resolution images of the surfaces were recorded in air using the contact mode available on the Nanowizard IV XP AFM (Bruker, USA), with MLCT cantilevers (Bruker, nominal spring constant of 0.01 N/m). Images were recorded at a resolution of 512 lines using an applied force of 0.8 nN. The cantilevers spring constant were determined prior to imaging using the thermal noise method [26]. For each surface, 12 images recorded on areas of 1 x 1 µm were analyzed using the Data Processing software (Bruker, USA) to determine the arithmetic average roughness (Ra).

**AFM tip functionalization.** Functionalized tips were produced according to a procedure described in Jauvert et al. [27]. For that, MLCT cantilevers (Si₃N₄, Bruker, nominal spring constant of 0.01 N/m) were first cleaned using a plasma cleaner (Diener Electronics, Germany). They were then immersed in a solution containing 3.3 g of ethanolamine hydrochloride (reference E6133 Sigma-Aldrich) dissolved in 6.6 mL of DMSO (5 M, reference 472,301 Sigma-Aldrich), and incubated overnight. The tips were then washed three times in DMSO, three times in ethanol, and dried under nitrogen flow. Phosphorus dendrimers containing 96 aldehyde groups (DENG4-CHO, Dendris) were then attached

to the amino-tips previously produced by incubating them in a solution containing the dendrimer dissolved (58 µM) in tetrahydrofuran (THF, reference 34865, Sigma-Aldrich) for 5 h. After that, the tips were washed three times in THF, three times in ethanol, and dried under a nitrogen flow. These so-called “dendritips” were then incubated in a solution of In (0.1 mg·mL⁻¹) in phosphate buffer (50 mM, pH 8) for 1 h. A reduction step was then performed by adding a solution of NaBH₃CN (20 mM final concentration, in NaOH 5 M, reference 918,830 Sigma-Aldrich) for 15 min. Finally, the tips were washed three times in phosphate buffer and used right away for the AFM experiments.

**Single molecule Force Spectroscopy experiments.** Force spectroscopy experiments were carried out using a Nanowizard IV XP (Bruker, USA), in phosphate buffer (50 mM, pH 8). All the cantilevers used were first calibrated using the thermal noise method [26], their spring constant ranged between 0.020 and 0.022 N/m. In these experiments, the interactions between In-functionalized AFM tips and PDMSr surfaces functionalized with $Jo_{cys}$ were probed. For that, force-distance curves were recorded in buffer solution with an approach-retraction speed of 1 µm/s, a z-length of 1 µm, and at an applied force of 250 pN, used for all the measurements. For specific blocking of the interactions, the PDMSr surface functionalized with $Jo_{cys}$ was incubated with a solution of In at 1 mg·mL⁻¹ during 1 h. The surface was then rinsed with phosphate buffer and used to probe the interactions with In-functionalized AFM tips. For dynamic force spectroscopy experiments, the same experimental conditions were used, only the force curves were acquired using different retraction speeds, ranging between 1 and 10 µm/s. For contact time experiments, the same experimental conditions were used, only the contact time between the tip and the sample during force measurements varied in a range from 0 to 2 s.

## 2.6. Statistical tests

Data analysis was performed using GraphPad Prism 8 Software (La Jolla, CA, USA) and independent replicates data were plotted and expressed as mean ± SD (standard deviation). Unless noted, data were statistically analyzed using one-way ANOVA to compare all conditions together. In some cases, a simple Student's test was performed to compare data by pairs. Statistics are expressed relative to the control condition. ns: non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

## 3. Results and discussion

The desired specifications for the grafted surface were the following: readily accessible elastomer, possibly commercially, covalent binding of the enzyme, single point immobilization, high enough density of immobilization in order to have a strong variation upon stretching. In an ideal case, a common chemistry could also be used for different kinds of elastomer surfaces. Consequently, we decided to evaluate two types of commercially available elastomers, based on polyisoprene (PI) and polydimethylsiloxane (PDMS) structures. Both are readily available and well known for their high elasticity. In order to ensure a covalent binding of the enzymes, thiol-maleimide click reaction was chosen, as it is widely used in chemical transformations for biological molecules, and known to be quick and quantitative. Bearing in mind that we wanted to retain the intrinsic properties of the xylanase while ensuring the most rigid covalent linker possible, we used the Jo-In protein welding system [13]. The use of the Jo-In unit also allowed us to move the desired enzyme away from the surface, with the idea of ensuring full access to the substrate [11]. Therefore, based on all these considerations, the planned immobilization scheme (Fig. 1) proposed a surface activation through an argon plasma, followed by the introduction of maleimide functions on the surface. These could subsequently be used for the grafting of a cysteine-modified $Jo_{cys}$, followed by that of the In-NpXyn11A xylanase exhibiting the In entity for the efficient Jo-In coupling.
### 3.1. Immobilization of $Jo_{Cys}$ on the elastomer surfaces

Given the significance of understanding their surface morphology, PI and PDMS samples underwent characterization via Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) (Fig. 2) after a washing procedure. Based on the analysis of high-resolution height AFM images, the measured average roughness (Ra) was respectively ca. 48 nm and 27 nm for PI and PDMS. PI images clearly revealed the presence of particles. Further analysis by Scanning Electron Microscopy energy-dispersive X-ray spectroscopy (SEM-EDX) (Fig. S4) showed that these were probably mineral charges such as calcium or magnesium carbonate or talc, coming from the PI formulation. Indeed, these mineral additives are often used in order to limit adhesiveness and friction. Attempts to eliminate them by extensive washing proved inefficient. Therefore, it was decided to continue using this surface for the concept demonstration, with the assumption that enzymes could probably be also grafted on these particles. This point will be further discussed later in the text. Compared to PI, the PDMS surface appeared smoother, although here also the presence of surface irregularities could be observed, most likely resulting from the manufacturing of the film. These two commercial surfaces were estimated to be relevant for the evaluation of the concept. However, linked to the presence of undesired entities or defects on their surface, two further similar surfaces were also evaluated, although not in a thorough manner. They consisted in a polybutadiene (PB) system, entirely synthetic, based on an already published procedure [28], and another PDMS commercial surface which was reinforced by polyester fibers (PDMSr) (see supplementary information). These two surfaces were free from any particles on their surface and presented a low roughness below 10 nm (Fig. S5). However, it was not possible to obtain the PB in large amounts to perform the grafting of the enzymes. As for the reinforced PDMS, the presence of the polyester fibers strongly limited its elasticity, and it was therefore not usable for high stretching experiments.

Because the grafting of $Jo_{Cys}$ was planned from a thiol-maleimide reaction, the surfaces had first to be functionalized with maleimide groups. For this, an argon plasma was used, followed by treatment with

water to generate hydroxyl groups. The subsequent reaction between these and a modified maleimidohexanoic acid led to the desired grafting of maleimide units on the surfaces (Fig. 3).

The functionalization can be best evidenced by the evolution of the contact angle (Fig. 4). Indeed, starting from original hydrophobic surfaces with contact angles respectively at 80.0 ± 2.5° and 112.1 ± 1.8° for PI and PDMS surfaces, plasma treatment led to angles close to zero, followed by new angles of respectively 66.0 ± 5.0 and 82.2 ± 7.4 after addition of the maleimidohexanoic acid derivative (Table 1). Fig. 4A and B show that both plasma and chemical treatment are necessary to reach these new statistically different contact angles. The same evolution was observed for PB and PDMSr surfaces (Fig. S7-A, B). It is noteworthy that these measurements were performed after 15 min of the chemical treatment, ensuring that the surface had not enough time to rearrange yet. Indeed, elastomer surfaces are prone to spontaneous evolution [29,30], owing to the mobility of the polymeric chains. This phenomenon has also been evidenced in our case, both for hydroxyl- and maleimide- functionalized samples (Fig. S6).

The next step consisted in the thiol-maleimide reaction between the surfaces and $Jo_{Cys}$. Again, the reaction was evidenced by a new evolution of the contact angle (Fig. 4C and D), as the grafting of a protein is expected to decrease it. In order to assess the grafting of $Jo_{Cys}$ through the thiol-maleimide reaction, a modified $Jo_{W}$ protein with the cysteine residue changed into tryptophan was also used as a control. On the PI surface (Fig. 4C), $Jo_{W}$ had a tendency to adsorb, as evidenced by the lower contact angle for the sample without any maleimide treatment (column - Maleimide + $Jo_{W}$). This tendency explains the decrease of the contact angle for the column with maleimide treatment and with $Jo_{W}$ addition (column + Maleimide + $Jo_{W}$). The lowest contact angle is however obtained for the subsequent maleimide treatment and addition of $Jo_{Cys}$, corroborating that the covalent grafting was efficient. The case of PDMS is not straightforward (Fig. 4D). Indeed, spontaneous adsorption of both $Jo_{Cys}$ and $Jo_{W}$ was not observed on the initial surface. However, a significant adsorption of $Jo_{W}$ was observed on the PDMS surface modified by the maleimide groups (column + Maleimide + $Jo_{W}$). This difference of behavior of $Jo_{W}$ between initial and functionalized

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Surface characterization of the initial elastomer surfaces after washing. A, B: SEM of respectively PI and PDMS surfaces. The bars correspond to 5 µm. C, D: AFM height images of respectively PI and PDMS surfaces 10 × 10 µm areas, recorded in contact mode, with an applied force of 0.8 nN (MLCT cantilevers, k = 0.01 N/m), in air.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Fig. 3. Schematic diagram summarizing the various stages of surface functionalization with maleimide groups.</figcaption></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 4. Contact angle measurements for PI (A) and PDMS (B) surfaces for the maleimide functionalization, and after $Jo_{cys}$ grafting for PI (C) and PDMS (D).</figcaption></figure>

<table><caption>**Table 1**
Contact angles (°) of the different surfaces. n = 10, results given as mean ± standard deviation.</caption><thead><tr><th>Surface</th><th>Initial</th><th>After maleimide functionalization</th><th>After Jo<sub>cys</sub> grafting</th></tr></thead><tbody><tr><td>PI</td><td>80.0 ± 2.5</td><td>66.0 ± 5.0</td><td>44.2 ± 3.9</td></tr><tr><td>PDMS</td><td>112.1 ± 1.8</td><td>82.2 ± 7.4</td><td>38.4 ± 5.9</td></tr></tbody></table>

surfaces is not clear presently. Finally, here also, the lowest contact angle was observed for the surface functionalized with the maleimide groups and addition of $Jo_{cys}$ (44.2 ± 3.9° and 38.4 ± 5.9° for PI and PDMS, respectively), the difference with the $Jo_{W}$ control being still statistically different. The same trend was observed for the PB and PDMSr surfaces (Fig. S7 and Table S1).

While there is an extensive literature on the many strategies for protein immobilization on solid surfaces or elastomers, whether non-

specific or covalent, to our knowledge very few papers in practice confirm the physical reality of interactions between a protein of interest and a surface. In general, the experimental results obtained provide a reasonable confirmation of the theory. We attempted to clarify our results using AFM approaches on PDMSr as this surface was free from any particles with a roughness below 10 nm (Fig. S5). For this, we functionalized AFM tips with In following a well-established protocol to graft $NH_2$-containing molecules onto AFM tips [27]. We then probed the interactions directly with $Jo_{cys}$-functionalized PDMSr (maleimide + $Jo_{cys}$, Fig. 5A) in single-molecule force spectroscopy experiments. The results obtained showed that approximately 50 % of the force curves obtained presented adhesion events, for which the maximum force recorded was of 62.5 ± 23.1 pN (Fig. 5B). The force curves obtained presented one single peak (inset in Fig. 5A), confirming that only one interaction was probed. To examine whether these interactions are specific, we next performed blocking experiments by saturating the Jo proteins present on
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Single molecule force spectroscopy experiments. (A) Schematic representation of the experiment performed: AFM tips were functionalized with In (0.1 mg/mL) and used to probe the interactions with PDMSr surfaces functionalized with $Jo_{cys}$. (B) Histogram showing the distribution of the adhesion forces recorded for the interactions between In-functionalized tips and $Jo_{cys}$-functionalized PDMSr at a loading rate of 10,000 pN/s. (C) Schematic representation of the experiment performed: AFM tips were functionalized with In (0.1 mg/mL) and used to probe the interactions with PDMSr surfaces functionalized with $Jo_{cys}$ and saturated with an excess of In (1 mg/mL). (D) Histogram showing the distribution of the adhesion forces recorded for the interactions between In-functionalized tips and $Jo_{cys}$-functionalized PDMSr saturated with an excess of In at a loading rate of 10,000 pN/s. (E) Dynamic force spectrum of In-functionalized tips towards $Jo_{cys}$-functionalized PDMSr or n(F) maleimide-PDMSr. These graphs report the adhesion force vs the logarithm of the critical loading rates.</figcaption></figure>

the surface with In added in an excess concentration (1 mg·mL⁻¹, Fig. 5C). In this case, the adhesion frequency decreased from 50 % to 34.7 %, while the adhesion force recorded was also reduced from 62.5 to 45.4 pN (Fig. 5D). These results indicate two things: the interaction between $Jo_{cys}$ on the surface and In on the tip is indeed specific, as the saturation of $Jo_{cys}$ proteins by an excess of In resulted in the reduction of both the adhesion frequency and the adhesion force. However, the fact that interactions still take place (the adhesion probability is reduced, but not to 0) indicates that another interaction is also taking place. To understand this and to map the energy landscape of all the interactions taking place, we performed dynamic force spectroscopy (DFS) experiments in which the functionalized tip is approached and retracted from the surface at increasing loading rates, by varying the tip retraction speed. The results obtained are presented in Fig. 5E, where each dot on the graph corresponds to the critical loading rate, i.e. the loading rate at the exact rupture of the interaction. On this graph, two non-linear trends are observed, one taking place at loading rates comprised between 1000 and 40,000 pN/s, and the second one taking place at loading rates comprised between 40,000 and 200,000 pN/s. These non-linear trends are typical of intermolecular interactions between different entities as described by Friddle et al. [31]. Thus, these results indeed confirm that two different interactions are taking place when probing the interactions between $Jo_{cys}$-functionalized PDMSr and In-functionalized AFM tips. As described before, $Jo_{cys}$ proteins are immobilized via maleimide functions on the PDMSr, which could also perhaps interact with the In-functionalized tips. To test this hypothesis, DFS experiments were repeated on PDMSr surfaces presenting only the maleimide functions. The results obtained here show only one non-linear trend comprised between 1000 and 40,000 pN/s (Fig. 5F), indicating that on $Jo_{cys}$-functionalized surface, the tip could interacts both with Jo proteins and with maleimide functions if those become exposed. However, the presence of the 1000–40000 trend in Fig. 5E could also be linked to possible $Jo_{cys}$ multilayers, which will be further discussed. Indeed, in such case, the functionalized tip interacting with a $Jo_{cys}$ present in a multilayer

would not be covalently linked to the surface. This would lead to weaker interactions. To evaluate the specificity of both interactions, we performed additional experiments in which the interactions were probed in force spectroscopy using increasing contact time between the tip and the surface. The results obtained, presented in Supplementary Fig. S8 show that while the adhesion probability increases with the contact time for $Jo_{cys}$-In interactions (from 50 % at a contact time of 0 s to almost 80 % at a contact time of 2 s), this is not the case for maleimide-In interactions. The dependence of the probability of adhesion to the contact time in the case of the $Jo_{cys}$-In interaction further confirms that this interaction is specific. On the contrary, the fact that this dependence does not exist for weak maleimide-In interactions (note that In has no cysteine residue) or $Jo_{cys}$ multilayers-In indicates that this interaction is a non-specific interaction.

Jo and In are two small proteins (10.6 and 16.5 kDa, respectively) with no Cys residue but similar amino acid composition. Several recent papers have clearly demonstrated the specificity of the Jo-In association [32,33] and that this approach is suitable to immobilize NpXyn11A on silica [11]. However, non-specific interactions are inherent to protein immobilization on surfaces. In our case, we hypothesize a non-homogeneous immobilization, the possibility of forming multilayers or non-specific immobilization of $Jo_{cys}$ on particles present on surfaces (Fig. 4A). Indeed, the use of PB free form particles showed a slight decrease in the contact angle when $Jo_w$ was used as control instead of $Jo_{cys}$ (Fig. S7C) compared to PI and PDMS (Fig. 4C, 4D), which were much dirtier surfaces. Although composition is different, this stressed the importance of using clean, homogeneous surfaces.

However, from a general standpoint, grafting of subsequent maleimide functions and $Jo_{cys}$ significantly decreased the contact angle for all surfaces (Table 1, Table S1 and Fig. 6 and S9). Even if AFM experiment did not prove it for sure and underlined possible unspecific interactions, we made the reasonable assumption that $Jo_{cys}$ is mostly bound to the surface through a covalent thiol-maleimide bond.
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Overall evolution of the contact angles of PI and PDMS surfaces after each process step. Statistical tests were performed on the indicated pairs, using Student's test.</figcaption></figure>

## 3.2. Immobilization of In-NpXyn11A on the elastomeric surfaces

Glycoside hydrolases from family 11 (GH11) are broadly used to hydrolyze xylose-based polymers such as wheat arabinoxylan (WAX).

But the 4-nitrophenyl-β-D-xylotrioside ($pNP-X_3$) is widely used as chromogenic substrate to assess GH11 activity. The xylanase Xyn11A from *Neocallimastix patriciarum* (*NpXyn11A*) was previously immobilized on commercially available porous paramagnetic beads of 10 µm diameter functionalized with Jo [11], using In fused to the N-terminus of the amino acid sequence of the xylanase. In this study, a similar strategy was used to immobilize In-NpXyn11A onto different elastomers. We first confirmed that the presence of the In domain in fusion with the catalytic one did not modify the activity of the enzyme toward both $pNP-X_3$ and WAX (Table S2).

Initial attempts were made to quantify the amount of In-NpXyn11A immobilized on elastomer. However, a standard approach consisting of measuring the remaining amount of enzyme in the supernatant after the immobilization procedure [11] was unsuccessful, mainly due to the low specific surface area of our elastomer compared to porous silica beads and the very low amount of immobilized enzyme, making difference measurement with loading amount uncertain. As previously demonstrated on solid surface [11], we hypothesized that the immobilization of the xylanase on elastomer would not modify its intrinsic activity. Thus, the amount of immobilized In-NpXyn11A was assessed indirectly by comparing enzymatic activity on the functionalized surfaces to a calibration curve established with increasing amount of enzymes in solution (Fig. S10A). For this, $pNP-X_3$, was used and its hydrolysis was monitored by UV-vis spectroscopy on the basis of the characteristic absorption at 401 nm of the phenolate. The small molecular weight of $pNP-X_3$ (535 g.mol⁻¹), making this substrate much smaller than the enzyme (42,500 g.mol⁻¹), can reach the pocket of the enzyme active site independently of steric hindrance or deleterious interactions with the surface. This method initially allowed us to verify that hydrolysis of $pNP-X_3$ occurred independently of enzyme density. Indeed, hydrolysis of $pNP-X_3$ was conducted with In-NpXyn11A immobilized on a PB surface at an initial grafting density of 18.6 nmol.m⁻² (Table S3), followed by two stretching stages (25 % and 131 %), leading to a density of grafting of 13 nmol.m⁻² and 6.5 nmol.m⁻², respectively. The measured specific activity was comparable to the free enzymes (3916 ± 430 IU.mg⁻¹, Fig. S10B).

With the active modified surfaces in hand, the next step consisted in evaluating the hydrolysis of a large substrate, WAX, with a molecular weight of 323,000 g.mol⁻¹. As described in the experimental part, the developed set-up enabled monitoring the enzymatic reaction at different stretching states for the surface. The stretching was chosen in order to be far from the breaking conditions, which are typically close to 1 000 % for elastomers [34]. Since In-NpXyn11A has been covalently immobilized on the elastomer via a single-point grafted $Jo_{cys}$, we assume that

<figure><img src="image_11.png" /><figcaption>**Fig. 7.** A: Enzymatic activity of a PI surface with increasing stretching step. Black columns represent the activity from the PI surface with no stretch (PI-0) and increasing stretching of 8 % (PI-1), 34 % (PI-2), 111 % (PI-3) and 224 % (PI-4). Green columns represent the activity in the solutions from which the PI surface was removed (from S1 to S5). B: SDS-PAGE of concentrated proteins present in solutions S1 to S5 (lane 3 to 7) released from the PI surface (PI-0 to PI-4, respectively). Protein $Jo_{cys}$ and In-NpXyn11A are used as internal markers (lane 1 and 2, respectively). The theoretical molecular weight of each putative protein is indicated and a black arrow locates $Jo_{cys-cys}$, In-NpXyn11A complex. Lane M stands for molecular mass markers. 1 Da = 1 g.mol⁻¹. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
stretching the surface would not affect the enzyme's structural conformation and thus would not impact its activity. The protocol facilitated the monitoring of enzyme kinetics sequentially on the same surface at different stretch levels, using fresh reaction medium after each stretch change, as well as monitoring this kinetics in the reaction medium once the membrane has been removed. A first experiment on PI surface led to the following results regarding the enzymatic activity (Fig. 7A). On this graph, black columns indicate the enzymatic activity from the surface at increasing stretching step (see Table S4), and the green ones the enzymatic activity measured in the solution (after removing the PI). The enzymatic activity from PI increased slightly with increasing surface stretch (from PI-0 at 22.9 µmol.L⁻¹.min⁻¹ to PI-4 at 36.9 µmol.L⁻¹.min⁻¹), although maximum stretch had the lowest activity (PI-5 at 18.2 µmol.L⁻¹.min⁻¹). If no enzymes were released, there should not have been any activity observed in the solution. Our results evidenced that a form of the enzyme was present in solutions S1 to S5 after increased stretching, even if the activity is decreasing from 12.4 µmol.L⁻¹.min⁻¹ to 0.6 µmol.L⁻¹.min⁻¹, respectively) SDS-PAGE analysis of the different solutions (Fig. 7B) revealed a single band for S1 to S3 whose apparent molecular weight could correspond to a $Jo_{cys-cys}Jo-In-NpXyn11A$ complex (10,600 + 10,600 + 16,500 + 26,000 = 63,700 g.mol⁻¹). This complex may result from a possible covalent dimerization of non-covalently bound $Jo_{cys}$ resulting from multilayers during the grafting process. Such behavior is not rare during protein immobilization process, intended or not [35], as overloading can cause protein multi-layers to form. This observation mirrored the dynamic force spectroscopy data, as the In functionalized AFM tip may have simulated the behavior of our enzyme. No bands were observed for S4 and S5 (Fig. 7B), reflecting a much lower specific activity (6.7 µmol.L⁻¹.min⁻¹ and 0.6 µmol.L⁻¹.min⁻¹, respectively) compared to S1, S2 and S3 (12.4 µmol.L⁻¹.min⁻¹, 13.7 µmol.L⁻¹.min⁻¹ and 9.1 µmol.L⁻¹.min⁻¹, respectively). Even though we did not appreciate this unexpected result in the experiment above on pNP-X₃, it will be taken into account below. However, the absence of any detectable band corresponding to $Jo_{cys-In-NpXyn11A}$ (53,100 g.mol⁻¹), or In-NpXyn11A (42,500 g.mol⁻¹) in the solution at this time may confirm a dominant association of In-NpXyn11A with immobilized $Jo_{cys}$ on the PI surface.

In light of this observation, subsequent experiments were conducted to ascertain the specific enzymatic activity of PI surface in question, contingent on their degree of stretching (Fig. 8). Additionally, a systematic measurement of the enzymatic activity of the solution was undertaken following the removal of the elastomers, with a view to assessing the release of enzymes from the surfaces in question. The principal objective was to take into account the amount of released enzyme in order to make any requisite adjustments in the amount of

enzyme immobilized on the surface, and therefore grafting density (Table 2).

Upon initial observation of Fig. 8A, it becomes evident that the specific activity of immobilized xylanase increased as the surface was stretched and the enzyme surface density decreased (Table 2), to approach the specific activity of In-NpXyn11A free in solution (3917 IU. mg⁻¹). This behavior is in accordance with previously published findings on a solid surface at varying grafting densities of In-NpXyn11A [11]. The explanation depends on steric effects relative to the dimensions of the substrate and the average center-to-center distance between neighboring enzymes. (the denser the graft, the smaller the center-to-center distance). When the distance in question is relatively small in comparison to the dimensions of the polymer, a chain that interacts with one enzyme on the surface may occupy space in a manner that precludes enzymes in immediate proximity from being available to other polymer chains or even from properly interacting with other regions of the same polymer chain. Consequently, these enzymes would become less efficient for a certain time period, which would in turn result in a reduction in the overall activity of the system. As shown in Fig. 8B, the specific activity of the immobilized enzyme seemed to be proportional to the surface density of grafting.

Table 2 illustrates additional aspects of this experience. The PI surface underwent multiple consecutive stretches, with a specific activity measurement performed on the same completely relaxed surface for each stretch. It can be observed that the surface grafting density values remain equivalent before and after stretching, although there is a compensatory loss of immobilized enzyme. We hypothesized that the released enzymes might correspond to the $Jo_{cys-cys}Jo-In-NpXyn11A$ complex described above and that the amount of covalently immobilized enzymes would remain constant during stretching. By subtracting the enzymatic activity of the released enzymes from the background, we can determine the amount of covalently immobilized enzyme, and thus, the grafting density, which decreases as the elastomer is stretched. Although these experiments have not been repeated, the results indicate that in one case the specific activity is not retained (PI-1, from 1321 IU. mg⁻¹ to 2143 IU.mg⁻¹), while in another it is (PI-2, from 2804 IU.mg⁻¹ to 2845 IU.mg⁻¹). The second aspect is the total amount of released enzyme (54.4 ng), representing 75 % of the initial amount immobilized on PI (72.5 ng). A comparison should be made between the aforementioned result and the estimated surface area occupied by talc on the PI surface, as observed under a scanning electron microscope (Fig. 2A). The PI surface exhibited an average charge size of $15.6 \times 10^{-2} \mu m^2$ over approximately 35 % of the surface area. Following stretching and relaxation, this value decreased to an average charge size of $7.3 \times 10^{-2} \mu m^2$ over 12 % of the surface area (data not shown). Similar experiments

<figure><img src="image_12.png" /><figcaption>**Fig. 8.** A: Specific activity of In-NpXyn11A immobilized on PI surface at different enzymatic densities obtained by stretching the surface (PI-0 to PI-3, black columns) compared to the specific activity of In-NpXyn11A free in solution (FE, green column). This series has been performed by allowing the surface to relax before a new stretching was applied. Amount of immobilized enzyme has been corrected from the amount of enzyme recovered on solution (Table 2). B: Corresponding relationship between surface enzymatic density and specific activity. A trend curve has been added for eyes. Enzymatic activities were assessed on wheat arabinoxylan 1 % w/v, in 50 mM phosphate, 1 mg/mL BSA, pH 6, in a reaction volume of 1.7 mL. (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_13.png" /></figure>
<caption>**Table 2**</caption>

Amount of immobilized on PI and released In-NpXyn11A based on enzymatic activity. These values were used to determine the grafting surface density on the PI surface at different stretches.

<table><thead><tr><th rowspan="2"></th><th colspan="10">Samples</th></tr><tr><th>PI-0</th><th>S-0</th><th>PI-1</th><th>S1-1</th><th>PI-1</th><th>S1-2</th><th>PI-2</th><th>S2-1</th><th>PI-2</th><th>S2-2</th><th>PI-3</th><th>FE</th></tr></thead><tbody><tr><td>Streching (%)</td><td>0</td><td>-</td><td>29</td><td>-</td><td>0</td><td>-</td><td>36</td><td>-</td><td>0</td><td>-</td><td>183</td><td>-</td></tr><tr><td>Surface<sup>a</sup> (10<sup>-4</sup> m<sup>2</sup>)</td><td>2.95</td><td>-</td><td>3.81</td><td>-</td><td>2.95</td><td>-</td><td>4.00</td><td>-</td><td>2.95</td><td>-</td><td>8.33</td><td>-</td></tr><tr><td>Enzyme concentration<sup>b</sup> (nmol.L<sup>-1</sup>)</td><td>1.05</td><td>0.21</td><td>0.84</td><td>0.18</td><td>0.66</td><td>0.15</td><td>0.51</td><td>0.08</td><td>0.43</td><td>0.17</td><td>0.26</td><td>0.75</td></tr><tr><td>Amount of released enzyme (ng)</td><td>-</td><td>14.1</td><td>-</td><td>12.5</td><td>-</td><td>10.4</td><td>-</td><td>5.6</td><td>-</td><td>11.8</td><td>-</td><td>52.2</td></tr><tr><td>(10<sup>-3</sup> nmol)</td><td></td><td>0.34</td><td>-</td><td>0.30</td><td>-</td><td>0.25</td><td>-</td><td>0.13</td><td>-</td><td>0.29</td><td>-</td><td></td></tr><tr><td>Amount of immobilized enzyme (ng)</td><td>72.5</td><td>-</td><td>58.4</td><td>-</td><td>45.9</td><td>-</td><td>35.5</td><td>-</td><td>29.9</td><td>-</td><td>18.1</td><td>-</td></tr><tr><td>(10<sup>-3</sup> nmol)</td><td>1.79</td><td>-</td><td>1.44</td><td>-</td><td>1.13</td><td>-</td><td>0.87</td><td>-</td><td>0.73</td><td>-</td><td>0.45</td><td>-</td></tr><tr><td>Grafting density (nmol/m<sup>2</sup>)</td><td>6.08</td><td>-</td><td>3.79</td><td>-</td><td>3.81</td><td>-</td><td>2.16</td><td>-</td><td>2.48</td><td>-</td><td>0.53</td><td>-</td></tr></tbody></table>

PI stands for activity measured on elastomer surface and S stands for activity measured in solution, after removing the surface. PI-1 and PI-2 are the original surface PI-0 stretched and relaxed. FE stands for enzyme free in solution.

were conducted on PDMS and PDMSr (Fig. S11 and S12, Table S5 and S6), even though PDMSr, as a reinforced elastomer, cannot be stretched by more than 20 %.

The investigation of the influence of enzyme proximity on catalytic activity represents a novel research avenue. Our previous studies [11] have demonstrated the potential value of this approach, particularly in elucidating the impact of enzyme proximity on the product profile of a family 11 xylanase acting on a polysaccharide. To this end, we devised an innovative immobilization technique on silica beads, which we sought to adapt to elastomers. The objective was to facilitate the manipulation of enzyme proximity. Our current work has identified several aspects of protein immobilization on surfaces that have been previously described in the literature, particularly with regard to non-specific interactions. However, our data demonstrates the efficacy of our immobilization strategy. Indeed, when the specific activities conducted on three distinct elastomer types are gathered, a correlation between graft density and enzymatic activity is evident (Fig. 9). The greater the distance between enzymes, the closer their activity is to that of the free enzyme in solution. However, the various elastomers may have physical and mechanical properties that could marginally interfere with our data. Additionally, our data illustrates the challenges associated with immobilization on surfaces with high levels of contamination, especially regarding particules. This final factor has a significant influence on the effective quantity of immobilized enzymes, which in turn affects the homogeneity of the grafting. This underlines the importance of working with controlled elastomers, such as PB. The convergence of multiple research disciplines offers promising avenues for understanding the dynamics of enzyme interactions during catalysis. This encompasses the study of enzyme distances in metabolic pathways and natural complexes such as cellulosome [36], where one or more enzymes with diverse and complementary activities are involved. Additionally, it provides a potential development pathway for industrial processes, enabling enhanced control over product characteristics through adjustments in enzyme-substrate distances and interactions.

**CRediT authorship contribution statement**

**Maxime Artico:** Writing – review & editing, Investigation, Formal analysis. **Cécile Formosa-Dague:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Dimitri Kamak:** Investigation. **Frédéric Peruch:** Writing – review & editing, Methodology, Formal analysis. **Anne-Françoise Mingotaud:** Writing – review & editing, Writing – original draft, Methodology, Funding acquisition, Formal analysis. **Clément Roux:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Cédric Y. Montanier:** Writing – review & editing, Writing – original draft, Methodology, Funding acquisition,

<figure><img src="image_14.png" /><figcaption>**Fig. 9.** Grouping all paired points (Specific activity/Grafting enzymatic density) from the different surfaces investigated in this study. PI in red, PDMS in green and PDMSr in blue. The horizontal dash line is the. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

Formal analysis, Conceptualization.

**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.

**Acknowledgements**

This work is financially supported by Université Fédérale Toulouse Midi-Pyrénées, INSA Toulouse, Région Occitanie and the authors would like to thank the FERMaT Research Federation for its financial support in the form of a starter pack grant.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.org/10.1016/j.apsusc.2025.163554.

<footnote>a The surface area as determined as described in the supplementary information</footnote>

<footnote>b The enzyme concentration was determined as described, based on the equivalent concentration of free enzyme.</footnote>
# Data availability

Data will be made available on request.

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