Louise Badruna

Vincent Burlat

Pierre Roblin

Thomas Enjalbert

Guy Lippens

Immacolata Venditto

Michael J O'donohue

Cédric Y Montanier
email: cedric.montanier@insa-toulouse.fr

The Jo-In protein welding system is a relevant tool to create CBM-containing plant cell wall degrading enzymes

Keywords: GH family 11 endo-1,4-β-xylanase, CBM family 2, CBM family 3, Bio Molecular Welding, spatial proximity ray scattering, MST -Microscale Thermophoresis, ITC -Isothermal titration calorimetry, NMR -Nuclear Magnetic Resonance, RC -regenerated cellulose, CNcellulose nanocrystals

Irrespective of their biological origin, most proteins are composed of several elementary domains connected by linkers. These domains are either functionally independent units, or part of larger multidomain structures whose functions are defined by their spatial proximity. Carbohydrate-degrading enzymes provide examples of a range of multidomain structures, in which catalytic protein domains are frequently appended to one or more noncatalytic carbohydrate-binding modules which specifically bind to carbohydrate motifs. While the carbohydrate-binding specificity of these modules is clear, their function is not fully elucidated. Herein, an original approach to tackle the study of carbohydrate-binding modules using the Jo-In biomolecular welding protein pair is presented. To provide a proof of concept, recombinant xylanases appended to two different carbohydrate-binding modules have been created and produced. The data reveal the biochemical properties of four xylanase variants and provide the basis for correlating enzyme activity to structural properties and to the nature of the substrate and the ligand specificity of the appended carbohydrate-binding module. It reveals that specific spatial arrangements favour activity on soluble polymeric substrates and that activity on such substrates does not predict the behaviour of multimodular enzymes on insoluble plant cell wall samples. The results highlight that the Jo-In protein welding system is extremely useful to design multimodular enzyme systems, especially to create rigid conformations that decrease the risk of intermodular interference. Further work on Jo-In will target the introduction of varying degrees of flexibility, providing the means to study this property and the way it may influence multimodular enzyme functions.

Introduction

Domains constitute the key building blocks of proteins, conferring their structural integrity and/or functionalities. They can form independent structural and or functional units, but are often combined in multidomain organizations in which neighboring domains associate to define protein structures and/or functions [START_REF] Wang | Protein domain identification methods and online resources[END_REF]. The majority of proteins from all taxa are composed of several domains connected by linkers [START_REF] Apic | Domain combinations in archaeal, eubacterial and eukaryotic proteomes[END_REF][START_REF] Apic | Multi-domain protein families and domain pairs: comparison with known structures and a random model of domain recombination[END_REF]. This quite complex organization is probably the source of functional diversity and also responsible for functional fine-tuning [START_REF] Altschuh | Modulation of the enzymatic activity of papain by interdomain residues remote from the active site[END_REF], including that of enzymes whose catalytic sites are often formed at the interface of several domains [START_REF] Valentini | The Allosteric Regulation of Pyruvate Kinase[END_REF].

Plant cell wall-degrading enzymes provide excellent examples of multidomain proteins.

This large group is characterized by a multitude of structures and functions, their diversity matching the complexity of plant cell wall (PCW) structures [START_REF] Lombard | The carbohydrate-active enzymes database (CAZy) in 2013[END_REF]. Lignified PCWs are macromolecular networks, composed of cellulose, hemicelluloses, pectins, proteins and lignins that interact and, in some cases, crosslink to form insoluble, three dimensional matrices [START_REF] Amos | Critical review of plant cell wall matrix polysaccharide glycosyltransferase activities verified by heterologous protein expression[END_REF]. The main enzymes involved in the breakdown of PCWs are glycoside hydrolases (GHs), a large and diverse group including cellulases, hemicellulases and pectinases. Most GHs display modular architecture, containing catalytic and non-catalytic domains [START_REF] Wu | Multimodularity of a GH10 Xylanase Found in the Termite Gut Metagenome[END_REF][START_REF] Henrissat | Glycoside Hydrolases and Glycosyltransferases. Families, Modules, and Implications for Genomics[END_REF]. Regarding the latter, carbohydrate binding modules (CBMs) are prominent.

When appended to a catalytic module, CBMs target specific regions in polysaccharides, consequently increasing the local concentration of catalytic domains and favoring intimate contacts between the substrate and the enzyme. In certain cases, CBMs also disrupt the surface of tightly packed polymers, such as cellulose or starch, thus facilitating enzyme action [START_REF] Shoseyov | Carbohydrate binding modules: biochemical properties and novel applications[END_REF].

In PCW-degrading enzymes, domain organizations are numerous and quite varied. In some cases, the catalytic GH domain and the CBM are combined in a single prolonged domain, while in others the CBM is appended to the GH via a linker peptide of variable size (from 4 to 158 residues), composition and structure [START_REF] Sammond | Cellulase linkers are optimizedbBased on domain type and function: insights from sequence analysis, biophysical measurements, and molecular simulation[END_REF]. Considering the number of known GH and CBM domains and the number of possible combinations, the organizational diversity of PCW-degrading enzymes is considerable [START_REF] Sidar | Carbohydrate binding modules: diversity of domain architecture in amylases and cellulases from filamentous microorganisms[END_REF].

Previous studies described the creation of synthetic multimodular GH constructs [START_REF] Pasari | Impact of Module-X2 and Carbohydrate Binding Module-3 on the catalytic activity of associated glycoside hydrolases towards plant biomass[END_REF][START_REF] Cuskin | How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity[END_REF][START_REF] Herve | Carbohydratebinding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects[END_REF] designed to investigate enzymatic activity. Often, a domain fusion strategy was adopted, using short linker sequences to associate different domains in recombinant proteins. While this strategy is frequently employed, its success hinges on expression of the fusion protein and requires the cloning and expression of each studied domain combinations. An alternative route to obtain a range of different domain combinations is to express single domains separately and then perform module linkage in a second in vitro step. Several strategies to achieve this have also been described [START_REF] Shah | Inteins: nature's gift to protein chemists[END_REF][START_REF] Pinto | An expanded library of orthogonal split inteins enables modular multi-peptide assemblies[END_REF][START_REF] Zhao | Assembly of branched ubiquitin oligomers by click chemistry[END_REF][START_REF] You | Multivalent chelators for spatially and temporally controlled protein functionalization[END_REF][START_REF] Singh | Self-Assembly of a Functional Triple Protein: Hemoglobin-Avidin-Hemoglobin via Biotin-Avidin Interactions[END_REF][START_REF] Fierobe | Design and Production of Active Cellulosome Chimeras[END_REF]. One of the more recent additions to the protein engineer's toolbox is genetically encoded click chemistry (GECC), based on a naturally occurring phenomenon identified in bacterial pili, where certain protein subunits are linked together via an isopeptide bond [START_REF] Sun | Unleashing chemical power from protein sequence space toward genetically encoded "click" chemistry[END_REF]. Exploiting this for protein engineering has led to the development of SpyTag-SpyCatcher [START_REF] Zakeri | Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin[END_REF] and the Biomolecular Welding tool [START_REF] Bonnet | Autocatalytic association of proteins by covalent bond formation: a Bio Molecular Welding toolbox derived from a bacterial adhesin[END_REF]. The latter comprises two proteins, designated Jo and In (10.6 and 16.5 kDa, respectively), which spontaneously form an intramolecular isopeptide bond when mixed in solution, leading to a two-domain protein measuring 6 nm in length. When Jo and In are individually fused to other protein domains, it is possible to create domain combinations in vitro, with the Jo-In intramolecular complex acting as the linker. Accordingly, Jo-In were recently used to combine two different GHs, producing bifunctional enzymes [START_REF] Enjalbert | Characterisation of the effect of the spatial organisation of hemicellulases on the hydrolysis of plant biomass polymer[END_REF].

In the current study, focusing on the well characterized xylanase Xyn11A from Neocallimastix patriciarum [START_REF] Vardakou | Understanding the structural basis for substrate and inhibitor recognition in eukaryotic GH11 xylanases[END_REF] Jo-In is used to link this GH to two different, non-cognate CBMs of bacterial origin either targeting the substrate of Xyn11A (xylan) or another PCW polymer (cellulose), the aim being to understand how the specific properties of these CBMs affect enzyme activity on simple substrates and complex PCW networks.

Materials and methods

Gene cloning

Plasmid constructs used are summarized in Supplementary Table S1. For some cloning purposes, PCR (Phusion™ High-Fidelity DNA Polymerase, ThermoFischer Scientific, Waltham, MA, USA) was used to amplify target sequences, generally introducing restriction enzyme target sequences, and to introduce PCR amplicons into linearized plasmid vectors by homologous recombination (In-Fusion® HD cloning kit, Clonetech, Mountain View, CA, USA). Alternatively, target sequences were synthesized by Genscript HK limited (Piscataway, NJ, USA) introducing appropriate restriction enzyme target sequences for subsequent plasmid construction.

Protein expression and purification

Proteins were expressed in Escherichia coli strain BL21 (DE3) or Tuner (DE3) harbouring the relevant plasmids and purified using standard methods described in Supplementary Table S2.

Covalent chimeric protein complexes

To prepare chimeric protein complexes, purified Jo and In fusion proteins were mixed, using a slightly lower concentration for the xylanase than for the CBMs (typically 2 µmoles of xylanase for 3 µmoles of CBM, 8.3 and 8.8 mg/ml of protein, respectively), for 1 h at 21°C and then stored overnight at 4 °C. Protein complexes were isolated from solution using a XK16 Hiload 16/600 Superdex S75 prep-grade gel filtration column (GE Healthcare Life Sciences, Chicago, IL, USA) connected to an Äkta Pure system. Elution was performed at 1 mL/min using 50 mM sodium phosphate buffer pH 7.4 supplemented with 150 mM NaCl.

Subsequently, NaCl was removed by dialysis and purified chimeric complexes were judged homogenous by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE).

Enzymatic activity measurements

The apparent kinetic parameters KMapp, Vmax, kcat, and kcat/KM app of Jo-NpXyn11A were measured using the dinitrosalicylic acid (DNSA) assay as previously described [START_REF] Enjalbert | Characterisation of the effect of the spatial organisation of hemicellulases on the hydrolysis of plant biomass polymer[END_REF], using various concentrations (0.3 to 30 mg/mL) of beechwood xylan (BWX, Megazyme) in activity assay buffer (50 mM sodium phosphate, 12 mM sodium citrate pH 6, supplemented with 1 mg/mL of bovine serum albumin (BSA, Merck KGaA, Darmstadt, Germany). DNSA assays were performed to determine the specific activity (SA) of xylanase derivatives (5 nM) on 0.5% (w/v) wheat arabinoxylan (WAX), 0.5% (w/v) rye arabinoxylan (RAX) or 1% (w/v) BWX respectively (all from Megazyme, Bray, Ireland) in 50 mM Tris-HCl pH 7.5, supplemented with 1 mg/mL of BSA as previously described [START_REF] Montanier | Changing surface grafting density has an effect on the activity of immobilized xylanase towards natural polysaccharides[END_REF]. SA of the xylanases (100 nM) was also determined using 4-nitrophenyl-β-D-xylotrioside (pNP-X3, LIBIOS, France) using 5 mM of substrate in activity assay buffer as previously described [START_REF] Enjalbert | Characterisation of the effect of the spatial organisation of hemicellulases on the hydrolysis of plant biomass polymer[END_REF]. SA were expressed in µmoles of product formed per min per µmole of enzyme (IU/µmole) in order to integrate the differences of mass concentration [START_REF] Enjalbert | Characterisation of the effect of the spatial organisation of hemicellulases on the hydrolysis of plant biomass polymer[END_REF]. To investigate enzyme activity on complex substrates, 5 g destarched wheat bran and 10 g wheat straw (0.5 mm) (both from ARD, Pomacle, France), were each washed in 2 L of deionized water for 1 h at 4 °C and recovered by filtration (0.45 µm) before drying at 50 °C for 3 d. For enzyme assays, wheat bran or straw (2 % w/v) were incubated overnight in 1.9 mL of activity assay buffer before the addition of enzyme (final concentration 1 µM). Reactions were conducted at 37 °C under constant mixing at 1200 rpm (ThermoMixer ® C, Eppendorf, Hamburg, Germany). Reaction progress was monitored by regular sampling as previously described [START_REF] Enjalbert | Characterisation of the effect of the spatial organisation of hemicellulases on the hydrolysis of plant biomass polymer[END_REF]. All experiments were performed in triplicate, and the reported values are the means of three experiments ± SD. Kinetic parameters were derived from the data using the Michaelis-Menten equation embedded in SigmaPlot 11.0 (Systat Software,San Jose, CA, USA).

Isothermal titration calorimetry (ITC)

ITC measurement was carried out using a MicroCal VP-ITC titration calorimeter (Malvern Panalytical, Malvern, UK) as detailed in Supporting Information. Integrated heat effects were analyzed by non-linear regression using a single-site binding model (Microcal ORIGIN software, version 7.0, Microcal Software), yielding values for the association constant Ka (M - 1 ) and the binding enthalpy ΔH (J.mol -1 ). Other thermodynamic parameters were calculated using the standard thermodynamic equation:

-RTlnKa= ΔG=ΔH-TΔS.

Microscale Thermophoresis (MST)

MST measurement was carried out using a Monolith NT115 (NanoTemper Technologies GmbH, München, Germany) at 25 °C, 20% LED power and 40 % MEST power as detailed in Supporting Information. Data analysis was performed with MO Affinity software 2.1 (Nanotemper). The Hill equation was chosen to determine a value for EC50 [START_REF] Brandizzi | [END_REF]. EC50 is the half-maximal effective concentration, meaning the more affinity for the substrate is, the smaller the value of the EC50 will be.

Nuclear Magnetic Resonance (NMR)

NMR experiments were performed on a Bruker Avance III HD 800 MHz spectrometer equipped with a 5 mm quadruple resonance QCI-P (H/P-C/N/D) cryogenically cooled probe head (Bruker, Billerica, MA, USA) as detailed in Supporting Information. Analysis of the data and fitting of the chemical-shift perturbation to the standard equation for a saturation isotherm was performed using SigmaPlot 11.0 (Systat Software, San Jose, CA, USA).

Small angle X-ray scattering (SAXS)

SAXS measurements were performed at Laboratoire de Génie Chimique, Toulouse, on the XEUSS 2.0 bench (Xenocs, Grenoble, France) equipped with a copper internal source (Genix3D) that produces a X-ray beam (8 keV and 30.10 6 ph.s -1 ) providing a size resolution of approximately 500 × 500 μm. Proteins were concentrated to approximately 10 mg/mL using a centrifugal filter device (Amicon® Ultra 30 or 50K, Merck KGaA). To remove aggregates and obtain a monodisperse solution, samples (50 µL) were injected onto a size exclusion column mounted on a HPLC coupled to the SAXS. For direct analysis, sample aliquots (40 µL) were transferred from the sample holder (maintained at 18 °C using a circulating water bath) to the measurement cell placed under vacuum to limit air absorption.

Data were collected on a 150 × 150 mm area DECTRIS detector (Pilatus 1M) at a sample-detector distance of 1.216 m, thus procuring a measurement range from 0.005 to 0.5 Å -1 .

Each sample dataset is an average of at least 6 measurements with a data collection time of 1,800 s. The averaged curves obtained using direct injection and SEC-HPLC were merged to obtain a composite curve devoid of an aggregation contribution at small angles and displaying low noise at high angles. Finally, to obtain the absolute scattering intensity I(q) for the solutes, the background buffer solution contribution was subtracted from the total SAXS profile. Data integration and reduction were performed using FOXTROT software. The biophysical parameters, such as gyration radius (Rg), maximal distance (Dmax) and Porod volume were calculated using PRIMUS [START_REF] Konarev | PRIMUS : a Windows PC-based system for small-angle scattering data analysis[END_REF] from the ATSAS suite. Low resolution shapes were calculated with DAMMIF and rigid body molecular modelling using the SAXS data was performed using CORAL.

Paraffin embedding, microtomy, on-section enzymatic digestion and double immunofluorescence labelling.

Sample preparation was performed essentially as previously described [START_REF] Badruna | CBMs as probes to explore plant cell wall heterogeneity using immunocytochemistry[END_REF] with minor modifications. Briefly, wheat straw (1 cm long) and wheat bran were fixed in acetic acid/ethanol without aldehyde, infiltrated in paraplast and assembled as tissue arrays in paraplast. Tissue arrays corresponded to hundreds of wheat bran fragments and at least four wheat straw cross sections. Tissue array blocks were soaked for several weeks in acetic acid/ethanol softening solution at 4 °C [START_REF] Brandizzi | [END_REF] and 14 µm thick serial sections were displayed on silane coated slides. Individual slides were dewaxed and covered with a microincubation chamber (22×40×0.2 mm deep; (200 μL), #70324-20, Electron Microscopy Science, Hatfield, PA, USA) containing either 50 mM sodium-phosphate 12 mM citrate buffer pH 6 alone or containing enzymes (300 nM final concentration). Slides were incubated for 24 h at 37 °C in a humid atmosphere and then recombinant enzymes were digested with proteinase K (5 µg/ml in 0.1 M Tris-HCl pH 8-50 mM EDTA pH 8) for 30 min at 37 °C. Double immunofluorescence labelling was performed as previously described [START_REF] Francoz | Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains[END_REF] using xylanspecific LM11 monoclonal antibody (PlantProbes, Leeds, UK) and cellulose specific His6-Tagged CtCBM3a recombinant protein (PlantProbes, Leeds, UK) as primary probes. These were labelled with goat anti-rat IgG-Alexa Fluor 488 (ThermoFischer, Waltham, MA, USA) and His6-Tag monoclonal antibody (4E3D10H2/E3)-Alexa Fluor 555 (ThermoFischer, Waltham, MA, USA), respectively. Slides were mounted in ProLong™ Gold Antifade mounting medium (ThermoFischer, Waltham, MA, USA) and scanned with a Nanozoomer 2.0 RS scanner (Hamamatsu photonics, Hamamatsu City, Japan) using a 40x objective and 7×1 µm Z stacks. Scans were analysed with NDP view (Hamamatsu photonics). The lignin autofluorescence observed for the A488 channel in all the cell walls from the untreated/unlabelled sections was set to a minimum threshold to observe the LM11/A488 specific labelling on the other sections. No such an autofluorescence is seen for the A555 channel. Figures were assembled using Photo-paint (Corel draw graphics suite 2018, Corel, Ottawa, Canada).

Results and Discussion

Investigating the impact of alternative multidomain arrangements on biological activity

In previous work [START_REF] Enjalbert | Characterisation of the effect of the spatial organisation of hemicellulases on the hydrolysis of plant biomass polymer[END_REF] it was demonstrated how Jo and In can be used to conveniently link proteins together [START_REF] Bonnet | Autocatalytic association of proteins by covalent bond formation: a Bio Molecular Welding toolbox derived from a bacterial adhesin[END_REF]. Therefore, Jo and In were exploited to create a series of GH-CBM chimeric proteins based on NpXyn11A. The expression of His6-tagged NpXyn11A linked to either Jo or In at its N-terminus [START_REF] Montanier | Changing surface grafting density has an effect on the activity of immobilized xylanase towards natural polysaccharides[END_REF] yielded active enzymes. Data regarding In-NpXyn11A is already available [START_REF] Montanier | Changing surface grafting density has an effect on the activity of immobilized xylanase towards natural polysaccharides[END_REF] so work was restricted to the comparison of the kinetic parameters of Jo-NpXyn11A with those of wild type NpXyn11A (Supplementary Table S3). Values of KM app for In-NpXyn11A and Jo-NpXyn11A (1.8 mg.mL -1 and 2.76 mg.mL -1 ) are 2.4-and 3.7fold higher than that of the wild type enzyme (0.75 mg.mL -1 ), indicating that the affinity for BWX is lowered. As discussed previously [START_REF] Enjalbert | Characterisation of the effect of the spatial organisation of hemicellulases on the hydrolysis of plant biomass polymer[END_REF], additions at the N-terminal extremity of the NpXyn11A probably hinder the flexible loops that connect the β-sheets and form the catalytic pocket that accommodates glycone moieties. This is inferred by the fact that even subtle modifications in this highly conserved region lead to significant alterations in enzyme activity [START_REF] Song | Engineering better biomass-degrading ability into a GH11 xylanase using a directed evolution strategy[END_REF]. SAXS data (see Figure 3 below) revealed that in solution In-NpXyn11A, displayed a partially unfolded conformation compared to NpXyn11A. These observations can possibly be correlated with the relatively high value of the standard deviation of the kinetic parameters.

Despite changes to KMapp and kcat, the overall catalytic efficiency of the two chimeric enzymes and NpXyn11A were similar due to compensatory effects on kcat and KMapp.

To use the Jo-In system to assess rapidly the effect of covalent linkage of NpXyn11A to CtCBM3a and CfCBM2b-1, the same strategy was used to prepare CBM chimeras, yielding the proteins Jo-CBM3a, In-CBM3a, Jo-CBM2b-1 and In-CBM2b-1. A pull down assay [START_REF] Crouch | The Contribution of noncatalytic carbohydrate binding modules to the activity of lytic polysaccharide monooxygenases[END_REF] performed using insoluble cellulose confirmed the binding ability of CtCBM3a and its Jo and

In derivatives (see supporting information for detailed protocol, Supplementary Figure S1).

Further investigation using Isothermal titration calorimetry (ITC) and either regenerated cellulose (RC) or cellulose nanocrystals (CN) confirmed that CtCBM3a showed similar Ka values in the case of both ligands (see supporting information for detailed protocol, Supplementary Figure S2). It is noteworthy that ITC data also indicate that the CtCBM3aligand interaction is enthalpy-driven, with the entropic component being unfavorable, consistent with previous data related to CBMs binding to RC [START_REF] Boraston | The interaction of carbohydrate-binding modules with insoluble noncrystalline cellulose is enthalpically driven[END_REF]. Having demonstrated that CNs constitute a suitable ligand for CtCBM3a, they were used to evaluate the binding properties of Jo-CBM3a and In-CBM3a using microscale thermophoresis (MST) (see supporting information for detailed protocol, Table 1 and Supplementary Figure S3). Since Jo-and In-CBM3a are optimally stable in different buffers, to compare them with CtCBM3a it was necessary to determine CN EC50 values for the latter in both buffers. Comparing Jo-CBM3a with CtCBM3a revealed that the EC50 values were highly similar (0.21 g.L -1 and 0.14 g.L -1 , respectively). However, the EC50 value characterizing the interaction of In-CBM3a with CN was 60-fold lower than that of CtCBM3a (0.003 g.L -1 and 0.18 g.L -1 , respectively), indicating a higher affinity of In-CBM3a for CN. In this respect, it is noteworthy that CtCBM3a binding to CN is only moderately sensitive to buffer changes, because the difference between the two EC50 values was only 1.28-fold. Therefore, accounting for buffer effects, while the appendage of Jo to the N-terminal extremity of CtCBM3a has a relatively minor impact on ligand binding, the appendage of In significantly reinforces it.

To investigate the ligand binding properties of CfCBM2b-1 and its Jo and In derivatives for soluble oligosaccharides, NMR was used (see supporting information for detailed protocol).

This revealed that CBM binding to X6 significantly perturbed the chemical shift of the side chains NH ε signals of two solvent exposed Trp residues (Supplementary Figure S4).

Monitoring these shifts provided a Kd value for the CfCBM2b-1/X6 interaction (Table 2), with data being in the same order of magnitude as a previously reported value [START_REF] Simpson | A family IIb xylan-binding domain has a similar secondary structure to a homologous family IIa cellulose-binding domain but different ligand specificity[END_REF].

Determination of the Kd value for X6 binding to In-CBM2b-1 gave a similar value, but in identical assay conditions no interaction (i.e. Kd > 10 mM) between Jo-CBM2b-1 and X6 was evidenced. The reason for this difference is unknown, but clearly Jo engages in unfavorable interactions with CBM2b-1 that possibly lead to the steric hindrance of one or more of the CBM's ligand binding determinants [START_REF] Simpson | A family IIb xylan-binding domain has a similar secondary structure to a homologous family IIa cellulose-binding domain but different ligand specificity[END_REF].

In summary, the attachment of Jo or In to the N-terminus of NpXyn11A yielded an active xylanase. However, attachment of these elements to the N-termini of CtCBM3a or CfCBM2b-1 yielded variable results. The attachment of In increased the binding affinity for both CBMs (significantly for CBM3a) while the attachment of Jo decreased both binding affinities (drastically for CBM2b-1). Nevertheless, despite this it was decided to proceed with the creation of Jo-In linked multimodular chimeras, because previous work has shown that the covalent association of Jo and In leads to a stable complex devoid of flexibility [START_REF] Bonnet | Autocatalytic association of proteins by covalent bond formation: a Bio Molecular Welding toolbox derived from a bacterial adhesin[END_REF].

Similarly, it was postulated that any deleterious effects arising from the linkage of either Jo or In to a CBM might be attenuated once the Jo-In complex is formed.

Creation and biochemical characterization of multimodular chimeric enzymes

Using the different Jo and In derivatives, four multimodular chimeric xylanases were prepared and purified (Figure 1A-B S2).

Measurement of hydrolytic activity of multimodular chimeric xylanases on pNP-X3, a

substrate small enough to avoid major interference from the CBM, showed that all the enzymes display activities in the same order of magnitude as recombinant NpXyn11A (Table 3). The activity of NpXyn11A-[In-Jo]-CBM2b-1 was reduced by 36%, even though In-NpXyn11A displayed almost the same activity as NpXyn11A. Conversely, while the attachment of Jo to NpXyn11A led to a 20% reduction in activity on pNP-X3, adding In-CBM2b-1 restored activity to a level almost identical to that of NpXyn11A. Moreover, the addition of In-CBM3a was not deleterious (Table 3). These results are consistent with current knowledge that the presence of CBM appendages does not enhance the activity of GHs on soluble substrates such as pNP-glycosides or short oligosaccharides [START_REF] Tomme | Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis[END_REF].

Regarding the activities of the four chimeric multimodular xylanases on soluble polymeric substrates (RAX, WAX and BWX), the enzymes fall into one of two categories. When In-NpXyn11A is linked to either of the Jo-CBMs the activity on all three substrates is mostly lower than NpXyn11A, with CBM2b-1 having the most deleterious effect of up to 66% loss of activity. This result could be related to the loss of binding properties of Jo-CBM2b-1 (Table 2) and does not follow examples in the literature suggesting that activity enhancement could be expected [START_REF] Kittur | Fusion of family 2b carbohydrate-binding module increases the catalytic activity of a xylanase from Thermotoga maritima to soluble xylan[END_REF]. Nevertheless, when Jo-NpXyn11A is linked to either In-CBMs, activity on the different substrates is mostly increased, with the exception of NpXyn11A-[Jo-In]-CBM2b-1 on BWX (10% activity loss) (Table 3). Remarkably, the activity of NpXyn11A-[Jo-In]-CBM3a on RAX was increased by 135% compared to that of NpXyn11A on the same substrate (2.92 ± 0.04 mM xylose equivalent and 1.25 ± 0.16 mM xylose equivalent after 15 min of reaction, respectively). Clearly, activity increases related to the presence of CBM2b-1 can be tentatively attributed to the specific ligand binding ability of the CBM. However, the significant increase correlated with the presence of the cellulose-targeting CBM3a is less intuitive. Nevertheless, a recent study also revealed that the appendage of a cellulose-specific CBM family 1 to xylanase NpXyn11C [START_REF] Zhang | Synergistic effect of acetyl xylan esterase from Talaromyces leycettanus JCM12802 and xylanase from Neocallimastix patriciarum achieved by introducing carbohydrate-binding module-1[END_REF] increased catalytic efficiency by 21% on BWX.

The underlying reasons for such activity enhancements is unclear and are often treated cautiously [START_REF] Hoffmam | Xylan-specific carbohydrate-binding module belonging to family 6 enhances the catalytic performance of a GH11 endo-xylanase[END_REF]. The fold and architecture of a xylanase core was proposed to explain the positive effect of a CBM targeting xylan on the catalytic activity of GH family 11 towards soluble xylan [START_REF] Kittur | Fusion of family 2b carbohydrate-binding module increases the catalytic activity of a xylanase from Thermotoga maritima to soluble xylan[END_REF]. In the case of the present results, accounting for the relatively large MW of the xylan polymers (⁓ 350 kDa) [START_REF] Montanier | Changing surface grafting density has an effect on the activity of immobilized xylanase towards natural polysaccharides[END_REF] and the shape of the chimeric enzyme (see SAXS data below, Figure 5B), it may be postulated that non-specific interactions cannot be excluded, although with no evidence for this.

The enzymatic activity of NpXyn11A derivatives was also evaluated using destarched wheat bran (DWB), which displays a high arabinoxylan:cellulose ratio [START_REF] Bouraoui | The GH51 α-l-arabinofuranosidase from Paenibacillus sp. THS1 is multifunctional, hydrolyzing main-chain and side-chain glycosidic bonds in heteroxylans[END_REF] and wheat straw (WS), which conversely displays a low arabinoxylan:cellulose ratio [START_REF] Motte | Morphological structures of wheat straw strongly impacts its anaerobic digestion[END_REF]. After 23 h, chimeric xylanases had released ⁓7.5-fold more reducing sugars from DWB than from WS (Figure 2), consistent with the greater availability of arabinoxylan in the former and also the greater structural and chemical complexity of WS. Moreover, the presence of either CBM clearly enhanced final reducing sugar yield (by ⁓17% in the case of DWB), even with the presence of CtCBM3a that apparently reduced the initial reaction rate (Figure 2A). The impact of the nature of the Jo-In linkage was also significant with NpXyn11A-[Jo-In]-CBM2b-1 displaying a faster initial rate than NpXyn11A-[In-Jo]-CBM2b-1. On WS, the presence of CtCBM3a proved to be a severe handicap, because activity was ⁓75% lower than that of the catalytic domain alone (Figure 2B). Instead, the impact of CtCBM2b-1 was imperceptible, since the activities of the CtCBM2b-1 chimeras were nearly identical to that of the catalytic domain alone. However, the discriminating nature of the Jo-In linkage was again perceptible, because despite its faster initial rate, NpXyn11A-[In-Jo]-CBM2b-1 generated ⁓15 % less reducing sugars when compared to NpXyn11A-[Jo-In]-CBM2b-1. These results demonstrate the importance of substrate targeting by CBMs, especially in complex environments such as PCWs [START_REF] Herve | Carbohydratebinding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects[END_REF]. DWB provides CfCBM2b-1 with an abundant source of highly accessible ligand binding sites, whereas the cellulose-specific CtCBM3a probably hinders the early progression of the enzyme in this matrix. In contrast, WS provides CtCBM3a with abundant crystalline cellulose. Thus, binding of CtCBM3a chimeras to cellulose sequesters the enzyme and prevents it from reaching its arabinoxylan target substrate [START_REF] Herve | Carbohydratebinding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects[END_REF][START_REF] Gao | Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis[END_REF]. It is noteworthy that although MST and NMR measurements revealed that In-CBM3a and Jo-CBM2b-1 display impaired ligand binding (Tables 1 and2) and experiments using purified xylan substrates suggest that the activities of NpXyn11A chimeras are sensitive to the exact nature of the Jo-In linkage (Table 3), these factors did not appear to be major determinants of activity on complex insoluble substrates.

SAXS and NMR analysis of the multimodular xylanases

As previously described [START_REF] Bonnet | Autocatalytic association of proteins by covalent bond formation: a Bio Molecular Welding toolbox derived from a bacterial adhesin[END_REF], the anti-parallel organization of the stable complex Jo-In and the possibility to link Jo or In to the N-and C-termini of proteins of interest [START_REF] Enjalbert | Characterisation of the effect of the spatial organisation of hemicellulases on the hydrolysis of plant biomass polymer[END_REF] offers the ability to create chimeric proteins and modulates the relative spatial orientation of linked protein domains. To examine the structures of the protein chimeras created in this work, SAXS data (Figure 3) were recorded and biophysical parameters were extracted (Figure 3C).

The single domain NpXyn11A generated a SAXS curve typical of a globular, folded protein (Figure 3A) that fits well with the theoretical curve calculated using CRYSOL and crystal structure data (PDB id: 2C1F) (data not shown). However, addition of the In domain resulted in a modified solution structure, with the Rg and Dmax values (46.2Å and 160Å respectively) being considerably higher than those of NpXyn11A (18.5Å and 60Å respectively), in agreement with the increase of the MW. It appears that the In domain of In-NpXyn11A is present as a long unfolded tail, while the NpXyn11A maintains its globular structure. SAXS curves of Jo and In derivatives of CfCBM2b-1 displayed the characteristics of unfolded proteins with a constant decreased at medium angles (0.008 to 0.1Å -1 ). The proton NMR spectrum of the isolated CfCBM2b-1 is characteristic of a well-folded protein domain, with several methyl resonances below 0 ppm and well-defined Trp side chain signals (Figure 4).

The Jo-CBM2b-1 and In-CBM2b-1 constructs display similar resonances devoid of chemical shifts or spectral broadening, indicating that the CBM domain maintains its 3D fold within these constructs. Indeed, calculation of a difference spectrum (i.e. subtracting the spectrum of the isolated CfCBM2b-1 domain from that of the Jo/In derivatives) confirmed that the spectra of the Jo/In -attached CfCBM2b-1 are simple composites of the spectra of the constituent proteins domains. This implies that when linked to CfCBM2b-1, neither Jo nor In intrinsically alter the structure of the CBM. However, the fact that Jo-CBM2b-1 binds less effectively than CfCBM2b-1 to X6 (Table 2) suggests that the unstructured Jo domain obstructs access to the CBM's ligand binding site. A similar conclusion possibly explains the lower activity of In-NpXyn11A against BWX, since the SAXS data (Figure 3) indicates that the In domain is partially unstructured and might obstruct access to the catalytic site (Supplementary Table S3 and Table 3).

The second set of SAXS curves presented in Figure 3B are those of the multimodular xylanases. These display a similar profile, with intensity decreasing to a plateau at small angles (q <0.01 Å -1 ), corresponding to the Guinier region. At higher angles (0.01<q<0.08 Å -1 and 0.08<q<0.2 Å -1 ) the curves decay, consistent with the power law function I(q) = q -p , with p value ≈ 2 and 4 respectively. This is characteristic of elongated proteins. showing the impact of enzymatic on-section treatment on xylan and cellulose immunolabelling. Serial sections of wheat straw were incubated for 24 h using the recombinant xylanase derivatives as labelled on the images and further used for double indirect immunofluorescence using LM11 (xylan specific antibody) and His6-CBM3a (cellulose specific CBM) as primary probes and anti-rat IgG-Alexa 488 and anti-His6-Alexa 555, respectively. The individual fluorescence channels are shown as labelled for a wide field view (two left rows) and for a vascular bundle magnified view (two right rows). Note, that to enable fair comparison between the treatment/controls, the same zones on the different serial sections are displayed. Note also that an intermediate magnification is shown for this vascular bundle in the ROI of Supplementary Figure S7, as well as two additional ROIs. bsf, bundle sheath fibres; cp, cortical parenchyma zones; ep, epidermis; if, intervascular fibres; mx, metaxylem; p, pith; ph, phloem; pp, pith parenchyma; px, protoxylem; s, sclerenchyma; vb, vascular bundle. Bars: 250 µm (two left rows); 25 µm (two right rows).

Table 1: Binding affinity of CtCBM3a and derivates against cellulose nanocrystals. Buffer 1: 50 mM Tris HCl pH 7.4, 150 mM NaCl, 10 mM MgCl2, 0.05 % Tween 20. Buffer 2: 50 mM sodium phosphate buffer, pH 7 and 0.05 % pluronic acid. See Supplementary Figure S3 for chart. EC50 is the half-maximal effective concentration, i.e. the higher the affinity for the substrate, the smaller the value of the EC50 .

  ). As shown by SDS-PAGE, the apparent molecular weight (MW) of NpXyn11A-[Jo-In]-CBM3a, NpXyn11A-[In-Jo]-CBM3a, NpXyn11A-[Jo-In]-CBM2b-1 and NpXyn11A-[In-Jo]-CBM2b-1 are consistent with predicted values, obtained by summing the MWs of the individual modules (Figure 1B, Supplementary Table

  For the multimodular xylanases, NpXyn11A-[In-Jo]-CBM2b-1 and NpXyn11A-[Jo-In]-CBM2b-1, the plots of P(r) versus r are very similar (Figure 5A) and reflect a multidomain, elongated, global form (Figure 5B). In contrast, although the P(r) profiles of NpXyn11A-[In-Jo]-CBM3a and NpXyn11A-[Jo-In]-CBM3a are also highly similar, they nevertheless differ from those of NpXyn11A-[In-Jo]-CBM2b-1 and NpXyn11A-[Jo-In]-CBM2b-1, displaying more marked oscillations that reflect the larger size of CtCBM3a (17.1 kDa compared to 9.1 kDa for CBM2b-1). Despite this difference, the curves are also indicative of elongated shapes composed of distinct domains (Figure 5B). The comparison of experimental SAXS data acquired for NpXyn11A-[In-Jo]-CBM2b-1 and NpXyn11A-[Jo-In]-CBM2b-1 with that of model curves generated using crystallographic data revealed that these were highly similar, with a goodness of fit χ² = 1.25 and 1.57 respectively (Figure 6A). For each chimera, modelling and superimposing the theoretical structure that displayed the best χ² value provided low-resolution hypothetical structures (Figure 6B-C). Irrespective of the Jo-In configuration, the distance between NpXyn11A and CfCBM2b-1 is quite similar in both models (118.0 ± 9.6Å for NpXyn11A-[In-Jo]-CBM2b-1 and 120.6 ± 5.5 Å for NpXyn11A-[Jo-In]-CBM2b-1). However, due to the axial asymmetry in the Jo-In complex, the torsion angle is + 66.3 ± 27.6° for NpXyn11A-[In-Jo]-CBM2b-1 and -145.4 ± 21.1° for NpXyn11A-[Jo-In]-CBM2b-1 (Supplemnetary Figure S6). Predictive structural modelling of NpXyn11A-[In-Jo]-CBM3a and NpXyn11A-[Jo-In]-CBM3a using CORAL was not possible because of significant structural variability (i.e. χ² > 2) that prevented fitting to the molecular envelope of the SAXS data.Differences in targeting multimodular xylanases in wheat strawTo further test the possibility of using the chimeric xylanases on raw substrate, experiments were performed in situ on wheat bran and wheat straw PCW sections and the accessible xylans and cellulose localization were monitored using immunological labelling. Following treatment of the wheat bran PCW sections with multimodular enzymes (NpXyn11A-[Jo-In]-CBM3a was omitted from this study), LM11/A488-specific xylan labelling between the pericarp and the nucellar epidermis (Figure7C1-F1) and intense continuous CBM3a/A555specific labelling of the nucellar epidermis (Figure7C2-F2) were observed. This contrasts with the untreated sections that displayed no LM11/A488 labelling and only faint CBM3a/A555 labelling (Figure7B). Although increased labelling after enzymatic treatment appears counter intuitive, it is almost certainly a consequence of PCW complexity. The action of the chimeric xylanases leads to the removal of xylan and concomitant exposure of hitherto masked PCW components, such as cellulose and xylan, which constitute new ligands for antibody or CBM binding. However, experiments performed on DWB failed to reveal any

Figure 2 .

 2 Figure 2. Degradation of complex substrates by NpXyn11A and derivatives thereof. (A) Wheat bran. (B) Wheat straw. Enzyme reactions were conducted in 50 mM sodium phosphate, 12 mM sodium citrate pH6, supplemented with 1 mg/ml BSA, at 37°C. Enzyme loading was at 1 µM. Substrate concentration was at 2% w/v.

Figure 3 :

 3 Figure 3: (A) SAXS data recorded for NpXyn11A, In-NpXyn11A, In-CBM2b-1 and Jo-CBM2b-1. (B) SAXS data recorded for NpXyn11A-[In-Jo]-CBM2b-1, NpXyn11A-[Jo-In]-CBM2b-1, NpXyn11A-[In-Jo]-CBM3a and NpXyn11A-[Jo-In]-CBM3a. (C) Tablesummarizing all parameters extracted from the Guinier plot and pair-atom distribution function P(r) such as gyration radius Rg and maximum internal distance Dmax. The folding state deduced from the parameters and the shape of the curves are also mentioned in the table for each fragment.

Figure 4 :

 4 Figure 4: 1D proton spectrum of (A) isolated CfCBM2b-1 (bottom, black), of In-CBMb2-1 (middle, red) and the difference spectrum (CfCBM2b-1 -In-CBMb2-1) (top, green). (B) Isolated CfCBM2b-1 (bottom, black), of Jo-CBMb2-1 (middle, red) and the difference spectrum (CfCBM2b-1 -Jo-CBMb2-1) (top, orange).

Figure 5 :

 5 Figure 5: (A) Pair distribution function calculated from SAXS data of NpXyn11A-[In-Jo]-CBM2b-1 and NpXyn11A-[Jo-In]-CBM2b-1 (blue and red curves respectively) and from SAXS data of NpXyn11A-[In-Jo]-CBM3a and NpXyn11A-[Jo-In]-CBM3a (green and yellow curves respectively). The P(r) function is plotted as P(r)/Pmax(r) vs distance r in order to compare the different curves by normalizing with Pmax(r). (B) Low resolution shape calculated with the DAMMIF program from ATSAS suite for the four constructs. The shapes are built with Pymol in mesh representation and filled with transparent spheres.

Figure 6 :

 6 Figure 6: (A) Comparison of experimental curves generated using NpXyn11A-[In-Jo]-CBM2b-1 and NpXyn11A-[Jo-In]-InCBM2b-1 and the theoretical curves (dotted black line) calculated using CRYSOL and crystallographic data. (B) Models of NpXyn11A-[In-Jo]-CBM2b-1 and (C) NpXyn11A-[Jo-In]-InCBM2b-1 were calculated using the CORAL program. In both models, Jo-In are coloured red and green respectively. NpXyn11A is in cyan and CtCBM2b-1in yellow. Catalytic residues of NpXyn11A and residues involved in ligand binding recognition of CtCBM2b-1 are represented by red lines. The domains and the linker are modelled with Pymol using cartoon and grey sphere representations respectively.

Figure 7 :

 7 Figure 7: Double immunofluorescence of paraffin-embedded wheat bran serial sections showing the impact of enzymatic on-section treatment on accessible xylan and cellulose immunolabelling. Serial sections of wheat bran were incubated for 24 h using the xylanase derivatives as labelled on the images and further used for double indirect immunofluorescence using LM11 (xylan specific antibody) and His6-CBM3a (cellulose specific CBM) as primary probes and anti-rat IgG-Alexa 488 and anti-His6-Alexa 555, respectively. The individual fluorescence channels are shown in the two first rows as labelled, and the merge of both fluorescence channels with the bright field channel is shown is the third row. Arrowheads: pericarp/nucellar epidermis interface; Bars: 300 µm.

Figure 8 :

 8 Figure 8: Double immunofluorescence of paraffin-embedded wheat straw serial sections

Table 2 :

 2 Ligand affinities of CfCBM2b-1, In-CBM2b-1 and Jo-CBM2b-1 for xylohexaose as measured by 1D NMR by titrating the resonances of the NHε of Trp 259 and Trp 291. Experiments were conducted in 50 mM sodium phosphate pH 7 at 298 °K.

Table 3 :

 3 Specific activity of NpXyn11A and derivatives, as single enzymes or in complex.Reactions were performed in 50 mM sodium phosphate, 12 mM sodium citrate pH 6, supplemented with 1mg/mL BSA, at 37°C. Substrate concentrations were 5 mM 4-nitrophenyl-β-D-xylotrioside (pNP-X3), 0.5% w/v wheat arabinoxylan (WAX), 0.5% w/v rye arabinoxylan (RAX) and 1% w/v beechwood xylan (BWX). The values are shown as means ± standard deviation of replicate n = 3.

Table 1 :

 1 Protein EC50 (g.L -1 )

		Buffer 1	Buffer 2
	In-CBM3a	-	0.003 ± 0.0001
	CtCBM3a	0.14 ± 0.01	0.18 ± 0.04
	Jo-CBM3a	0.21 ± 0.12	-

Table 2 :

 2

			Kd (mM)
	Protein
		This work (Trp 259)	This work (Trp 291)	[35]
	CfCBM2b-1	0.84	1.02	0.29
	In-CBM2b-1	0.22	0.52	-
	Jo-CBM2b-1	> 10	> 10	-

Table 3 :

 3

			Specific Activity
	Single enzymes	pNP-X3 (IU/µmole)	WAX 10 3 (IU/µmole)	RAX 10 3 (IU/µmole)	BWX 10 3 (IU/µmole)
	NpXyn11A	155.78 ± 4.07	114.59 ± 2.67	83.44 ± 8.21	65.03 ± 2.19
	In-NpXyn11A	153.17 ± 1.68	-	-	-
	Jo-NpXyn11A	124.63 ± 3.64	-	-	-

Acknowledgements

We thank Pr. Harry Gilbert (ICMB, Newcastle University) for providing access and expertise in ITC experiments. We also thank Dr. Jean Jacques Bono and Virginie Gasciolli (LIPM, Toulouse) for providing access and expertise in MST experiments. The imaging using Nanozoomer RS was performed at the FR-AIB imaging platform of TRI-genotoul facility (Toulouse). We thank Dr. Gianluca Cioci for technical assistance with FPLC provided by the ICEO facility dedicated to enzyme screening and discovery, part of the Integrated Screening Platform of Toulouse (PICT, IBiSA).

Supplementary Figures S7 andS8). The use of either probe on untreated sections produced homogenous labelling of both PCW components (Figure 8B). However, after treatment with NpXyn11A (Figure 8C) and NpXyn-[Jo-In]-CBM2b-1 (Figure 8D), LM11/A488 labelling was repeatedly found to be diminished when observing different vascular bundles (Supplementary Figure S7). This suggests that the presence of CBM2b-1 did not enhance the activity of NpXyn11A on WS. Additionally, compared to NpXyn11A, NpXyn-[Jo-In]-CBM2b-1 was apparently less active on intervascular fibres. Finally, diminution of the CBM3a/A555 labelling (cellulose-specific probe) was correlated with xylan hydrolysis.

Conversely, treatment with

generally did not affect LM11/A488 labelling intensity (Figure 8E-F; Supplementary Figure S7), although this was strongly enhanced in patches around the phloem, the protoxylem and in vascular bundle cell corners. In distal zones, intense patches of LM11/A488 labelling were also observed in sclerenchyma cell corners and around the pith parenchyma intercellular spaces (Supplementary Figure S8). For PCW sections treated with NpXyn11A-[In-Jo]-CBM2b-1, additional patches of LM11/A488 labelling were also observed in the intervascular fibres, but this was not the case when NpXyn11A-[In-Jo]-CBM3a was used (Figure 8E-F; Supplementary Figure S7). The impact on cellulose labelling was also more evident for

Previously, both CtCBM3a and CfCBM2b-1 were shown to potentiate the activity of a cognate xylanase on tobacco PCWs, presumably by improving its substrate targeting capability [START_REF] Herve | Carbohydratebinding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects[END_REF]. However, the present results do not confirm this and may signify limited usefulness for the Jo-In system when studying enzyme activity on complex matrices, even though the structural environment of xylan and xylan-cellulose interactions are different in tobacco PCW [START_REF] Busse-Wicher | The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana[END_REF]. It can be postulated that Jo-In introduces a high degree of rigidity compared to natural linkers found in PCW-degrading enzymes. While rigidity might be undesirable in certain circumstances, it can be useful in others. Specifically, greater protein rigidity will be useful in investigating different spatial organizations in multidomain proteins, locking chimeric protein isoforms in different domain configurations.

Conclusions

A previous study on CtCBM3a linked to endoglucanase CelA of Clostridium thermocellum revealed that the CBM must be correctly oriented to potentiate enzymatic activity, especially on insoluble substrates [START_REF] Sajjad | Influence of positioning of carbohydrate binding module on the activity of endoglucanase CelA of Clostridium thermocellum[END_REF]. In this regard, the nature of the linker is certainly important because its structure will strongly contribute to the spatial orientations of linked protein domains. Here, the Jo-In complex has been used to link protein domains, the resultant linker complexes being stable, rather rigid protein structures. The first implication of this rigidity is the likelihood that there will be no physical interference between the attached protein domains. In the case of NpXyn11A linked to CBMs, this is certainly the case because the solution structures are elongated and characterized by well separated GH and CBM domains. The second implication is that the spatial orientation (torsion angle) of the two linked proteins domains is locked. If this is optimal then synergy should be possible and enhancement of the activity of the GH domain will be a likely outcome. Conversely, if this is suboptimal, the chimera will be definitively impaired, lacking sufficient linker flexibility to allow alternative solution conformers. Accordingly, it is proposed that data related to the hydrolysis of insoluble substrates described herein reflects this fact, revealing that NpXyn11A-[Jo-In]-CBM2b-1 has a more optimal configuration for hydrolysis than NpXyn11A-[In-Jo]-CBM2b-1. The third implication of linker rigidity is that protein chimeras are likely to encounter difficulty in penetrating structurally complex three-dimensional matrices. In this work, the fact that the addition of CBMs to NpXyn11A failed to potentiate the hydrolysis of wheat straw possibly supports this hypothesis.

In summary, this work confirms the usefulness of the Jo-In system in creating multidomain GHs. However, one caveat is the intrinsic rigidity of Jo-In. When designing chimeric proteins, this property must be considered with respect to the intended purpose.

Author contributions

LB, VB, CYM conceived and designed the experiments. LB, PR, TE, GL, IV and CYM performed the experiments. LB, VB, PR, GL, MJO and CYM analyzed the data. LB, MJO and CYM wrote the paper.


domain: chim spi

text:
L Massot

H Meskine

M Gibilaro

P Chamelot

Electrochemical co-deposition of Erbium and Ytterbium in molten LiF-CaF2: an original way for lanthanides extraction on inert electrode

Keywords: Electrochemical co-reduction, Erbium, Ytterbium, molten fluorides, lanthanides co-extraction

Electrochemical study of Er(III)/Er and Yb(III)/Yb systems was independently investigated in molten LiF-CaF2 on inert electrode at 840°C using cyclic voltammetry and square wave voltammetry. Experimental results showed that two different behaviours were observed: Er(III) was reduced into metal in a one-step and diffusion controlled process and Yb(III) was first reduced into Yb(II) then into Yb. The electrochemical study of LiF-CaF2 containing both Er(III) and Yb(III) on inert Mo electrode highlighted a co-deposition of Er and Yb leading to Er-Yb surface alloys formation. Alloys formation was evidenced using open-circuit chronopotentiometry and SEM-EDS. Finally, co-extraction of Er and Yb from molten salt was carried out by electrolysis with high extraction efficiency (> 99.5 %). These results pave the way to the Lanthanides extraction without involving cathode material nor another ion as for reactive electrodeposition.

1/ Introduction

Pyrochemical processes are mainly envisaged for the reprocessing of nuclear spent fuels in the frame of a sustainable development of nuclear energy production [START_REF] Ackerman | Chemical basis for pyrochemical reprocessing of nuclear fuel[END_REF][START_REF] Inoue | Development of pyroprocessing and its future direction[END_REF]. The interest of pyroprocesses is based on the properties of the molten salt phase: high radiation and thermal resistance and relative high transparency to neutrons. Pyrometallurgical processes are also attractive for the dissolution of fuels or inert matrices, which are very difficult or impossible in aqueous media. A key-step for spent nuclear fuel reprocessing is the separation of actinides (Ans) from lanthanides (Lns), due to their similar properties. Electrochemical processes in molten salts are very promising by using precipitation with oxide ions [START_REF] Lambertin | Temperature dependence and effect of oxide anion on the americium chemistry in the molten LiCl-KCl eutectic[END_REF], electrodeposition [START_REF] Iizuka | Actinides Recovery from Molten Salt/Liquid Melt System by Elecrochemical Methods[END_REF][START_REF] Chamelot | Feasibility of the electrochemical separation of An, Ln, solvent in molten fluorides[END_REF] or reductive extraction [START_REF] Rault | Test of Actinide-Lanthanide Separation in an Aluminum-Based Pyrochemical System[END_REF]. The ultimate step is to extract Lns from the solvent to recycle it. In the last twenty years, the study on the electrodeposition process in molten salts have attracted great interest. Two ways are possible, depending on the Ln reduction potential ELn:

-if the Lns ions can be reduced into metal at a potential significantly higher than the solvent cations reduction one, then Lns can be electrodeposited on an inert cathode; -if the Lns ions reduction into metal cannot be observed on a voltammogram (reduction occurring at a lower potential than the cathodic electrochemical window), UnderPotential Deposition (UPD) has to be implemented.

UPD is obtained by reactive electrodeposition with the cathode substrate or by co-reduction with another species (AlF3). It has been successfully carried out in our laboratory to extract NdF3, GdF3, CeF3, EuF3 and SmF3 [START_REF] Gibilaro | Study of neodymium extraction in molten fluorides by electrochemical co-reduction with aluminium[END_REF][START_REF] Taxil | Lanthanides extraction processes in molten fluoride media. Application to nuclear spent fuel reprocessing[END_REF][START_REF] Gibilaro | Co-reduction of aluminium and lanthanide ions in molten fluorides: application to cerium and samarium extraction from nuclear waste[END_REF][START_REF] Nourry | Neodymium and gadolinium extraction from molten fluorides by reduction on a reactive electrode[END_REF][START_REF] Gibilaro | Electrochemical extraction of europium from molten fluoride media[END_REF] from molten LiF-CaF2 in the 840-950°C temperature range with an extraction efficiency higher than 99%.

These techniques lead to Ln-Ni, Cu, Al alloys, and the major drawback of these methodologies is that Lns are not deposited into pure metal form, leading to the consumption of the cathode material, or Al ions content in the bath. An additional step is also needed to recover pure Ln metals from alloys. This article proposes an original way to extract Lns from a molten fluoride salt (LiF-CaF2) without involving reactive species. The methodology proposed consists in the co-reduction of two lanthanides which have different reduction pathways (one or two reduction steps) on inert electrode. Erbium (Er) and Ytterbium (Yb) were selected to demonstrate the feasibility of this approach. Studies in molten chloride media are available and are resumed below:

-Er(III)/Er system: Castrillejo et al. [START_REF] Castrillejo | Electrochemical behaviour of erbium in the eutectic LiCl-KCl at W and Al electrodes[END_REF], Cao et al. [START_REF] Cao | Electrochemical behaviour of erbium and preparation of Mg-Li-Er alloys by codeposition[END_REF] and Tang et al. [START_REF] Tang | Electrochemistry of ErCl3 and morphology of erbium electrodeposits produced on Mo substrate in early stages of electrocrystallization from LiCl-KCl molten salts Electrochim[END_REF] studied the electrochemical behaviour of Er(III) into molten LiCl-KCl on inert electrode and the obtained results showed that Er(III) reduction into Er is a one-step process, exchanging 3 electrons and limited by diffusion into the solution. Reactive electrodeposition of Er(III) has also been investigated by Sun Yi et al. with the formation of Al-Li-Er alloys into molten LiCl-KCl-AlCl3-Er2O3 [START_REF] Sun | Electrochemical behaviour and codeposition of Al-Li-Er alloys in LiCl-KCl-AlCl3-Er2O3 melts[END_REF] and Wang et al. who prepared Er-Mg alloys on Mg electrodes [START_REF] Wang | Electrochemical extraction and separation of praseodymium and erbium on reactive magnesium electrode in molten salts[END_REF].

-Yb(III)/Yb system: Smolenski et al. [START_REF] Smolenski | Electrochemistry of ytterbium (III) in molten alkali metal chlorides[END_REF], Novoselova et al. [START_REF] Novoselova | Thermodynamic properties of thulium and ytterbium in fused NaCl-KCl-CsCl eutectic[END_REF] and Castrillejo et al. [START_REF] Castrillejo | Chemical and Electrochemical Extraction of Ytterbium from Molten Chlorides in Pyrochemical Processes[END_REF]

2/ Experimental

The cell was made of a vitreous carbon crucible placed in a refractory steel cylindrical vessel closed by a lid cooled by circulating water and protected against fluoride vapours by a graphite liner on its inner part. Experiments were performed under argon atmosphere (99.999%). The operating temperatures were measured using a thermocouple placed into the molten salt. The solvent consisted of a LiF-CaF2 (Merck 99.99%) mixture (eutectic composition: 79.5/20.5 molar ratio), initially dehydrated by heating under vacuum to its melting point.

Erbium and Ytterbium ions were introduced in the form of ErF3 and YbF3 powder (Merck 99.99%) respectively.

The working and auxiliary electrodes were a Molybdenum wire (Goodfellow 99.99%, 1mm diameter) and a vitreous carbon rod (5 mm diameter) respectively. The working electrode surface area was accurately determined by measuring the immersion depth when the electrode was taken out of the furnace after each experiment.

A platinum wire (Alfa Aesar 99.95%, 1mm diameter) immersed in the electrolyte was used as quasi-reference electrode Pt/PtOx/O 2-. In order to compare the electrochemical systems, whatever the oxide ions content, all the potentials were referred to the equilibrium potential of LiF/Li system, acting as intern reference. This equilibrium potential was firstly measured using cyclic voltammetry on Mo electrode and then was subtracted to the potentials.

The electrochemical study and the electrolyses were performed by cyclic and square wave voltammetries and open-circuit chronopotentiometry with an Autolab PGSTAT 302N potentiostat / galvanostat controlled with NOVA 2.1 software.

The electrodes were observed and characterized after electrolysis by Scanning Electron

Microscopy coupled to Energy Dispersive Spectroscopy probe (Phenom XL SEM-EDS).

Inductively coupled plasma -atomic emission spectroscopy (ICP -AES Horiba Ultima 2)

was used for Er(III) and Yb(III) content determination after sampling of the melt.

3/ Results and discussion

3-1-1/ Electrochemical behaviour determination

Figures 1a and 1b present typical cyclic voltammograms plotted on Mo electrode at 840°C

and 100 mV.s -1 for LiF-CaF2-ErF3 (0.139 mol.kg -1 ) and LiF-CaF2-YbF3 (0.143 mol.kg -1 ) systems respectively. On both voltammograms, it can be observed a single cathodic peak at around 0.07 V vs. LiF/Li for Er(III) and around 0.9 V vs. LiF/Li for Yb(III). For Er(III) system, the cathodic peak is correlated to one reoxidation peak with a stripping shape characteristic of solid phase dissolution, whereas reoxidation peak shape observed for Yb(III) system indicates the formation of soluble compound during cathodic scan.

As presented in the Fig. 1a and Fig. 1b insets, the quasi-reversibility of both systems, characterized by Er(III) and Yb(III) reduction peak potentials independent of the scan rate, is verified. Additional reduction peak was observed at around 0.5V. As this reduction peak was not observed in pure LiF-CaF2, and its current density was very low, this system was attributed to the presence of impurity in the ErF3 powder.

A linear variation of the cathodic peak current densities as function of molality is presented in

where jp the peak current density, [Yb(III)] and [Er(III)] the lanthanide molality (mol kg -1 ).

These equations allow the in situ determination of Er(III) and Yb(III) content in the molten medium at 840°C.

In order to determine the number of exchanged electrons involved into their reduction, square wave voltammetry was performed and the voltammograms obtained on Mo electrode at 25Hz

for LiF-CaF2-ErF3 and LiF-CaF2-YbF3 are presented on Fig. 3a and 3b respectively.

These SWV voltammograms highlight one reduction peak at around 0.07 and 0.9 V vs. LiF/Li for Er(III) and Yb(III) systems respectively, as observed on the CV voltammograms of Fig. 1a and1b. As the linearity of differential peak current density versus the square root of the frequency is verified for both systems (Fig 3a and 3b insets), the SWV voltammograms were deconvoluted using the methodology previously published in our laboratory [START_REF] Nourry | Data acquisition in thermodynamic and electrochemical reduction in a Gd(III)/Gd system in LiF-CaF2 media[END_REF]. This signal treatment allows the number of exchanged electrons to be determined using equation (3) [START_REF] Bard | Electrochemistry: principles, methods and applications[END_REF]:

/ = 3.52 (3)
where n is the number of exchanged electrons, F the Faraday constant (96500 C), T the absolute temperature in K and R the ideal gas constant (J mol -1 K -1 ).

The obtained number of exchanged electrons are 3±0.1 for Er(III) reduction and 1±0.1 for Yb(III) reduction.

With these results, the following reactions can thus be assumed, similarly to molten chloride media:

Er(III) + 3 e -= Er

where S the electrode surface area in cm², D the diffusion coefficient in cm² s -1 , c0 the solute concentration in mol cm -3 and v the potential scan rate in V s -1 .

The slope of the linear equations at 840°C (insets of Figs. 1a and1b), proving that reactions are controlled by Ln(III) diffusion into the solution, are:

-For Er(III) reduction:

" # $ / = -. %& ( ) / * + / ,-+ (8)
-For Yb(III) reduction:

" # $ / = -. . ( ) / * + / ,-+ (9)
Diffusion coefficients are determined from eqs. ( 8) and ( 9

where DEr(III) and DYb(III) are in m² s -1 and T in K.

3-2/ Electrochemical study of LiF-CaF2-ErF3-YbF3 system

The electrochemical behaviour of each Ln ions was thus determined in molten fluoride salt and their mixing in the same solution is presented in this part.

Experiments were carried out on Mo electrode at 840°C using cyclic voltammetry and square wave voltammetry, and the obtained plots are presented in Fig. 5a and Fig. 5b respectively.

The observations on both CV and SWV voltammograms are similar:

-two reduction peaks are observed at around 0.08 and 0.9 V vs. Li(I)/Li. In agreement with the results presented above, these two peaks are attributed to Er(III)/Er and Yb(III)/Yb(II) systems respectively, -in between these two potentials, a significant reduction current density is evidenced.

As no signal was observed at these potentials when only one species was present in the solution, this new cathodic current can be attributed to the Er-Yb co-reduction, as previously observed in our Laboratory for Al-Lns co-reduction [START_REF] Gibilaro | Co-reduction of aluminium and lanthanide ions in molten fluorides: application to cerium and samarium extraction from nuclear waste[END_REF].

Moreover, this cathodic current is observed for potentials lower than the peak potential of Yb(III) reduction into Yb(II), indicating a co-reduction between Er(III) and Yb(II) species.

The following overall reaction can be written:

x Er(III) + y Yb(II) + (3x + 2y) e -= ErxYby [START_REF] Tang | Electrochemistry of ErCl3 and morphology of erbium electrodeposits produced on Mo substrate in early stages of electrocrystallization from LiCl-KCl molten salts Electrochim[END_REF] In order to evidence alloys formation and to confirm Er and Yb co-deposition on inert Mo electrode, open-circuit potentiometry was performed. After a short cathodic polarization at -400 mA cm -2 during 15 s (see Figure 6), some potential plateaus can be observed:

-two plateaus at around 0. It can be noted that Er-Yb metallic compounds are detected on each micrography. Different compositions are observed (ErYb2, Er2Yb3 and Er2Yb) and the higher the potential is, the richer in Yb the compound is. These results clearly prove that Er-Yb electrochemical codeposition occurred. Moreover, deposits seem to be adherent enough to envisage Er and Yb extraction from the molten solution by potentiostatic electrolyses.

3-3/ Electrochemical extraction of Er and Yb

The erbium and ytterbium co-extraction from molten LiF-CaF2 was realised by potenstiostatic electrolyses on Mo plate at 840°C with the following methodology:

-the electrolysis potential was chosen equal to 0.4 V vs. Li(I)/Li, -several electrolyses of relatively short durations were performed and after eah electrolysis, cyclic voltammogram was plotted on Mo electrode and the cathode was renewed after each measurement, -the progress of the extraction can classically be followed by in situ measurements of residual species content in the bath, using cyclic voltammetry and the calibration relationships (eqs. 1 and 2). Er(III) reduction peak on Mo electrode was selected due to its lower potential and higher intensity than Yb(III) ones. The extraction yield was thus expressed as the ratio of Er(III) reduction peak current density measured after electrolysis and the initial one, -a normalized time t*, defined in refs [START_REF] Nourry | Neodymium and gadolinium extraction from molten fluorides by reduction on a reactive electrode[END_REF][START_REF] Massot | Electrochemical study of the Eu(III)/Eu(II) system in molten fluoride media[END_REF], was applied in the present work:

F * = F H IJ K LMJ ( 15
)
where t is the time (h), Sel is the cathode surface area (cm²), Vsol is the volume of the solution (cm 3 ) and t* is the normalized time (h cm -1 ).

Typical square wave voltammograms are presented in Figure 8 where a significant decrease of the current density can be observed during the extraction process, correlated to the decrease of Er(III) and Yb(III) ions concentration in the bath. As showed in Figure 9, representing the extraction progress versus the normalized time, the concentration decreased more quickly at the beginning of the electrolysis.

The maximum progress was reached after 4 h cm -1 of electrolysis. At the end of experiments, the cathodic current density value was approaching zero on the square wave voltammogram and the extraction efficiency was calculated with ICP analysis of Er and Yb in the salt. The obtained value is equal to 99.5 % for Er(III) and Yb(III), showing a quasi-total extraction of both Ln from the salt on an inert electrode.

4/ Conclusion

In the frame of molten fluoride solvent recycling in spent nuclear fuel pyrochemical reprocessing, lanthanides extraction was performed by co-reduction on inert metallic substrate and thus, the solvent could be recycled. To make this co-deposition possible, at least two lanthanide species are initially needed: one Ln ion which is reduced into metal in a one-step process and another one with a two-step reduction mechanism, as in this work.

The Er(III) and Yb(III) electrochemical behaviour was investigated in the LiF-CaF2-ErF3 and LiF-CaF2-YbF3 systems on inert electrode. This study evidenced the two different electrochemical reduction pathways, as mentioned above.

Diffusion coefficients were also determined in the 840-930°C temperature range and the results show a dependence of ln D with the inverse of the temperature.

Electrochemical experiments in the LiF-CaF2-ErF3-YbF3 system evidenced the co-reduction of both ions leading to Er-Yb alloys formation on Mo electrode. The obtained alloys are ErYb2, Er2Yb3 and Er2Yb.

Potentiostatic electrolyses were carried out on Mo plate and a complete Ln extraction was achieved (99.5% for both ions) after a normalized duration of 4 h cm -1 .

As a perspective, the methodology can be applied to others lanthanides elements which are known to be reduced into metal in a two-step process in molten fluorides: samarium and europium. ) 1000/T (K -1 )

Legend of figures

  investigated Yb(III) electrochemical behaviour on inert W electrode into molten LiCl-KCl, NaCl-KCl, CsCl and NaCl-KCl-CsCl in the 450 -800 °C temperature range. All the studies showed that Yb(III) was reduced into Yb(II) in a one-step process limited by diffusion into the solution. Castrillejo et al. [19] also evidenced the formation of Al-Yb alloys by UPD on Al electrode, proving that the reduction of Yb(II) into Yb occurred at a potential lower than solvent reduction. As no publication can be found on the electrochemical behaviour of Er(III) and Yb(III) in molten fluorides, the first part of the present article is dedicated to the electrochemical behaviour of ErF3 and YbF3 into molten LiF-CaF2 on inert Mo electrode. Then the coreduction of Er(III) and Yb(III) on inert electrode is examined by using cyclic voltammetry, square wave voltammetry and open-circuit chronopotentiometry in the 840-930°C temperature range. The final part concerns the simultaneous extraction of both elements and the extraction efficiency has been determined.

3- 1 /

 1 Electrochemical study of LiF-CaF2-ErF3 and LiF-CaF2-YbF3 systems Cyclic and square wave voltammetries were carried out to investigate the electrochemical behaviour of Er(III) and Yb(III) on Mo electrode in the LiF-CaF2-ErF3 and LiF-CaF2-YbF3 systems at 840°C.

Figure 2 for

 2 Figure 2 for Yb(III) reduction (grey) and Er(III) reduction (black). These relationships are

( 4 ) 5 )

 45 Yb(III) + e -= Yb(II) (It can be noted that Yb(II) reduction into Yb metal is not observed in the electrochemical window, meaning that the extraction of Yb from molten LiF-CaF2 in a pure metallic form is not possible, as previously observed for Eu and Sm in molten LiF-CaF2 [22;23]. 3-1-2/ Diffusion coefficient Er(III) and Yb(III) reduction peak current densities variations versus the square root of the CV scan rate are presented in the Fig. 1a and Fig. 1b insets. Berzins Delahaye and Randles Sevcik relationships, valid for reversible and diffusion-controlled soluble/insoluble (Er(III)/Er system) and soluble/soluble (Yb(III)/Yb(II) system) systems respectively are given in eqs.

  333) = (2.57 ± 0.02)x10 +? cm²s +[START_REF] Gibilaro | Electrochemical extraction of europium from molten fluoride media[END_REF] All these experiments were carried out in the 840 -930 °C temperature range and the obtained values, plotted in Fig.4, show that the variation of diffusion coefficient with the temperature obeys Arrhenius' like law expressed as:

  08 and 1.4 V vs. Li(I)/Li, corresponding to Er(III)/Er and Yb(III)/Yb(II) potential respectively, -three supplementary ones, at around 0.3, 0.4 and 0.8 V vs. Li(I)/Li. In agreement with CV and SWV results, these plateaus can be attributed to Er-Yb co-reduction leading to the formation of alloys or defined compounds, proving the Er and Yb simultaneous co-deposition on inert Mo cathode. The number of plateaux should correspond to the number of compounds produced. Electrolyses were realized on Mo electrodes in order to observe and characterize the produced Er-Yb compounds using SEM-EDS, where the imposed potentials correspond to the potential plateaus observed in Fig.6. The micrographs and SEM analyses of Mo electrodes surfaces after polarisation during 1800s at 840°C are gathered on Fig. 7a (E=0.8 V), 7b (E=0.4 V) and 7c (E=0.3 V).

Figure 1a :Figure 2 :Figure 4 :Figure 6 :

 1a246 Figure 1a:

Figure 8 :

 8 Figure 8:

Figure 9 :

 9 Figure 9:

  Figure 1a

  Figure 5b

Figure 6 Figures 7a, b and c 25 Figure 8 Figure 9 - 1 SEM

 625891 Figure 6