Unraveling the Supramolecular Structure and Nanoscale Dislocations of Bacterial Cellulose Ribbons Using Correlative Super-Resolution Light and Electron Microscopy.

Cellulose is a structural linear polysaccharide that is naturally produced by plants and bacteria, making it the most abundant biopolymer on Earth. The hierarchical structure of cellulose from the nano- to microscale is intimately linked to its biosynthesis and the ability to process this sustainable resource for materials applications. Despite this, the morphology of bacterial cellulose microfibrils and their assembly into higher order structures, as well as the structural origins of the alternating crystalline and disordered supramolecular structure of cellulose, have remained elusive. In this work, we employed high-resolution transmission electron and atomic force microscopies to study the morphology of bacterial cellulose ribbons at different levels of its structural hierarchy and provide direct visualization of nanometer-wide microfibrils. The non-persistent twisting of cellulose ribbons was characterized in detail, and we found that twists are associated with nanostructural defects at the bundle and microfibril levels. To investigate the structural origins of the persistent disordered regions that are present along cellulose ribbons, we employed a correlative super-resolution light and electron microscopy workflow and observed that the disordered regions that can be seen in super-resolution fluorescence microscopy largely correlated with the ribbon twisting observed in electron microscopy. Unraveling the hierarchical assembly of bacterial cellulose and the ultrastructural basis of its disordered regions provides insights into its biosynthesis and susceptibility to hydrolysis. These findings are important to understand the cell-directed assembly of cellulose, develop new cellulose-based nanomaterials, and develop more efficient biomass conversion strategies.

Electrochemical Nano-Roughening of Gold Microstructured Electrodes for Enhanced Sensing in Biofluids.

A key challenge for sensor miniaturization is to create electrodes with smaller footprints, while maintaining or increasing sensitivity. In this work, the electroactive surface of gold electrodes was enhanced 30-fold by wrinkling followed by chronoamperometric (CA) pulsing. Electron microscopy showed increased surface roughness in response to an increased number of CA pulses. The nanoroughened electrodes also showed excellent fouling resistance when submerged in solutions containing bovine serum albumin. The nanoroughened electrodes were used for electrochemical detection of Cu2+ in tap water and of glucose in human blood plasma. In the latter case, the nanoroughened electrodes allowed highly sensitive enzyme-free sensing of glucose, with responses comparable to those of two commercial enzyme-based sensors. We anticipate that this methodology to fabricate nanostructured electrodes can accelerate the development of simple, cost-effective, and high sensitivity electrochemical platforms.

Efficient Labeling of Nanocellulose for High-Resolution Fluorescence Microscopy Applications.

The visualization of naturally derived cellulose nanofibrils (CNFs) and nanocrystals (CNCs) within nanocomposite materials is key to the development of packaging materials, tissue culture scaffolds, and emulsifying agents, among many other applications. In this work, we develop a versatile and efficient two-step approach based on triazine and azide-alkyne click-chemistry to fluorescently label nanocelluloses with a variety of commercially available dyes. We show that this method can be used to label bacterial cellulose fibrils, plant-derived CNFs, carboxymethylated CNFs, and CNCs with Cy5 and fluorescein derivatives to high degrees of labeling using minimal amounts of dye while preserving their native morphology and crystalline structure. The ability to tune the labeling density with this method allowed us to prepare optimized samples that were used to visualize nanostructural features of cellulose through super-resolution microscopy. The efficiency, cost-effectiveness, and versatility of this method make it ideal for labeling nanocelluloses and imaging them through advanced microscopy techniques for a broad range of applications.

Bioinspired Thermoresponsive Xyloglucan-Cellulose Nanocrystal Hydrogels.

Thermoresponsive hydrogels present unique properties, such as tunable mechanical performance or changes in volume, which make them attractive for applications including wound healing dressings, drug delivery vehicles, and implants, among others. This work reports the implementation of bioinspired thermoresponsive hydrogels composed of xyloglucan (XG) and cellulose nanocrystals (CNCs). Starting from tamarind seed XG (XGt), thermoresponsive XG was obtained by enzymatic degalactosylation (DG-XG), which reduced the galactose residue content by ∼50% and imparted a reversible thermal transition. XG with native composition and comparable molar mass to DG-XG was produced by an ultrasonication treatment (XGu) for a direct comparison of behavior. The hydrogels were prepared by simple mixing of DG-XG or XGu with CNCs in water. Phase diagrams were established to identify the ratios of DG-XG or XGu to CNCs that yielded a viscous liquid, a phase-separated mixture, a simple gel, or a thermoresponsive gel. Gelation occurred at a DG-XG or XGu to CNC ratio higher than that needed for the full surface coverage of CNCs and required relatively high overall concentrations of both components (tested concentrations up to 20 g/L XG and 30 g/L CNCs). This is likely a result of the increase in effective hydrodynamic volume of CNCs due to the formation of XG-CNC complexes. Investigation of the adsorption behavior indicated that DG-XG formed a more rigid layer on CNCs compared to XGu. Rheological properties of the hydrogels were characterized, and a reversible thermal transition was found for DG-XG/CNC gels at 35 °C. This thermoresponsive behavior provides opportunities to apply this system widely, especially in the biomedical field, where the mechanical properties could be further tuned by adjusting the CNC content.

Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-Like Membrane.

The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the different proteins in this family and their relative abundance literally determine the fate of the cells. These interactions, however, are difficult to quantify, as they occur in a lipid membrane and involve proteins with multiple conformations and stoichiometries which can exist both in soluble and membrane. Here we focus on the interaction between two core Bcl-2 family members, the executor pore-forming protein Bax and the truncated form of the activator protein Bid (tBid), which we imaged at the single particle level in a mitochondria-like planar supported lipid bilayer. We inferred the conformation of the proteins from their mobility, and detected their transient interactions using a novel single particle cross-correlation analysis. We show that both tBid and Bax have at least two different conformations at the membrane, and that their affinity for one another increases by one order of magnitude (with a 2D-KD decreasing from ≃1.6µm-2 to ≃0.1µm-2) when they pass from their loosely membrane-associated to their transmembrane form. We conclude by proposing an updated molecular model for the activation of Bax by tBid.

Xyloglucan Structure Impacts the Mechanical Properties of Xyloglucan-Cellulose Nanocrystal Layered Films-A Buckling-Based Study.

Interactions between polysaccharides, specifically between cellulose and hemicelluloses like xyloglucan (XG), govern the mechanical properties of the plant cell wall. This work aims to understand how XG molecular weight (MW) and the removal of saccharide residues impact the elastic modulus of XG-cellulose materials. Layered sub-micrometer-thick films of cellulose nanocrystals (CNCs) and XG were employed to mimic the structure of the plant cell wall and contained either (1) unmodified XG, (2) low MW XG produced by ultrasonication (USXG), or (3) XG with a reduced degree of galactosylation (DGXG). Their mechanical properties were characterized through thermal shrinking-induced buckling. Elastic moduli of 19 ± 2, 27 ± 1, and 75 ± 6 GPa were determined for XG-CNC, USXG-CNC, and DGXG-CNC films, respectively. The conformation of XG adsorbed on CNCs is influenced by MW, which impacts mechanical properties. To a greater degree, partial degalactosylation, which is known to increase XG self-association and binding capacity of XG to cellulose, increases the modulus by fourfold for DGXG-CNC films compared to XG-CNC. Films were also buckled while fully hydrated by using the thermal shrinking method but applying the heat using an autoclave; the results implied that hydrated films are thicker and softer, exhibiting a lower elastic modulus compared to dry films. This work contributes to the understanding of structure-function relationships in the plant cell wall and may aid in the design of tunable biobased materials for applications in biosensing, packaging, drug delivery, and tissue engineering.

Dynamically Evolving Surface Patterns through Light-Triggered Wrinkling Erasure.

For many applications, it is imperative that changes in polymer surface topography, especially periodic patterns, can be triggered on command by a well-defined remote signal. In this contribution, we report a light-induced cascade of changes in wrinkling wavelengths on thin polymer layers supported by an elastomeric substrate under tensile stress. Through the applied supramolecular design, the effect of varying the ratio of light-active and light-passive components can be easily assessed, and it is shown that both the cascade type as well as the rate of the progress of the dynamic light-induced changes can be tuned by this ratio as well as by the light intensity. Furthermore, for the reported phenomena to occur, nominally only every 20th polymer repeat unit needs to be occupied by a chromophore, which makes the conversion of the sub-nanometer photoisomerization reaction into 10 µm scale changes of periodic surface patterns extremely efficient.

Membrane Cholesterol Reduces Polymyxin B Nephrotoxicity in Renal Membrane Analogs.

Polymyxin B (PmB) is a "last-line" antibiotic scarcely used due to its nephrotoxicity. However, the molecular basis for antibiotic nephrotoxicity is not clearly understood. We prepared kidney membrane analogs of detergent-susceptible membranes, depleted of cholesterol, and cholesterol enriched, resistant membranes. In both analogs, PmB led to membrane damage. By combining x-ray diffraction, molecular dynamics simulations, and electrochemistry, we present evidence for two populations of PmB molecules: peptides that lie flat on the membranes, and an inserted state. In cholesterol depleted membranes, PmB forms clusters on the membranes leading to an indentation of the bilayers and increase in water permeation. The inserted peptides formed aggregates in the membrane core leading to further structural instabilities and increased water intake. The presence of cholesterol in the resistant membrane analogs led to a significant decrease in membrane damage. Although cholesterol did not inhibit peptide insertion, it minimized peptide clustering and water intake through stabilization of the bilayer structure and suppression of lipid and peptide mobility.

Influence of Polymer Electronics on Selective Dispersion of Single-Walled Carbon Nanotubes.

The separation and isolation of semiconducting and metallic single-walled carbon nanotubes (SWNTs) on a large scale remains a barrier to many commercial applications. Selective extraction of semiconducting SWNTs by wrapping and dispersion with conjugated polymers has been demonstrated to be effective, but the structural parameters of conjugated polymers that dictate selectivity are poorly understood. Here, we report nanotube dispersions with a poly(fluorene-co-pyridine) copolymer and its cationic methylated derivative, and show that electron-deficient conjugated π-systems bias the dispersion selectivity toward metallic SWNTs. Differentiation of semiconducting and metallic SWNT populations was carried out by a combination of UV/Vis-NIR absorption spectroscopy, Raman spectroscopy, fluorescence spectroscopy, and electrical conductivity measurements. These results provide new insight into the rational design of conjugated polymers for the selective dispersion of metallic SWNTs.

Modeling enzymatic hydrolysis of lignocellulosic substrates using fluorescent confocal microscopy II: pretreated biomass.

In this study, we extend imaging and modeling work that was done in Part I of this report for a pure cellulose substrate (filter paper) to more industrially relevant substrates (untreated and pretreated hardwood and switchgrass). Using confocal fluorescence microscopy, we are able to track both the structure of the biomass particle via its autofluorescence, and bound enzyme from a commercial cellulase cocktail supplemented with a small fraction of fluorescently labeled Trichoderma reseii Cel7A. Imaging was performed throughout hydrolysis at temperatures relevant to industrial processing (50°C). Enzyme bound predominantly to areas with low autofluorescence, where structure loss and lignin removal had occurred during pretreatment; this confirms the importance of these processes for successful hydrolysis. The overall shape of both untreated and pretreated hardwood and switchgrass particles showed little change during enzymatic hydrolysis beyond a drop in autofluorescence intensity. The permanence of shape along with a relatively constant bound enzyme signal throughout hydrolysis was similar to observations previously made for filter paper, and was consistent with a modeling geometry of a hollowing out cylinder with widening pores represented as infinite slits. Modeling estimates of available surface areas for pretreated biomass were consistent with previously reported experimental results.

Modeling enzymatic hydrolysis of lignocellulosic substrates using confocal fluorescence microscopy I: filter paper cellulose.

Enzymatic hydrolysis is one of the critical steps in depolymerizing lignocellulosic biomass into fermentable sugars for further upgrading into fuels and/or chemicals. However, many studies still rely on empirical trends to optimize enzymatic reactions. An improved understanding of enzymatic hydrolysis could allow research efforts to follow a rational design guided by an appropriate theoretical framework. In this study, we present a method to image cellulosic substrates with complex three-dimensional structure, such as filter paper, undergoing hydrolysis under conditions relevant to industrial saccharification processes (i.e., temperature of 50°C, using commercial cellulolytic cocktails). Fluorescence intensities resulting from confocal images were used to estimate parameters for a diffusion and reaction model. Furthermore, the observation of a relatively constant bound enzyme fluorescence signal throughout hydrolysis supported our modeling assumption regarding the structure of biomass during hydrolysis. The observed behavior suggests that pore evolution can be modeled as widening of infinitely long slits. The resulting model accurately predicts the concentrations of soluble carbohydrates obtained from independent saccharification experiments conducted in bulk, demonstrating its relevance to biomass conversion work.

Fabrication of conductive polymer nanofibers through SWNT supramolecular functionalization and aqueous solution processing.

Polymeric thin films and nanostructured composites with excellent electrical properties are required for the development of advanced optoelectronic devices, flexible electronics, wearable sensors, and tissue engineering scaffolds. Because most polymers available for fabrication are insulating, one of the biggest challenges remains the preparation of inexpensive polymer composites with good electrical conductivity. Among the nanomaterials used to enhance composite performance, single walled carbon nanotubes (SWNTs) are ideal due to their unique physical and electrical properties. Yet, a barrier to their widespread application is that they do not readily disperse in solvents traditionally used for polymer processing. In this study, we employed supramolecular functionalization of SWNTs with a conjugated polyelectrolyte as a simple approach to produce stable aqueous nanotube suspensions, that could be effortlessly blended with the polymer poly(ethyleneoxide) (PEO). The homogeneous SWNT:PEO mixtures were used to fabricate conductive thin films and nanofibers with improved conductivities through drop casting and electrospinning. The physical characterization of electrospun nanofibers through Raman spectroscopy and SEM revealed that the SWNTs were uniformly incorporated throughout the composites. The electrical characterization of SWNT:PEO thin films allowed us to assess their conductivity and establish a percolation threshold of 0.1 wt% SWNT. Similarly, measurement of the nanofiber conductivity showed that the electrospinning process improved the contact between nanotube complexes, resulting in conductivities in the S m(-1) range with much lower weight loading of SWNTs than their thin film counterparts. The methods reported for the fabrication of conductive nanofibers are simple, inexpensive, and enable SWNT processing in aqueous solutions, and offer great potential for nanofiber use in applications involving flexible electronics, sensing devices, and tissue engineering scaffolds.

Benchtop Fabricated Nano-Roughened Microstructured Electrodes for Electrochemical and Surface-Enhanced Raman Scattering Sensing.

Tailoring a material's surface with hierarchical structures from the micro- to the nanoscale is key for fabricating highly sensitive detection platforms. To achieve this, the fabrication method should be simple, inexpensive, and yield materials with a high density of surface features. Here, using benchtop fabrication techniques, gold surfaces with hierarchically structured roughness are generated for sensing applications. Hierarchical gold electrodes are prepared on pre-stressed polystyrene substrates via electroless deposition and amperometric pulsing. Electrodes fabricated using 1 mm H[AuCl4] and roughened with 80 pulses revealed the highest electroactive surface area. These electrodes are used for enzyme-free detection of glucose in the presence of bovine serum albumin and achieved a limit of detection of 0.36 mm, below glucose concentrations in human blood. The surfaces nanoroughened with 100 pulses also showed excellent surface-enhanced Raman scattering (SERS) response for the detection of rhodamine 6G, with an enhancement factor of ≈2 × 106 compared to detection in solution, and for the detection of a self-assembled monolayer of thiophenol, with an enhancement factor of ≈30 compared to the response from microstructured gold surfaces. It is envisioned that the simplicity and low fabrication cost of these gold-roughened structures will expedite the development of electrochemical and SERS sensing devices.

Thermobifida fusca cellulases exhibit limited surface diffusion on bacterial micro-crystalline cellulose.

Elucidation of cellulase-cellulose interactions is key to modeling biomass deconstruction and in understanding the processes that lead to cellulase inactivation. Here, fluorescence recovery after photobleaching and single molecule tracking (SMT) experiments are used to assess the surface diffusion of Thermobifida fusca cellulases on bacterial micro-crystalline cellulose. Our results show that cellulases exhibit limited surface diffusion when bound to crystalline cellulose and that a large fraction of the cellulases remain immobile even at temperatures optimal for catalysis. A comparison of our experimental results to Monte Carlo (MC) simulations, which use published diffusion coefficients to model cellulase displacements, shows that even those enzymes that are mobile on the cellulose surface exhibit significantly slower diffusive motions than previously reported. In addition, it is observed that the enzymes that show significant displacements exhibit complex, non-steady surface motions, which suggest that cellulose-bound cellulases exist in molecular states with different diffusive characteristics. These results challenge the notion that cellulases can freely diffuse over cellulose surfaces without catalyzing bond cleavage.

Observing and modeling BMCC degradation by commercial cellulase cocktails with fluorescently labeled Trichoderma reseii Cel7A through confocal microscopy.

Understanding the depolymerization mechanisms of cellulosic substrates by cellulase cocktails is a critical step towards optimizing the production of monosaccharides from biomass. The Spezyme CP cellulase cocktail combined with the Novo 188 ß-glucosidase blend was used to depolymerize bacterial microcrystalline cellulose (BMCC), which was immobilized on a glass surface. The enzyme mixture was supplemented with a small fraction of fluorescently labeled Trichoderma reseii Cel7A, which served as a reporter to track cellulase binding onto the physical structure of the cellulosic substrate. Both micro-scale imaging and bulk experiments were conducted. All reported experiments were conducted at 50 °C, the optimal temperature for maximum hydrolytic activity of the enzyme cocktail. BMCC structure was observed throughout degradation by labeling it with a fluorescent dye. This method allowed us to measure the binding of cellulases in situ and follow the temporal morphological changes of cellulose during its depolymerization by a commercial cellulase mixture. Three kinetic models were developed and fitted to fluorescence intensity data obtained through confocal microscopy: irreversible and reversible binding models, and an instantaneous binding model. The models were successfully used to predict the soluble sugar concentrations that were liberated from BMCC in bulk experiments. Comparing binding and kinetic parameters from models with different assumptions to previously reported constants in the literature led us to conclude that exposing new binding sites is an important rate-limiting step in the hydrolysis of crystalline cellulose.

Investigation of the porous structure of cellulosic substrates through confocal laser scanning microscopy.

At the most fundamental level, saccharification occurs when cell wall degrading enzymes (CWDEs) diffuse, bind to and react on readily accessible cellulose fibrils. Thus, the study of the diffusive behavior of solutes into and out of cellulosic substrates is important for understanding how biomass pore size distribution affects enzyme transport, binding, and catalysis. In this study, fluorescently labeled dextrans with molecular weights of 20, 70, and 150 kDa were used as probes to assess their diffusion into the porous structure of filter paper. Fluorescence microscopy with high numerical aperture objectives was used to generate high temporal and spatial resolution datasets of probe concentrations versus time. In addition, two diffusion models, including a simple transient diffusion and a pore grouping diffusion models, were developed. These models and the experimental datasets were used to investigate solute diffusion in macro- and micro-pores. Nonlinear least squares fitting of the datasets to the simple transient model yielded diffusion coefficient estimates that were inadequate for describing the initial fast diffusion and the later slow diffusion rates observed; on the other hand, nonlinear least squares fitting of the datasets to the pore grouping diffusion model yielded estimations of the micro-pore diffusion coefficient that described the inherently porous structure of plant-derived cellulose. In addition, modeling results show that on average 75% of the accessible pore volume is available for fast diffusion without any significant pore hindrance. The method developed can be applied to study the porous structure of plant-derived biomass and help assess the diffusion process for enzymes with known sizes.

Fluorescent labeling and characterization of cellulose nanocrystals with varying charge contents.

Cotton-source cellulose nanocrystals (CNCs) with a range of surface charge densities were fluorescently labeled with 5-(4, 6-dichlorotriazinyl) aminofluorescein (DTAF) in a facile, one-pot reaction under alkaline conditions. Three CNC samples were labeled: (I) anionic CNCs prepared by sulfuric acid hydrolysis with a sulfur content of 0.47 wt %, (II) a partially desulfated, sulfuric acid-hydrolyzed CNC sample, which was less anionic with an intermediate sulfur content of 0.21 wt %, and (III) uncharged CNCs prepared by HCl hydrolysis. The DTAF-labeled CNCs were characterized by dynamic light scattering, atomic force microscopy, fluorescence spectroscopy and microscopy, and polarized light microscopy. Fluorescent CNCs exhibited similar colloidal stability to the starting CNCs, with the exception of the HCl-hydrolyzed sample, which became less agglomerated after the labeling reaction. The degree of labeling depended on the sulfur content of the CNCs, indicating that the presence of sulfate half-ester groups on the CNC surfaces hindered labeling. The labeling reaction produced CNCs that had detectable fluorescence, without compromising the overall surface chemistry or behavior of the materials, an aspect relevant to studies that require a fluorescent cellulose substrate with intact native properties. The DTAF-labeled CNCs were proposed as optical markers for the dispersion quality of CNC-loaded polymer composites. Electrospun polyvinyl alcohol fibers loaded with DTAF-labeled CNCs appeared uniformly fluorescent by fluorescence microscopy, suggesting that the nanoparticles were well dispersed within the polymer matrix.

Functionalization of 3D Printed Scaffolds Using Polydopamine and Silver Nanoparticles for Bone-Interfacing Applications.

The prevention of bacterial colonization and the stimulation of osseointegration are two major requirements for bone-interfacing materials to reduce the incidence of complications and promote the restoration of the patient's health. The present investigation developed an effective, two-step functionalization of 3D printed scaffolds intended for bone-interfacing applications using a simple polydopamine (PDA) dip-coating method followed by the formation of silver nanoparticles (AgNPs) after a second coating step in silver nitrate. 3D printed polymeric substrates coated with a ∼20 nm PDA layer and 70 nm diameter AgNPs proved effective in hindering Staphylococcus aureus biofilm formation, with a 3000-8000-fold reduction in the number of bacterial colonies formed. The implementation of porous geometries significantly accelerated osteoblast-like cell growth. Microscopy characterization further elucidated homogeneity, features, and penetration of the coating inside the scaffold. A proof-of-concept coating on titanium substrates attests to the transferability of the method to other materials, broadening the range of applications both in and outside the medical sector. The antibacterial efficiency of the coating is likely to lead to a decrease in the number of bacterial infections developed after surgery in the presence of these coatings on prosthetics, thus translating to a reduction in revision surgeries and improved health outcomes.

Efficient capture of recombinant SARS-CoV-2 receptor-binding domain (RBD) with citrate-coated magnetic iron oxide nanoparticles.

Several vaccines against COVID-19 use a recombinant SARS-CoV-2 receptor-binding domain (RBD) as antigen, making the purification of this protein a key step in their production. In this work, citrate-coated magnetic iron oxide nanoparticles were evaluated as nano adsorbents in the first step (capture) of the purification of recombinant RBD. The nanoparticles were isolated through coprecipitation and subsequently coated with sodium citrate. The citrate-coated nanoparticles exhibited a diameter of 10 ± 2 nm, a hydrodynamic diameter of 160 ± 3 nm, and contained 1.9 wt% of citrate. The presence of citrate on the nanoparticles' surface was confirmed through FT-IR spectra and thermogravimetric analysis. The crystallite size (10.1 nm) and the lattice parameter (8.3646 Å) were determined by X-ray diffraction. In parallel, RBD-containing supernatant extracted from cell culture was exchanged through ultrafiltration and diafiltration into the adsorption buffer. The magnetic capture was then optimized using different concentrations of nanoparticles in the purified supernatant, and we found 40 mg mL-1 to be optimal. The ideal amount of nanoparticles was assessed by varying the RBD concentration in the supernatant (between 0.113 mg mL-1 and 0.98 mg mL-1), which resulted in good capture yields (between 83 ± 5% and 94 ± 4%). The improvement of RBD purity after desorption was demonstrated by SDS-PAGE and RP-HPLC. Furthermore, the magnetic capture was scaled up 100 times, and the desorption was subjected to chromatographic purifications. The obtained products recognized anti-RBD antibodies and bound the ACE2 receptor, proving their functionality after the developed procedure.

Determination of the molecular states of the processive endocellulase Thermobifida fusca Cel9A during crystalline cellulose depolymerization.

Detailed understanding of cell wall degrading enzymes is important for their modeling and industrial applications, including in the production of biofuels. Here we used Cel9A, a processive endocellulase from Thermobifida fusca, to demonstrate that cellulases that contain a catalytic domain (CD) attached to a cellulose binding module (CBM) by a flexible linker exist in three distinct molecular states. By measuring the ability of a soluble competitor to reduce Cel9A activity on an insoluble substrate, we show that the most common state of Cel9A is bound via its CBM, but with its CD unoccupied by the insoluble substrate. These findings are relevant for kinetic modeling and microscopy studies of modular glycoside hydrolases.