Preferential Adsorption and Activity of Monocomponent Cellulases on

Mar 13, 2013 - and Orlando J. Rojas*. ,†,§. †. Department of Forest Products Technology, School of Chemical Technology, Aalto University, FI-0007...
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Preferential Adsorption and Activity of Monocomponent Cellulases on Lignocellulose Thin Films with Varying Lignin Content Raquel Martín-Sampedro,*,†,† Jenni L. Rahikainen,‡ Leena-Sisko Johansson,† Kaisa Marjamaa,‡ Janne Laine,† Kristiina Kruus,‡ and Orlando J. Rojas*,†,§ †

Department of Forest Products Technology, School of Chemical Technology, Aalto University, FI-00076 Aalto, Espoo, Finland VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Espoo, Finland § Departments of Forest and Biomaterials and Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695, United States ‡

S Supporting Information *

ABSTRACT: Understanding the enzymatic hydrolysis of cellulose and the influence of lignin in the process are critical for viable production of fuels and chemicals from lignocellulosic biomass. The interactions of monocomponent cellulases with cellulose and lignin substrates were investigated by using thin films supported on quartz crystal microgravimetry (QCM) resonators. Trichoderma reesei exoglucanase (CBH-I) and endoglucanase (EG-I) bound strongly to both cellulose and lignin but EG-I exhibited a distinctive higher affinity with lignin, causing a more extensive inhibition of the cellulolytic reactions. CBH-I was found to penetrate into the bulk of the cellulose substrate increasing the extent of hydrolysis and film deconstruction. In the absence of a cellulose binding domain (CBD) and a linker, the CBH-I core adsorbed slowly and was not able to penetrate into the film. Conversely to CBH-I, EG-I exhibited activity only on the surface of the lignocellulose substrate even when containing a CBD and a linker. Interestingly, EG-I displayed a clearly different interaction profile as a function of contact time registered by QCM.

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INTRODUCTION The search for alternative and renewable sources for the production of fuels and chemicals has increased an interest in the bioconversion of lignocellulosic biomass.1,2 A major ratelimiting step in biochemical processing of lignocellulose is the enzymatic depolymerization of the substrate into mono- and oligomeric sugars that are subsequently fermented and processed into fuels or other chemicals.3 Therefore, a better undestanding of the interactions between the enzymes and solid lignocellulosic substrates is required in order to develop an effective pretreatment and enzymatic hydrolysis and obtain cost-effective methods for bioconversion of cellulose. Enzymatic degradation of crystalline cellulose is catalyzed by a group of enzymes acting synergistically. Cellulases catalyze the cleavage of β-1,4-glycosidic bonds that link the glycosyl units of cellulose. However, different enzymes act on different sites of the cellulose polymer: (i) exo-β-(1,4)-D-glucanases or cellobiohydrolases (CBHs) hydrolyze cellulose chains from the chain ends to produce soluble cellobiose as a main product; (ii) endo-β-(1,4)-glucanases (EGs) hydrolyze internal nicks of the cellulosic chains and thereby introduce new chain ends for CBHs to act on, and (iii) β-D-glucosidases hydrolyze the soluble disaccharide cellobiose to monomeric glucose.4,5 Trichoderma reesei cellulases are well-characterized enzymes that are industrially relevant and widely exploited in many commercial enzyme products. The major exo- and endogluca© 2013 American Chemical Society

nases of T. reesei are composed of domains, which are structurally and functionally discrete units of proteins. The catalytic domain (CD) is connected to a cellulose binding domain (CBD) through an interdomain linker peptide.6 The CD is responsible for the hydrolysis reaction, whereas the primary role of CBDs is to concentrate the enzymes on the insoluble cellulosic surface;7 this often leads to improved hydrolytic performance. The presence and surface distribution of lignin have a detrimental effect on the enzymatic degradation of lignocellulosic biomass.8,9 Two possible mechanisms of inhibition have been suggested, namely, a physical, steric hindrance of the cellulosic surfaces,10,11 and the reversible/irreversible adsorption of cellulases onto lignin, which causes losses of active enzymes.8,12−14 Hydrophobic,15,16 electrostatic,8 and hydrogen bonding interactions8,13,17 are suggested to contribute to enzyme affinity with lignin. However, the exact mechanisms by which cellulases interact with lignin and become inhibited have yet to be fully elucidated. Due to the complexity of lignocellulose and cellulase systems, most studies in this field measure the overall production of sugar after enzymatic hydrolysis based on batch sampling and Received: February 11, 2013 Revised: March 10, 2013 Published: March 13, 2013 1231

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distilled water and neutralized with hydrochloric acid at 1% v/v. The precipitated product was rinsed several times with water and then freeze-dried for further use. This acetylated lignin is hereafter referred to as “AcL”. Enzymes. Three purified T. reesei cellulases were selected to study enzyme interactions with the films. Cellobiohydrolase I (CBH I, TrCel7A) and the corresponding catalytic core domain (CBH I core, TrCel7A core) were purified as described in Rahikainen et al.31 and Suurnäkki et al.,33 respectively. Endoglucanase I (EG I, TrCel7B) was purified as described in Suurnäkki et al.33 Enzyme purity was studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) using precast 10% gels applicable to stain-free protein visualization34 with a Criterion system provided by Bio-Rad Laboratories Inc. (Hercules, CA). Results are shown in Figure S1 of the Supporting Information. Film Preparation. Films were prepared on silica-coated QCM-D crystals (Q-sense AB, Västra Frölunda, Sweden) or 1 × 1 cm2 silicon wafers with Si 100 native oxide layer on top (Okmetic, Espoo, Finland). Substrates were exposed to ultraviolet radiation (UV/ozonator) prior to film deposition. Solutions of 10 g/L of CTA or AcL were prepared by using chloroform as solvent. These solutions were blended at different CTA:AcL ratios: 1:0, 5:1, 1:1, and 0:1. In all the solutions, the concentration of the major component was always kept constant at 5 g/L. Solutions were spin-coated onto the clean silica substrate at 4000 rpm for 1 min (acceleration speed of 2130 rpm/s). Conversion of CTA to cellulose, and AcL to lignin was carried out by exposing the films to an ammonium hydroxide atmosphere in a desiccator at room temperature for 3 days. The resultant bicomponent cellulose/ lignin (Ce/L) films were then rinsed with Milli-Q water, dried with nitrogen gas, and stored in the dark. Film Characterization. AFM imaging was used to characterize the changes in the structure and morphology of the cellulose, lignin, and bicomponent films, before and after deacetylation and enzymatic hydrolysis. A Nanoscope IIIa Multimode scanning probe microscope (Digital Instruments, Inc., Santa Barbara, CA) was used. The images were acquired in tapping mode using an E-scanner and silicon cantilevers (NSC15/AIBS from Micromasch, Tallinn, Estonia). The drive frequency of the cantilever was 325 kHz, and the radius of curvature of the AFM tip was less than 10 nm, according to the manufacturer. At least two different films obtained for each condition were prepared, and at least two different areas were analyzed on each of them. No image processing except flattening was made. Image analysis was performed using Nanoscope software (ver. V6.13 R1, Digital Instruments, Inc.) from which roughness and Z-sections in line profiles were determined. The changes in morphology of the films before and after deacetylation are reported in Supporting Information (AFM images in Figure S2), while changes induced by enzymatic treatment are introduced in the Results and Discussion section. Water contact angle measurements were performed to test the changes in hydrophobicity of the surfaces. A CAM-200 contact angle goniometer (KSV Instruments Ltd., Helsinki, Finland) was used to determine the water (7 μL) advancing contact angle on the surfaces. The measurements were conducted in ambient air at room temperature. The surface chemical composition of the thin films was quantified by using an X-ray photoelectron spectroscopy (XPS) electron spectrometer (AXIS 165, Kratos Analytical, Man-

offline measurements. However, time resolution in these methods is limited and therefore they fail to probe dynamic phenomena, especially in the initial stages of the process.18 To overcome these limitations, several in situ methods have been proposed, such as in situ UV−vis spectrophotometry18 or direct real-time visualization by high-speed atomic force microscopy (AFM).19 Likewise, cellulose thin films (regenerated cellulose, cellulose nanocrystals, or nanofibrillated cellulose) have been used with sensing techniques such as quartz crystal microbalance (QCM),20−26 ellipsometry,27−29 neutron reflectometry25,26 or surface plasmon resonance30 to monitor the binding and/or catalytic activity of cellulases, in situ and in real time. Recently, we used ultrathin lignin films to study interactions with cellulases by using the QCM technique.31 Despite the useful information that has been accessed from such studies, a more complete assessment could be achieved if lignin, cellulose, hemicelluloses, and other macromolecules from the cell wall were all present in the given model substrate. Therefore, we have previously used thin films made of lignocellulose nanofibrils in order to more closely monitor the interactions of cellulases.32 Moreover, we have used bicomponent films containing lignin and cellulose so as to gain control of the mass inventory, composition, and distribution of each component. They have allowed fundamental investigations on the inhibitory effect of lignin since other factors, such as hemicellulose content, and fibril dimensions can be decoupled.14 However, these initial attempts used commercial multicomponent enzymes systems, which may contain coadjuvants such as surfactants and preservatives that may influence the measurements, particularly when using the cited highly sensitive techniques.21 Therefore, the objective of this work is to study the enzymatic hydrolysis by monocomponent cellulases acting on bicomponent cellulose/lignin films on QCM sensors with adjustable polymer concentration. The synthesis of the bicomponent films was based on the work of Hoeger et al.14 with the difference being that the precursor cellulose polymer used consisted of cellulose triacetate (CTA) instead of trimethylsilyl cellulose (TMSC), which simplified the procedure for cellulose/lignin film preparation. Furthermore, the use of purified monocomponent cellulases facilitated our investigation on the nature of the surface interactions between the different types of cellulases and cellulose and lignin and to unveil aspects related to enzyme inhibition. Results from this study are expected to contribute to a better understanding of the inhibitory role of lignin and the nature of enzyme−substrate interactions, especially in the case of monocomponent enzymes that take part of the more complex systems. In addition, it will make possible a systematic screening of enzyme components for given characteristics of the substrate (mainly in relation to cellulose and lignin types and content).

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EXPERIMENTAL SECTION Materials. All reagents used were of analytical grade (Sigma-Aldrich). Milli-Q water (from a Millipore Direct-Q 3UV, Molsheim, France) was used to prepare all the solutions. CTA from Fluka was used as the precursor of cellulose. According to the manufacturer, the acetyl content of CTA was 43−49% (corresponding to a degree of substitution (DS) of ∼3). Organosolv lignin from aspen was kindly supplied by Lignol Innovations Ltd. (Burnaby, Canada). Lignin was acetylated by dissolving 100 mg in a mixture of pyridine and acetic anhydride (1:1 ratio) and subjecting it to microwave irradiation for 1 min at 750 mW. Afterward, it was diluted with 1232

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bicomponent films were monitored in situ using a QCM-D (QCM E4 model, Q-sense AB, Gothenburg, Sweden). The experiments were carried out at 40 °C in pH 5 (buffer acetate 0.1 M). The buffer solution was injected into the QCM flow module where the substrates (films) were mounted. After the unit was filled with the background solution, the flow rate was maintained at 0.1 mL/min using a peristaltic pump. The film swelled in contact with the buffer solution, until it reached equilibrium. Following, the respective monocomponent enzyme solution (CBH I, EG I or CBH I core with concentrations of 4.5 and 9 μM) was continuously injected at a flow rate of 0.1 mL/min for 15 min. The pump was then stopped and started again (0.1 mL/min) after 3 h (4 h in the case of EG I) of treatment to rinse the system with buffer solution. All measurements were recorded at 5 MHz fundamental resonance frequency and its overtones corresponding to 15, 25, 35, 55, and 75 MHz. The third overtone (15 MHz) was used for data processing.

chester, UK) with monochromated Al Kα X-ray source at 100W. Both low-resolution survey scans and high-resolution regions of carbon and oxygen 1s signals were recorded. For a more detailed chemical analysis, carbon high-resolution regions were further curve fitted using symmetric gaussians.35 At least three different areas were analyzed on each film. XPS results in Table S1 (Supporting Information) indicate that both CTA and AcL were converted to Cellulose (Ce) and lignin (L), respectively, after a single deacetylation step with ammonium hydroxide vapor. Complete deacetylation of CTA and AcL was confirmed by the depletion of COO− groups after 68 h deacetylation. Furthermore, reduction of the water contact angle (WCA) of the cellulose film (Ce:L of 1:0) from 61 to 26o further confirmed complete conversion of CTA to cellulose (Figure S2, Supporting Information). Figure 1 shows the surface concentration of C−C groups from XPS (carbon atoms bonded only to other carbon atoms

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RESULTS AND DISCUSSION Enzymatic Hydrolysis with CBH I. Cellulose, lignin, and bicomponent films containing given amounts of lignin were exposed to a solution of T. reesei cellobiohydrolase CBH I (Cel7A) in order to study the effect of lignin on binding and cellulolytic reactions. CBH I represents approximately 50−60% of total protein in commercial multicomponent enzyme products produced by T. reesei and is known to degrade cellulose from its reducing end groups.37 The adsorption of enzyme and hydrolysis of cellulose was followed in real-time by monitoring the shifts in QCM frequency −Δf 3 (proportional to the shift in mass on the sensor) (Figure 2). After the enzyme injection, a sharp increase in −Δf 3 was observed with all the films, irrespective to the lignin content. This indicates the dominant role of CBH I adsorption in the initial stages involving enzyme−substrate interaction. In the case of the pure lignin films, a plateau was achieved after less than 10 min upon enzyme injection. In films containing cellulose, the resonance frequency plateau was followed by gradual decrease in the −Δf 3, indicating that the enzyme catalyzed film degradation after its initial adsorption. Similar behavior was observed when bicomponent cellulose/lignin films were exposed to a multicomponent cellulase system.14 This can be explained by the fact that CBH I makes an important fraction of total protein composition in multicomponent enzyme cocktails, as indicated above. Importantly, we note that CBH I was adsorbed more extensively on films containing higher relative amounts of cellulose: the calculated maximum binding capacity (Table S2 in Supporting Information), in QCM Hz units, corresponded to 36, 32, 25, and 22 Hz for 1:0, 5:1, 1:1, and 0:1 Ce/L films, respectively. As a reference, the binding capacity determined for multicomponent enzymes were 57, 138, and 145 Hz for 10:1, 5:1 and 1:1 Ce/L films, respectively.14 Overall, our results suggests a distinctively higher affinity of CBH I with cellulose compared to lignin. After 20 min of treatment, the enzyme injection was stopped to study how the absence of enzyme in solution affects the hydrolysis of pure cellulose or bicomponent (Ce/L) films (Figure 2a). No significant changes in the −Δf 3 profiles were observed, indicating that the films had already been saturated with the enzyme. Results in Figure 2 corresponded to enzymatic treatment using a 9 μM CBH I solution, but similar results were observed for 4.5 μM CBH I solutions (see Figure

Figure 1. Correlation plot including the % C−C surface concentration versus the O/C atomic ratio calculated from the XPS data in Table S1. Results for bicomponent films before (crosses) and after deacetylation treatment (circles) are shown, as well as theoretical values (filled diamond symbols) of cellulose, milled wood lignin (MWL), and organosolv lignin (Org. Lignin).14,35.

or hydrogen) versus the atom oxygen/carbon ratio on the surface of bicomponent films before and after deacetylation. According to Johansson et al., 36 correlating the two independent variables for lignocellulose samples provide more reliable data than using either of them. Theoretical values for cellulose, milled wood lignin (MWL) and organosolv lignin14 are indicated in Figure 1, as a reference. It should be noticed that although theoretically pure cellulose does not have a C−C signal, there is always a small contribution that is attributed to adventitious surface contamination.35 This partially explains the differences between the values for cellulose film after deacetylation (Ce/L 1:0) and the respective reference. Lignin film after deacetylation (Ce/L 0:1) showed similar values to those of organosolv lignin, although the C−C % was slightly higher. The XPS values shown in Figure 1 for all the bicomponent films after deacetylation (Ce/L) were located on a line between the pure components, cellulose and lignin, following linear mixing rules. The increase in lignin content in the blends resulted in a decrease in the O/C ratio together with an increase in the C−C %, as expected. Enzymatic Treatments in Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). Enzyme binding and hydrolytic activity on the cellulose, lignin, and 1233

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surface is fully covered by enzymes.28 However, when a multicomponent enzyme system was applied, endoglucanases increased the number of free chain ends for the exoglucanase to act on, producing a synergistic effect.20,21 Therefore, the addition of fresh enzyme solution speeds up the hydrolysis process when multicomponent enzymes are used, as observed ́ by Martin-Sampedro et al.32 In the case of pure cellulose films (Ce/L 1:0), an increase in −Δf 3 and dissipation, resulting from the increased mass and viscoelasticity of the film, respectively, were observed in the initial adsorption-dominated phase. Afterward, the high hydrolytic activity of the enzyme on the surface of the film decreased the surface mass and thickness of the film, also causing a slight decrease in dissipation. Then, the dissipation leveled off and started to increase considerably, while the −Δf continued to decrease. This strong increase in dissipation indicates penetration of the enzyme into and digestion within the bulk of the film, causing water swelling and decreasing the stiffness throughout the bulk of the film.22,25,26 If the hydrolysis continues, the dissipation value will reach a maximum at a certain point after which it starts to decrease, because the film becomes denser, and the surface turns into cellulose clusters, which contain the less hydrolyzable residual cellulose chains.20,22,23,25,26 However, in the current study, the buffer rinsing of the film was carried out before reaching this point, so the results in Figure 2 correspond to the early stages of substrate hydrolysis. The decrease in −Δf and dissipation upon rinsing was mainly due to removal of reversibly adsorbed enzymes. XPS measurements support the occurrence of irreversible enzyme binding,30 as indicated by the nitrogen signal recorded in the case of films after enzyme treatment (Table S4, Supporting Information). In the case of the pure lignin films, the shift in frequency and dissipation followed the typical QCM adsorption profile. The irreversible, nonspecific binding of the enzymes on the lignin films was confirmed by the presence of nitrogen in the films (Table S4, Supporting Information). These results are in agreement with those recently reported by Rahikainen et al.31 who used lignin films isolated from different sources. As mentioned earlier, the enzymes have hydrophobic amino acid residues38,39 that likely contribute to hydrophobic interactions with the lignin substrate.15,17 Electrostatic and hydrogen bonding interactions also contribute to the adsorption of cellulases onto lignin.8,13,17 This irreversible adsorption causes a reduction of free enzyme available that otherwise would hydrolyze the cellulose material. XPS results indicated a slightly higher increase of nitrogen content in the films containing lignin after the CBH I treatment, which supported a more extensive irreversible adsorption. The inhibitory effect of lignin is clearly demonstrated in bicomponent substrates. When lignin content in the films was increased, a reduction in the extent of the hydrolysis occurred. The films containing 16.7% of lignin (Ce/L 5:1) produced frequency and dissipation profiles similar to those of pure cellulose films (Ce/L 1:0). However, although similar amount of enzyme was initially adsorbed on both types of substrates, the total amount of cellulose hydrolyzed was lower when lignin was present (as indicated by calculated parameter B, Table S3 of the Supporting Information, corresponding to 85 and 51 Hz for 1:0 and 5:1 Ce/L films, respectively). The increase in dissipation related to hydrolytic phase was less prominent with the bicomponent films containing higher amounts of lignin, indicating more limited deconstruction and swelling of the film.

Figure 2. Negative shift in QCM frequency (−Δf 3) and changes in dissipation (ΔD3) as a function of time after 15 min of continuous injection of cellobiohydrolase CBH I (9 μM, flow rate of 100 μL/min) followed by buffer (pH 5) rinsing as indicated by the vertical arrows. Cellulose/lignin films with different Ce/L ratios were used. Profiles in panels a and b display the change in frequency during the first 40 min and full time range (240 min), respectively. Data in panel c correspond to the dissipation shift in the full time range.

S3, Supporting Information). It has been reported previously that for small enzyme concentrations, equilibrium adsorption increases linearly with bulk enzyme concentration; eventually cellulase adsorption ceases for high bulk cellulase concentrations.27−29 The system has a limited amount of reducing chain ends for CBH I to act on, and it is expected that the available reactive sites are already occupied. Thus, in the adsorption-plateau region, higher solution concentrations of enzyme do not increase deconstruction rates because the 1234

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When a multicomponent enzyme mixture was used to treat similar bicomponent films (Ce/L 5:1),14 higher enzyme adsorption and inhibition were observed. A reasonable explanation is that exoglucanase CBH I has higher affinity to cellulose, as we have found and discussed before, limiting the amount of enzyme binding to lignin domains, while stronger inhibitory effect to multicomponent enzyme system is caused by other cellulase components with higher affinity with lignin.14 The shifts in frequency and dissipation registered for Ce/L 1:1 and Ce/L 0:1 substrates were very similar, indicating an almost complete inhibition (note Ce/L 1:1 and Ce/L 0:1 contain the same amount of lignin). There is indication that the enzymes adsorbed onto the lignin domains during the first minutes of treatment reduce the accessibility and prevents binding to cellulose, supporting the hypothesis of lignin inhibition due to physical or steric blocking.10,11 Enzymatic Hydrolysis with CBH I Core. QCM-D experiments were carried out with the CBH I core, that is, CBH I lacking the linker and the cellulose CBD (Figure 3). Clearly, the main differences with respect to native CBH I were the more limited adsorption onto the films (a maximum QCM binding capacity of 23 Hz for 1:0 and 5:1 Ce/L films, and 10 Hz for 1:1 and 0:1 Ce/L films was determined as reported in Table S2 in the Supporting Information) and the less extensive hydrolysis of cellulose, as indicated by the smaller overall change in frequency (parameter B in Table S3 of Supporting Information of 35, 26, 5, and 1 Hz for 1:0, 5:1, 1:1 and 0:1 Ce/ L films, respectively). Results from other work support these observations.12,26,29,31,40 The QCM dissipation of cellulose films increased upon cellulose adsorption and slowly decreased due to hydrolysis. Thus, similar but more limited and slower changes in dissipation and frequency were observed for the CBH I core compared with native CBH I, during the first minutes of treatment. The second phase observed for native CBH I, when the dissipation increased significantly, was not evident when CBH I core was used. As mentioned above, this phase is associated with the penetration of the enzyme into and digestion within the bulk of the film.25,26 In fact, it has been proposed that the enzyme penetrates by the CBD hopping from one cellulose binding site to another.26 Therefore, there is indication that the CBH I core is not able to penetrate, and its action is localized only on the surface of film. CBH I core was adsorbed onto lignin films slowly and to a lesser extent compared to native CBH I, due to the lack of binding domain. It has been suggested that CBH I binding onto isolated lignin films involves a two-phase process with a fast start and slower second phase, whereas CBH I core adsorption follows only one mode with slow adsorption.31 Thus, the CBD and/or linker are responsible for the initial fast phase of adsorption. Upon buffer rinsing, only a limited mass of enzyme is removed from both Ce/L 0:1 and 1.1 films, indicating that most CBH I core is irreversibly adsorbed to lignin-rich substrates. Compared to native CBH I, a limited degree of CBH I core removal was observed upon rinsing Cellulose-rich substrates (Ce/L 1:0 and 5:1). Furthermore, XPS analysis (Table S4 of Supporting Information) indicates an increase in nitrogen content upon CBH I core treatment. These results support the hypothesis that irreversible adsorption also happens in the absence of CBD and/or linker, causing a similar inhibition of cellulose hydrolysis, as was observed for native CBH I.

Figure 3. QCM negative shift of frequency and dissipation change as a function of time after 15 min of continuous injection of a cellobiohydrolase CBH I core (CBH I without the linker and cellulose binding domain) (9 μM, flow rate of 100 μL/min) followed by buffer (pH 5) rinsing as indicated by the vertical arrows. Cellulose/lignin films with different Ce/L ratios were used. Profiles in panels a and b show the change in frequency for the first 40 min and the full time range (240 min), respectively. Data in panel c correspond to the dissipation shift in the full time range.

Figure S4 (Supporting Information) includes the frequency and dissipation profiles for enzymatic treatment with CBH I core at lower concentrations (4.5 μM). Contrary to what was observed for native CBH I, a reduction in both hydrolysis and adsorption rates took place when the concentration of enzyme in solution was reduced. This indicates that the presence of CBD facilitates surface saturation on the films. Enzymatic Hydrolysis Using EG I. Pure cellulose, lignin, and bicomponent films were exposed to a solution containing 1235

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endoglucanase I (9 μM) (Figure 4). EGI makes up 12−15% of the multicomponent enzyme system of T. reesei.37 Remarkably,

number of free chains ends causes the synergistic effect when CBH I and EG I enzymes are present. For the pure cellulose film (Ce/L 1:0), a decrease in −Δf 3 was observed during the first minutes of treatment, clearly indicating a degradation of the film. It is only after a few minutes that the adsorption of the enzyme is registered. This means that initially hydrolytic reactions are dominant, in contrast to what was observed in the case of CBH I and multicomponent enzyme systems. This effect has also been observed for the hydrolysis of amorphous cellulose films with EG I, depending on the enzyme concentration.21 After the initial drop, −Δf 3 slightly increased indicating mass increase of the film caused by higher level of enzyme or water coupling (swelling) compared to film degradation. After 15−17 min, the hydrolysis became dominant (−Δf 3 started to decrease) with no clear effect upon stopping enzyme injection. At this point, dissipation started to decrease, signifying a reduction in viscoelasticity of the film. After ca. 50 min of treatment, the frequency reached a plateau, indicating equilibrium between hydrolysis and adsorption of enzyme or water (swelling). No further changes in viscoelasticity occurred as indicated by the plateau in dissipation. These results point out that after initial enzymatic action on the surface of the film, the enzyme did not penetrate onto the bulk of the film and both hydrolysis and swelling stopped. Neutron reflectometry experiments indicated mass loss upon treatment of regenerated cellulose films with endoglucanases, over several hours, although the QCM frequency registered was nearly constant after 1 h due to water uptake.21 Finally, upon rinsing with buffer, a large shift in frequency was observed, indicating a removal of material from the cellulose film. This mass decrease can be caused either by release of cellulose fragments, deswelling of the cellulose film or by enzyme desorption.20,26 We note that an increase in nitrogen content was measured upon EG I treatment (Table S4, Supporting Information), indicating irreversible adsorption of enzyme onto cellulose films. In contrast to cellulose films, EGI interaction with lignin produced a distinctive adsorption phase. After 20 min of treatment, the lignin film was not yet saturated: 2 h of continuous enzyme injection was necessary for QCM frequency to reach a plateau (data not shown). Furthermore, the adsorption of EG I onto lignin appeared to be irreversible, since no change in frequency was observed upon buffer rinsing of lignin films (Ce/L 0:1). The high affinity to lignin results in enzyme inhibition, as can be observed from the slight decrease in −Δf 3 (related to cellulose hydrolysis) that was measured with films containing 16.7% lignin (Ce/L 5:1) (see parameter B in Table S3 of Supporting Information, which was calculated after taking into account the different phases of the process: 38 and 13 Hz for 1:0 and 5:1 Ce/L films, respectively), leading to a calculated inhibition of 65% in this case. Comparing EG I and CBH I adsorption, larger amounts of CBH I were adsorbed onto cellulose films (maximum binding capacity of 36 and 11 Hz, respectively according to Table S2 in the Supporting Information). More extensive adsorption of EG I onto cellulose have been also reported.41 Maurer et al.24 reported that it is possible that the CBD of CBH I contacts the surface more readily because of the orientation of the enzyme in the surface region, producing a higher adsorption rate and affinity between CBH I and cellulose. The exterior of the cellulose catalytic domain may also interact with the surface of cellulose, providing additional binding interactions and guiding the binding of the CBD. The opposite is observed for lignin:

Figure 4. QCM negative shift of frequency and dissipation change as a function of time after 15 min of continuous injection of endoglucanase EG I (9 μM, flow rate of 100 μL/min) followed by buffer (pH 5) rinsing as indicated by the vertical arrows. Cellulose/lignin films with different Ce/L ratios were used. Profiles in panels a and b show the change in frequency for the first 40 min and the full time range (300 min), respectively. Data in panel c indicate the dissipation shift in the full time range.

different QCM frequency profiles were observed for EGI compared to CBH I and multicomponent enzymes mixtures. While CBH I hydrolyzes cellulose from the reducing end groups, EG I randomly cleaves the internal bonds at amorphous sites. Thus, EG I generates chain ends by decreasing the degree of polymerization and also causes swelling and an exposure of these chains to the surrounding medium.20 This increase in the 1236

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Figure 5. AFM 1 × 1 μm2 height images of bicomponent films with different Ce/L ratios (1:0, 5:1, 1:1, and 0:1 from left to right) before (first row) and after enzymatic treatment with CBH I (second row), CBH I core (third row) or EG I (fourth row). The height scale (bar at the far right of each row) corresponds to Z values between −5 and +5 nm. The roughness and contact angle of each film are also indicated.

multicomponent enzymes mixtures,14 an increase in roughness was observed in all Ce/L films after treatment with monocomponent enzymes. The hydrolysis of cellulose resulted in a higher AFM height difference between the lignin domains and the silica support. Furthermore, the irreversible adsorption of enzymes onto the remaining domains also contributed to the increase in roughness. Comparing films treated with native CBH I and CBH I core, larger increase in roughness was observed for the native enzyme. XPS data (Table S4, Supporting Information) includes the contribution of Si 2p signal from the solid support for films treated with native CBH I. This is taken as evidence of the removal of cellulose from the film. The percentage of silicon decreased with the increase lignin content in the films, confirming the trend of a reduced hydrolysis extent observed in QCM-D experiments. Interestingly, no Si 2p signal was observed after treatment with CBH I core, which indicate, in

larger adsorption of EG I was measured compared to CBH I (maximum binding capacity of 22 and 70 Hz, see Table S2 in the Supporting Inforamtion). Palonen et al.12 reported that endoglucanases may bind more efficiently on lignin due to its more open active site compared to the tunnel-shaped active site of cellobiohydrolases. The more open nature of the active site may reveal the aromatic residues to lignin, which may lead to adsorption through hydrophobic interactions.12 This high affinity of EG I with lignin causes a more extensive inhibition for cellulose hydrolysis. Finally, when the concentration of the EG I solution was reduced (4.5 μM), limited adsorption and hydrolysis rates were observed for all the films (Figure S5, Supporting Information). Morphological and Chemical Changes Induced by Cellulolytic Enzymes. Figure 5 indicates the morphology of bicomponent films before and after enzymatic treatment with CBH I, CBH I core, and EG I. As was the case with 1237

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Core, and EG I (Figures S3, S4 and S5, respectively); XPS data of bicomponent films enzymatically treated (Table S4); a correlation plot including the % C−C surface concentration versus the O/C atomic ratio calculated from XPS data after enzymatic hydrolysis (Figure S6); and AFM 0.5 × 0.5 μm2 height images of lignin films after enzymatic treatment with CBH I or EG I (Figure S7). This information is available free of charge via the Internet at http://pubs.acs.org.

agreement with QCM-D experiments, that the lack of binding domain and/or linker limited the hydrolysis to the surface of the films. Thus, when representing the XPS C−C percentage against the O/C atomic ratio for enzymatic treated films (Figure S6, Supporting Information) the loci of the films in the plot remain in a line between pure cellulose and pure lignin, except for Ce/L 1:0 and 5:1 films treated with native CBH I. In these films, the cellulose hydrolysis was more intensive, and a significant increase in O/C was observed, mainly due to the exposure of silica support. For EG I treated films, similar increase in roughness was observed, compared to native CBH I. Conversely, QCM-D results indicated lower cellulose hydrolysis with EG I, in agreement with the absence or small contribution of Si 2p signal EG I treated film, expecting lower increase in roughness. Nevertheless, the irreversible adsorption of EG I was higher (higher nitrogen content), causing an increase in roughness. Lignin films were very flat before enzymatic treatment and only enzyme adsorption occurred. Figure S7 (Supporting Information) shows AFM 0.5 × 0.5 μm2 height images of substrates after CBH I and EG I treatments. CBH I-treated film shows features of approximately 10−15 nm in size, very similar in shape and size to the individual enzymes observed by Hirsh et al.42 after immobilization of cellulases on polystyrene films. Conversely, EG I-treated films included larger features, despite having similar domain structure and dimensions (15 × 5 × 10 nm) than exoglucanases,28 suggesting enzyme aggregates. It is likely that the higher affinity of EG with lignin and the different hydrolysis behavior of this enzyme compared to CBH I is related to the formation of such enzyme aggregates.

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*(O.J.R.) E-mail address: [email protected]; Tel: +1-9195137494; fax: +1- 919-515 6302. (R.M-S.) E-mail address: [email protected]; Tel. +34 913476834; fax: +34 913476767. Present Address †

Cellulose and Paper Laboratories. Forestry Products Department, CIFOR-INIA, Ctra de La Coruña Km 7.5, 28040 Madrid, Spain Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial support from the Academy of Finland (Finland Distinguish Professorship, O.J.R.) and funding support from the Lignocel Project (TEKES, Finland). Dr J.M. Campbell is acknowledged for the XPS measurements.

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CONCLUSIONS Bicomponent films containing cellulose and lignin were used as novel sensing platforms to study in situ and in real-time cellulose binding and hydrolysis events. The effect of lignin content on the cellulose hydrolysis was determined by using monocomponent enzymes from T. reesei. On cellulose-rich films, CBH I showed first activity on the surface and then penetration and digestion within the bulk of the substrate. When CBH I core enzyme was used, the absence of CBD and/ or linker slowed down enzyme adsorption and no penetration was observed, limiting CBH I core action to the surface. Although EG I has a CBD and a linker, it was found to act only on the surface. CBH I (with or without CBD) presented high affinity with cellulose, while EG I adsorbed largely on lignin surfaces, causing higher hydrolysis inhibition. Inhibition due to the presence of lignin was observed for all enzymes assayed, mainly due to irreversible adsorption. The results obtained by using model bicomponent (cellulose/lignin) films advance our understanding of enzymatic activity on lignocellulose a step further; however, the detailed microstructure of the substrate, its porosity, and more complex chemical and spatial heterogeneity are factors that require consideration.

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AUTHOR INFORMATION

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information document includes data on the purity of the monocomponent cellulases (Figure S1); AFM images and XPS data of bicomponent films before and after deacetylation (Figure S2 and Table S1); results from empirical models related to binding and hydrolysis rate constants (Tables S2 and S3); the negative shift in QCM frequency as a function of time during enzymatic treatment with 4.5 μM CBH I, CBH I 1238

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