Direct in Situ Observation of Synergism between Cellulolytic Enzymes

Nov 6, 2013 - High-resolution atomic force microscopy (AFM) was used to image the real-time in situ degradation of crystalline by three types of T. re...
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Direct in Situ Observation of Synergism between Cellulolytic Enzymes during the Biodegradation of Crystalline Cellulose Fibers Jingpeng Wang,† Amanda Quirk,† Jacek Lipkowski,*,† John R. Dutcher,‡ and Anthony J. Clarke§ †

Department of Chemistry, ‡Department of Physics, and §Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada S Supporting Information *

ABSTRACT: High-resolution atomic force microscopy (AFM) was used to image the real-time in situ degradation of crystalline by three types of T. reesei cellulolytic enzymes TrCel6A, TrCel7A, and TrCel7Band their mixtures. TrCel6A and TrCel7A are exo-acting cellobiohydrolases processing cellulose fibers from the nonreducing and reducing ends, respectively. TrCel7B is an endoglucanase that hydrolyzes amorphous cellulose within fibers. When acting alone on native cellulose fibers, each of the three enzymes is incapable of significant degradation. However, mixtures of two enzymes exhibited synergistic effects. The degradation effects of this synergism depended on the order in which the enzymes were added. Faster hydrolysis rates were observed when TrCel7A (exo) was added to fibers pretreated first with TrCel7B (endo) than when adding the enzymes in the opposite order. Endo-acting TrCel7B removed amorphous cellulose, softened and swelled the fibers, and exposed single microfibrils, facilitating the attack by the exo-acting enzymes. AFM images revealed that exo-acting enzymes processed the TrCel7B-pretreated fibers preferentially from one specific end (reducing or nonreducing). The most efficient (almost 100%) hydrolysis was observed with the mixture of the three enzymes. In this mixture, TrCel7B softened the fiber and TrCel6A and TrCel7A were directly observed to process it from the two opposing ends. This study provides high-resolution direct visualization of the nature of the synergistic relation between T. reesei exo- and endo-acting enzymes digesting native crystalline cellulose.

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INTRODUCTION Cellulose is the most abundant biopolymer on earth, and thus it represents a major source of renewable carbon for the production of biofuel (cellulosic ethanol) and other valueadded bioproducts. It is composed of unbranched homopolymers of β-(1,4)-linked D-glucose; the actual repeating unit is β(1,4)-linked glucose dimer D-cellobiose. Despite this simple molecular composition, the complexity of the intra- and interhydrogen-bonding networks and resulting rigidity of crystalline cellulose fibrils make them ideal for their structural role as the major component of plant cell walls.1 However, the crystallinity of cellulose poses significant challenges for its efficient and thus economical conversion to a renewable source of bioenergy (reviewed in refs 2−4). The biodegradation of crystalline cellulose involves the synergistic action of cellulolytic enzymes with different specificities, and considerable effort has been expended to exploit this process for biomass utilization (reviewed in refs 1 and 5). Despite several decades of investigation, the molecular mechanism of this synergistic process remains unsolved, especially for the cooperation between cellobiohydrolases. This lack of fundamental knowledge reflects the technical complexity of studying the function of soluble enzymes acting on their insoluble substrate. Nonetheless, an understanding of the synergy between cellulolytic enzymes is essential to their efficient use in the production of cellulosic ethanol on the industrial level. © 2013 American Chemical Society

Recently, we developed a protocol involving high-resolution atomic force microscopy (AFM) to monitor the enzymatic digestion of never-dried crystalline cellulose both in situ and in real time.6,7 Herein, we present the application of this technique to provide a unique direct observation of the synergistic action of an industrially important cellulolytic system on the degradation of crystalline cellulose fibers. These measurements provide information that will contribute to the design of more efficient enzyme mixtures for biomass conversion. Most cellulolytic enzymes are glycoside hydrolases that can be categorized as cellobiohydrolases (CBHs), endoglucanases (EGs), or β-glucosidases.1 Generally, CBHs are exo-acting processive enzymes that hydrolyze cellulose from chain ends and release cellobiose as the product, whereas EGs are endoacting and thus randomly attack cellulose chains, producing cellooligosaccharides. β-Glucosidases hydrolyze cellobiose and soluble oligosaccharides to glucose and thereby relieve the product inhibition of CBHs. To date, the best-characterized cellulolytic system is that produced by industrially important fungus Trichoderma reesei8 (a clonal derivative of Hypocrea jecorina9). The major component of the T. reesei cellulolytic Received: September 4, 2013 Revised: October 28, 2013 Published: November 6, 2013 14997

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the exoexo synergy between TrCel6A and TrCel7A using chemically treated crystalline fibers (cellulose III) as the substrate. More recently, Ganner et al.26 successfully used AFM to study the synergy of cellulases in situ, but as with the former study, these investigations were restricted to observations made on a film of polymorphic (amorphous and crystalline) and dehydrated cellulose embedded in an epoxy resin. AFM was also used by Ding et al.27 to determine the microfibrillar structure and enzymatic solubilization of plant cell walls using samples pretreated with acid chlorite. In earlier work, we used high-resolution AFM to investigate in situ the interaction of Cel6A and TrCel7B with single native, never-dried bacterial cellulose (BC) fibers in real time.6,7 Changes in fiber volume, the root-mean-square roughness, and rates of hydrolysis of the single fibers were determined directly from the in situ AFM images acquired over time. Analyses based on the high-resolution AFM images revealed the molecular-level enzymatic action. In the case of TrCel7B, initial hydrolysis is followed by the swelling of the exposed individual microfibrils and bundles of microfibrils, resulting in the loosening of surface fibrils and the exposure of microfibril ends at the fiber surface.6 In the present study, we employed this AFM-based analysis to study the synergism between cellulolytic enzymes of T. reesei. There are several elements of novelty in this work. All previous AFM studies involved chemically pretreated samples. In contrast, using nanoscale AFM imaging, we observed the degradation of never-dried, native crystalline by three types of cellulases (TrCel6A, TrCel7A, and TrCel7B) alone and in combination. We succeeded in obtaining high-resolution images of single cellulose fibers. The high-resolution images allowed us to perform quantitative analyses of changes with incubation time in the fiber volume, width, length, and height. We demonstrated the real-time concerted actions between different cellulolytic enzymes and provided a visual, direct understanding of the underlying synergistic mechanism. Moreover, our studies with the mixture of the three enzymes provided direct visual evidence of the bidirectional activity of TrCel6A andTrCel7A digesting the opposing ends of the same single fiber.

system is glycoside hydrolase family 7 (GH7) CBH, TrCel7A (formerly CBH I).8 It has a modular structure consisting of a catalytic domain connected by a linker peptide to a carbohydrate binding module (CBM).10,11 The catalytic domain has a tunnel-shaped active site that is composed of 10 binding subsites.12 T. reesei also produces and secretes a second CBH, TrCel6A (formerly CBH II), and a number of EGs, including TrCel7B, TrCel5A, and TrCel12A in lower abundance.8 Compared to the active site of CBHs, that of EGs is more open and groove-shaped.13 It is well recognized that CBHs are more efficient in the degradation of crystalline cellulose, whereas EGs preferentially target less-ordered, amorphous cellulose regions.14 The complementary actions of different cellulolytic enzymes are thought to be responsible for the synergistic effects observed, whereby the combined enzymatic activity of a mixture of two or more enzymes is substantially higher than the sum of their individual activities. In the past, several types of synergism have been reported, yet only two types have been studied extensively, endoexo and exoexo synergy.5 The conventional explanation for the endoexo synergism mechanism proposes that randomly acting EG generates new glucan chain ends that serve as the starting points for processive actions of CBH.15 Recent studies provide additional explanations for endoexo synergy, suggesting that EG may preferentially remove obstacle-like amorphous cellulose in the glucan chains, thus assisting the processive movement of adsorbed CBH and accelerating the release of trapped CBH as described by Jalak et al. (and references therein).16 Cooperation between two CBHs (i.e., exoexo synergy) was first reported over 30 years ago,17 but the mechanism by which this occurs is less understood. Previously, it was shown by X-ray crystallography of the enzymes, electron microdiffraction analysis of cellulose microcrystals, and theoretical considerations that the two T. reesei CBHs, TrCel7A and TrCel6A, hydrolyze crystalline cellulose from the reducing and nonreducing ends, respectively. It was subsequently proposed that these differences in the chain end preference and directionality of actions were responsible for the apparent exoexo synergy (reviewed in Teeri et al.).18 However, controversy developed when TrCel6A was shown to possess some endo-processive hydrolytic activity on crystalline cellulose, suggesting that the exoexo synergism observed was actually the result of conventional endoexo synergism.19,20 Many of the synergism studies conducted in the past employed traditional biochemical assays and chromatography methods, and the synergistic effects were reported on the basis of the detection of bulk concentrations of the resulting products.5,16 Most of these studies did not resolve the area below mesoscopic length scales (micrometers or larger). However, cellulase−cellulose interactions occur, of course, on the molecular level, and they are very dependent upon the surface micro- and nanostructures and the composition of the fibers. Therefore, the mechanistic understanding of enzyme synergism obtained from in situ high-resolution microscopy studies is of critical importance. Atomic force microscopy (AFM) allows the direct visualization of the enzymatic degradation of crystalline cellulose fibers on the nanometer scale. It has been used to study the crystal structure and microfibril surface and assembly of cellulose21−23 and to probe the mechanism of cellulose−enzyme interactions (recently reviewed in Bubner et al.24). Recently, state-of-the-art, highspeed AFM has been used by Igarashi et al.25 to demonstrate

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EXPERIMENTAL SECTION

Materials. Single-side-polished silicon wafers (WaferNet, Inc., IC grade) were used as the supporting substrate in all AFM imaging experiments. Before each experiment, Si samples (1.2 cm × 0.9 cm) were ultrasonicated in HPLC-grade acetone (Fisher) for 15 min, followed by soaking in piranha solution for 4 h and finally thorough rinsing in water and methanol (HPLC grade). Unless otherwise indicated, all AFM imaging experiments were performed in 50 mM citrate buffer (pH 5.0), which was prepared from sodium citrate (99.9%, Alfa Aesar) and anhydrous citric acid (99.5%, Fluka). Millipore ultrapure water (resistivity 18.2 MΩ cm at 25 °C) was used for all aqueous solutions. Preparation of Cellulose Fiber Thin Films. Native bacterial cellulose (BC) fibers were obtained from Acetobacter xylinum, and they were prepared in thin films as described by Quirk et al.7 The immobilization of this never-dried crystalline cellulose onto silicon wafer substrates was performed using the Langmuir−Blodgett (LB) technique in which floating BC fibers were reproducibly transferred onto the substrate, resulting in a thin layer of isolated single fibers. The sample with a wet film was quickly transferred to the AFM cell that was subsequently filled with citrate buffer. Efforts were made to prevent drying the film during this sample preparation step. Each AFM sample imaged in this work contained approximately 10 μg of BC in the hydrated state.7 We have demonstrated in ref 6 that the amount of sugar released from this amount of sample is below the detection limit 14998

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function in Gwyddion (GPL free software). A mask was first applied to the fiber via the height threshold control, and then the volume was obtained using the grain minimum basis algorithm. On the basis of multiple attempts to measure the selected fiber volume and average height/width from each image carefully, we found that maximum errors of ±5, ± 7, and ±7% were associated with measurements of the volume, the average height, and the average width, respectively. Tip artifacts are an ever-present problem in AFM imaging studies. Several precautions were taken to minimize this problem. First, an AFM tip calibration kit containing colloid gold particles (5, 10, 15, 20, and 30 nm diameter, Pelco standard kit 16205, Ted Pella, Inc.) was used to characterize the tips used in this work. Second, new tips were always used for every trial. Third, at the beginning of each trial the scan direction was always rotated by 45 and 90° while keeping all other settings the same to ensure that the obtained images had minimal tip artifacts. Fourth, all of the topography images used in the quantitative analyses were corrected for the tip-broadening effect by employing a tip-deconvolution module (using a blind estimation and surface reconstruction method) in the commercial SPIP software package (ImageMetrology Ltd., Denmark). Scanning electron microscopy (SEM) was used as previously described6,7 to determine the true tip geometry instead of using the nominal specifications of the tip geometry provided by the manufacturer. In the SEM images, the AFM tips used for imaging were observed to have a conical shape with a radius of apex curvature determined to be 15 ± 5 nm.

of the reducing sugar assay. Therefore, no attempts were made to determine the amount of sugar released in AFM experiments. When greater amounts of cellulose were transferred to the support, the aerial density of fibers was too large, preventing high-resolution imaging and quantitative analysis of single fiber digestion as shown in our previous paper.7 The freshly prepared cellulose samples were used immediately in AFM imaging experiments. Purification of T. reesei Enzymes (TrCel7A, TrCel6A, and TrCel7B). Each cellulolytic enzyme was isolated and purified from a commercial preparation of the T. reesei enzyme secretome (Iogen Corporation) by anion-exchange chromatography on DEAE-Sepharose as described by Bhikhabhai et al.28 Ultrafiltration was used both to concentrate the purified Cel7B and exchange the purification buffer with 50 mM sodium citrate (pH 5.0) prior to dividing into aliquots and storing at −20 °C. Stock enzyme solutions used for each AFM imaging experiment were prepared for immediate use by thawing an aliquot of enzyme in 10 mL of a 50 mM citrate buffer (pH 5.0). Protein concentrations were determined using the method of Bradford et al.,29 SDS-PAGE was performed using the method of Laemmli30 with Commassie blue staining, and quantification by scanning densitometry was performed using a Chemigenius2 (Syngene) imaging system. To ensure that these preparations were devoid of other contaminating cellulolytic enzymes, samples were analyzed by Western immunoblotting using component-specific polyclonal antisera from rabbit31 as previously described6 (data not shown). AFM Imaging and Data Analysis. AFM images were collected using a Pico SPM microscope (Molecular Imaging, now Agilent Technologies) with an AFMS 182 scanner and the PicoScan 5.3 software system or the Agilent 5500 system with the small scanner and PicoView software. All imaging experiments were performed in magnetic ac (MAC) mode using tip B of type I MAClevers (Agilent Technologies). The average thickness, width, length, and nominal force constant of this cantilever were approximately 1.0 μm, 35 μm, 90 μm, and 1.75 N/m, respectively. The feedback loop was adjusted to 20−25% amplitude reduction (amplitude set point) to maintain the tip−sample interaction in the light-tapping regime. A homemade AFM liquid cell was equipped with capillary ports for the addition of enzyme solutions for in situ imaging experiments. The cell did not have flowcell capability, and hence after the addition of the second enzyme the first enzyme remained in the system. All imaging trials were carried out at room temperature (20 ± 2 °C). To ensure absolute stability, the AFM was located in a specially designed laboratory with an acoustic and vibration isolation cage. Changes in the cellulose surface structure during an ongoing hydrolysis process were monitored by a continuous time-lapse AFM imaging technique.6,7 In each trial, target cellulose fibers were chosen and scanned for a minimum of 30 min prior to adding enzyme to ensure that the system (temperature, mechanical drift, noise level, and firmness of fiber−substrate attachment) was stable. Then, a precise volume of enzyme stock solution was injected very carefully such that the original target fiber was not disturbed. Each real-time AFM image of the target fiber was obtained repetitively at a scan speed of 2 to 3 Hz, resulting in a total recording time of 2 to 4 min per image and up to 3 to 4 h of total enzyme incubation time. Movies consisting of a series of time-lapse AFM topography images are presented in the Supporting Information. Data analyses, including cross-section profiles, fiber lengths, heights and widths, and volume calculations, were all performed on AFM topography images using Gwyddion v2.28 software. Each topography image was processed with zeroth-order line flattening and secondorder polynomial background leveling excluding fiber regions. Cross sections of fiber surfaces were calculated using line profiles drawn perpendicular to the fiber with line widths of a few pixels to average the data. The average fiber width was calculated on the basis of 10 measurements of the width that were made at 10 equidistant points along the selected fiber segment. The average fiber height was obtained by averaging the total height of selected fiber segment with subtraction of the average background height. The volume of a selected fiber segment was determined by using the grain analysis

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RESULTS AND DISCUSSION We previously demonstrated that a continuous time-lapse AFM imaging technique can be used successfully and reliably to investigate enzyme−cellulose systems.6,7 These and other studies have demonstrated that prolonged AFM imaging (usually implemented in light tapping mode) does not cause a mechanical disruption to or a disintegration of cellulose fibers,6,7,32,33 and thus the observed changes in fiber morphology during incubation with a single cellulolytic enzyme (TrCel7A or TrCel6A) can be considered to be the result of enzymatic action. In the present study, we applied the timelapse AFM imaging technique to investigate the endoexo synergy in the digestion of crystalline, which is the most common synergy of cellulases.11,16 We describe the synergy in three cases: (i) exoendo on a fiber incubated first with TrCel7A, followed by the addition of TrCel7B; (ii) endoexo on a fiber incubated first with TrCel7B and then treated with TrCel7A; and (iii) endoexo-exo on a fiber incubated first with TrCel7B and then with the consecutive addition of TrCel6A and TrCel7A. In these three experiments, the enzymes were added sequentially to fibers following successive incubation periods. The results of the initial incubation with single enzymes were consistent with our previously published data6,7 (Supporting Information). The data presented in the main body of this article illustrate chiefly degradation due to combinations of the enzymes. To assist in the interpretation of AFM images, it is useful to recall that BC has predominantly an Iα crystalline structure:1 it consists of individual glucan chains with cross sections of ∼1 nm that stack in parallel with each other and adhere by means of intra- and intermolecular hydrogen bonding networks and van der Waals forces to form structurally rigid crystalline microfibrils with cross sections on the order of 4−6 nm. These microfibrils are the building blocks of ribbon-shaped macrofibrils with cross sections of ∼4 nm × 100 nm. The stacking of macrofibrils gives fibers with cross sections in the range of 0.1− 1 μm. Because of the parallel packing of glucan chains, their reducing ends are located at one end of the fiber and their nonreducing ends are at the other end of the fiber.34 14999

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Exoendo Synergy. The frame labeled “0 min” in Figure 1 presents a topography image of two BC fibers stacked side by

incubation times with the mixture of the enzymes after TrCel7B was added to the AFM cell. The fragments of microfiber ribbons that were initially present at the fiber surface are visibly degraded 59 min after the introduction of TrCel7B into the mixture, and at longer incubation times, the width of the fiber decreased with a gap appearing between the two fibers. We note that a substantial number of the target fibers remained after 214 min of incubation with TrCel7A and TrCel7B. The quantitative volume calculations presented in Figure 2 show changes in the fiber volume during initial digestion by

Figure 1. Time-lapse AFM topography images of crystalline cellulose degradation by TrCel7B following preincubation with TrCel7A (exoendo synergism). After 174 min of incubation at 20 °C for BC in 50 mM sodium citrate at pH 5.0 with 28.0 μg/mL TrCel7A (image labeled 0 min), TrCel7B (21.0 μg/mL) was added and incubation was continued for the times shown.

Figure 2. Changes in fiber volume upon digestion of crystalline cellulose with TrCel7A and TrCel7B (exoendo synergism). The volume changes were calculated from the topography images obtained during the 174 min preincubation with TrCel7A (closed symbols), as described in Figure 1, and from those obtained from images acquired after the addition of TrCel7B (open symbols).

side that had been incubated with TrCel7A for 174 min (thus hydrolyzing cellulose from their reducing ends). The selected target fibers possessed a variety of morphological features, such as naturally formed fiber ends and ribbons. The average height of the fragments of the ribbons seen in these images is ∼3 nm, which is the average height of a single ribbon of microfibrils in a BC cellulose fiber.34,35 Figure SI-1 illustrates that after the initial 3 h period of hydrolysis major portions of the fibers remained intact, indicating an incomplete digestion process by TrCel7A. However, the positions of the fragments of the ribbons of microfibrils and the separation between these fragments changed. The relative change in fiber width and height as a function of the incubation time are shown in Figure SI-2a,b. The decreases in the average fiber width and height are ∼13 and ∼30%, respectively. The absolute changes in the fiber height are shown in Figure SI-2c. Within the first 3 h of hydrolysis, the average fiber height decreased by ∼2 nm (average value of three independent trials), which is 30% less than the value expected for the digestion of a single ribbon. This result indicates that TrCel7A hydrolyzes slightly less than the single layer of microfibrils and that the enzymatic attack took place at the end of the microfibril ribbons. The results of three trials presented in Figure SI-2 indicated the high level of reproducibility of these experiments. The hydrolytic action of TrCel7A on a single BC fiber observed in these experiments is in good agreement with the previous results obtained from bulk bioanalytical assays reported in the literature, which suggested that the hydrolytic activity of CBHs, when acting alone, decreases after an initial period of time and that these enzymes are incapable of completely digesting cellulose fibers.5,14,36 After ∼3 h of incubation of the BC fibers with TrCel7A, a solution of TrCel7B (hydrolyzing amorphous regions of the fiber) was injected into the AFM cell and fiber images were collected for the next 214 min. The three additional frames in Figure 1 show a series of images acquired at different

single enzyme TrCel7A (from 0 to 174 min) and by mixture TrCel7A + TrCel7B (from 174 + 2 to 174 + 214 min). The volume of the TrCel7A-treated fiber decreased by 35% of its original volume (65% was undigested). With the injection of TrCel7B, degradation was renewed and the volume decreased linearly with time over the next 214 min such that only 30% of the initial volume remained undigested. With reference to the fiber volume at the time of injection of TrCel7B, the enzyme mixture digested approximately 50% of the fiber volume. Therefore, the half time for the fiber digestion by the enzyme mixture was ∼3.5 h. Figure 3 presents fiber height profiles taken at selected intervals of time during the incubation with TrCel7A (Figure 3A) and with the mixture of TrCel7A + TrCel7B. Incubation with the single enzyme caused only small changes in the height profile. In contrast, the height profile changed significantly with time during the incubation with the enzyme mixture. The hydrolysis was not uniform across the fibers because they were preferentially digested from the top, whereas the base of the fibers that were in contact with the solid support surface remained intact. This is consistent with a predominantly layerby-layer hydrolysis of the microfiber ribbons. The thinning of the top portions of the fibers suggests that the exterior fragments of the fiber are more disordered (amorphous) and hence more susceptible to attack by TrCel7B. It also indicates that the central core of the fiber that has predominantly crystalline structure was digested slowly. Because individual TrCel7A and TrCel7B enzymes at comparable concentrations did not have similar hydrolytic activity,6,7 these results demonstrate a distinct synergistic effect and the enhancement 15000

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methods. The polymorphic cellulose used in this earlier study was dehydrated and embedded in an epoxy resin.26 In contrast, our preparations of pure crystalline cellulose were maintained in a hydrated state and simply adsorbed onto the Si surface. This provides a striking example of the complexity associated with the study of cellulolytic enzymes acting on insoluble substrates and why an understanding of the process has been hard to achieve. Selected topography images obtained after adding TrCel7A to TrCel7B-pretreated fibers are presented in Figure 4.

Figure 4. Time-lapse AFM topography images of crystalline cellulose degradation by TrCel7A following preincubation with TrCel7B (endoexo synergism). BC fibers in 50 mM sodium citrate buffer at pH 5.0 were incubated with 7.0 μg/mL TrCel7B for 124 min (panel labeled 0 min) prior to the addition of 23.0 μg/mL TrCel7A and further incubated at 20 °C for the times shown (12−55 min). (A, B) The two crystalline fibers monitored in the experiment. The red arrow denotes the site of progressive directional degradation of fiber A.

Figure 3. Changes in the height profile of cellulose fibers upon digestion with TrCel7A and TrCel7B (exoendo synergism). Height profiles of the fibers digested over time were determined along the line marked in the first panel of Figure 1 (labeled 0 min). (A) Height profiles of fibers during the first 118 min of incubation with TrCel7A and (B) height profiles of fibers during the 214 min of incubation with both TrCel7A and TrCel7B following the 174 min preincubation with only TrCel7A.

Additional images are shown in Figure SI-4. These images show changes in the two fiber segments labeled A and B over a 55 min period of digestion by the enzyme mixture. Segments A and B were effectively digested after 55 and 47 min, respectively. The addition of TrCel7A to the fiber pretreated with TrCel7B digested the fiber much more quickly than the sample treated first with TrCel7A and then by the addition of TrCel7B. Quantitative volume analysis of a fragment of fiber A (Figure 5a) revealed that the injection of TrCel7A immediately led to increased digestion, leaving only small amounts of undigested cellulose fibers after 55 min. The data in Figure 5b show that the height of the fiber decreased much faster than its width so that no apparent reduction in the width of the fiber could be seen in the images. This behavior is in contrast to the exoendo case discussed above in which the addition of TrCel7B to the TrCel7A-pretreated cellulose caused a significant decrease in the width of the fiber. The digestion of the fiber in the endoexo case apparently proceeded in a ribbon-byribbon manner. This point is illustrated in Figure 6, which shows in the inset a high-resolution image of a fiber with a characteristic step of ∼6 nm in height corresponding to a ribbon end. The height of this step was not significantly changed during the 120 min incubation with TrCel7B. However, the profile lines taken along the length of the fiber shown in Figure 6 indicate that the step disappeared 12 min after the addition of TrCel7A to the mixture. Apparently, TrCel7B softened the fiber, exposed individual fibrils, and removed amorphous cellulose such that the ribbon was digested very quickly by the addition of TrCel7A. The highresolution single-fiber images in Figure 4 also revealed that the enzymatic attack of TrCel7A takes place preferentially from one end of the fiber. Fiber A was apparently progressively digested

of the activity of the two enzymes in the mixture. TrCel7B appeared to remove amorphous cellulose fragments, thereby exposing individual fibrils that were then digested from the reducing end by TrCel7A. Endoexo Synergy. Endoexo synergistic action has been shown to be reciprocal; not only does TrCel7A benefit from the action of TrCel7B but also the action of TrCel7A enhances the hydrolysis of the substrate pretreated with TrCel7B.14,16,37,38 To verify this property, the hydrolytic reaction of adding TrCel7B to TrCel7A-pretreated native BC fibers (described above) was compared to the reverse sequence of enzyme addition (viz. addition of TrCel7A to fibers pretreated with TrCel7B). AFM images recorded during the initial incubation of the fibers with TrCel7B (from 0 to 120 min) are presented in Figure SI-3a. Consistent with the results of our earlier work6 and the data shown in Figure SI-3 of the Supporting Information, TrCel7B caused swelling of the fiber and exposed individual fibrils by removing amorphous fragments. Because of the combined actions of swelling and degradation, the overall volume of the fiber decreased by only 5% after 120 min of incubation with TrCel7B. Thus, in contrast to what has been reported recently in another AFM-based study,26 TrCel7B appears to be capable of disrupting crystalline cellulose, albeit not completely. Ganner et al.26 observed only marginal activity of this enzyme on crystalline regions of polymorphic cellulose, but this difference may be accounted for by the different sample preparation 15001

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This is consistent with a parallel packing of glucan chains in crystalline cellulose and with the properties of TrCel7A that attacks the chains from only their reducing ends.12,38 This point is illustrated further in Figure 7, which shows cross sections in

Figure 5. Changes in fiber volume, average height, and width with time upon digestion of crystalline cellulose with TrCel7B and TrCel7A (endoexo synergism). (A) Determination of volume changes were made from the topography images obtained during the 120 min preincubation with TrCel7B (open symbols), as described in Figure 4, and from those obtained from images acquired at the times shown after the addition of TrCel7A (closed symbols). The arrow denotes the addition of TrCel7A. (Inset) Segment of the fiber analyzed, denoted by the red “mask.” (B) Corresponding height (closed symbols) and width (open blue symbols) profiles of the digested fiber.

Figure 7. Height profiles of a cellulose fiber during digestion with TrCel7A after TrCel7B pretreatment (endoexo synergism). The height profiles were measured at the times as indicated across the fiber along the line shown in the corresponding AFM images at its (A) top, (B) middle, and (C) lower end. The crystalline cellulose fiber was imaged after 120 min of pretreatment with TrCel7B and then at the times as indicated following the addition of TrCel7A and further incubation for 53 min.

the direction normal to the fiber taken at the top, middle, and bottom of the fiber at different digestion times. The cross sections demonstrate clearly that the height of the fiber decreased much faster at the top of the fiber than in the middle and at the bottom and that in the initial stages the middle is digested faster than the bottom of the fiber. Presumably, the reducing ends of glucan chains are oriented toward the top of the fiber in this image. This behavior is again different than in the exoendo case, where Figure 2 shows that the fibers were digested approximately uniformly along their full length. The synergy due to the addition of TrCel7A to TrCel7B-pretreated fibers was enhanced relative to the synergy after the addition of TrCel7B to TrCel7A-pretreated fibers. Apparently, the synergetic action is not fully symmetrical. The prolonged pretreatment with TrCel7B removed amorphous cellulose more effectively, swelled fibers, and exposed microfibril ends and in this way better assisted the attack of TrCel7A. Endoexo-Exo Case. The synergistic effect was also investigated for a mixture of three T. reesei cellulolytic enzymes, TrCel7B (removing amorphous cellulose and exposing

Figure 6. High-resolution imaging of crystalline cellulose digestion by TrCel7B followed by TrCel7A (endoexo synergism). Height profiles of the fiber shown in the inset were measured along the direction marked by the line after incubation for the times as indicated with TrCel7B followed by the addition of TrCel7A for 12 min. (Inset) High-resolution AFM image of a cellulose fiber incubated with TrCel7B.

in one direction, from the top toward the bottom of the fiber as seen in Figure 4. 15002

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individual microfibrils), TrCel6A (attacking nonreducing ends and limited amorphous regions), and TrCel7A (attacking reducing ends). The native BC fibers were initially incubated with TrCel7B for a period of 2.5 h. TrCel6A was then added, and the fibers were incubated for another 2.5 h. The image labeled as 0 min in Figure 8 shows the topography image of

Figure 9. Changes in fiber volume with digestion time of crystalline cellulose with TrCel7A following pretreatment with both TrCel7B and TrCel6A (endoexo-exo synergism). The determination of volume changes was made from the masked segment of the fiber shown in the inset over the 82 min time course following the inclusion of all three enzymes.

Figure 8. AFM topography images of crystalline cellulose digestion by TrCel7A following its pretreatment with both TrCel7B and TrCel6A (endoexo-exo synergism). The cellulose fibers in 50 mM sodium citrate buffer at pH 5.0 were preincubated at 20 °C with both 22 μg/ mL TrCel6A and 7.0 μg/mL TrCel7B for 2.5 h. TrCel7A (23.0 μg/ mL) was then introduced, and AFM images were obtained over the following 82 min at the times indicated.

selected fibers after this pretreatment with the two enzymes. It illustrates that a significant portion of the initial volume remained undigested, consistent with the results by Gunner et al.26 Finally, TrCel7A was injected into the mixture, and the fibers were imaged continuously over the next 82 min. The topography of the fibers at different times in this digestion is presented in Figure 8. A movie showing the continuous process of this fiber digestion is available in the Supporting Information, and it provides a very clear and direct visualization of the enzymatic attack of the fibers by this enzyme mixture. Quantitative analysis of volume changes for the central fragment of the fibers imaged in Figure 8 revealed that the degradation was very efficient, with almost 100% digestion occurring within 80 min (Figure 9). However, the most interesting point revealed by these images was that in the presence of both TrCel6A (attacking the nonreducing end) and TrCel7A (attacking the reducing end) the fiber was digested from both ends and digestion progressed toward the center. This is clearly illustrated in Figure 10 in which we show a plot of the height profiles taken at the top, middle, and bottom of the central fiber seen in the images of Figure 8. After 25 min of digestion, the top and the bottom of the fibers have been exhaustively digested whereas the height in the middle of the fiber was reduced by only ∼20%. In fact, a small section in the middle of the central fiber was still visible after 69 min of digestion (Figure 8). To compare the digestion rates at

Figure 10. Height profiles of a cellulose fiber during digestion with TrCel7A after TrCel7B and TrCel6A pretreatment (endoexo-exo synergism). The height profiles were measured at the times indicated across the fiber along the line shown in the corresponding AFM images (insets) at its (A) top, (B) middle, and (C) lower end. The crystalline cellulose fiber was imaged after 2.5 h of pretreatment with both TrCel6A and TrCel7B and then at the times indicated following the addition of TrCel7A.

different locations on the fiber, Figure SI-5 in the Supporting Information plots the relative areas under the height profiles in 15003

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observation of the mechanism for synergy among the three classes of T. reesei cellulolytic enzymes at the crystalline level, providing significant insights into the process of cellulose biodegradation.

Figure 10 as a function of digestion time. The results show clearly that fiber ends are processed quickly and that the middle is digested at a slower rate. The exo enzymes are processing glucan chain ends. Clearly more glucan chain ends are available at fiber ends than in the middle. Figure SI-5 also shows that the top and bottom fiber ends are digested at somewhat different rates. That result may reflect a different rate of chain processing by TrCel7A and TrCel6A as observed also by Gunner et al.26 This behavior is in contrast to the endoexo case (TrCel7B plus TrCel7A) presented in Figure 4 in which the fiber was softened by the action of TrCel7B and digested by TrCel7A from one end only. The images in Figure 8 show that a small chunk of the fiber remained undigested after 82 min. The brightness of this chunk changes with time. Figure SI-6 in the Supporting Information shows height profiles taken for this fragment at selected digestion times. The changes in the height profile indicate that the height of this fragment increases with time by about 2 to 3 nm. Such a change may be explained by the swelling of this recalcitrant fragment of the fiber.

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ASSOCIATED CONTENT

* Supporting Information S

Selected topography images of BC fibers incubated with TrCel7A. Changes in the BC fiber average height and width during incubation with TrCel7A. Selected topography images of BC fibers incubated with TrCel7B. Changes in the fiber volume and average height and width during incubation with TrCel7B. Additional topography images of fibers digested by a mixture of TrCel7B + TrCel7A. Plots showing different digestion rates at the ends and middle of a single fiber by the TrCel6A, TrCel7A, and TrCel7B mixture. Height profiles for a recalcitrant fiber not digested by the TrCel6A, TrCel7A, and TrCel7B mixture. Two video clips comprising time-lapse AFM topography images recorded from two imaging trials involving enzyme mixtures. This material is available free of charge via the Internet at http://pubs.acs.org.

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SUMMARY AND CONCLUSIONS We have used an in situ continuous time-lapse AFM imaging technique to monitor the degradation of bacterial cellulose fibers by three cellulolytic enzymes, TrCel6A, TrCel7A, and TrCel7B, from T. reesei. As is well documented in numerous studies involving a variety of indirect methods that monitored crystalline cellulose degradation (ref 16 and references therein), each of the three enzymes was incapable of substantial cellulose hydrolysis when acting alone. However, mixtures of two enzymes exhibited significant synergistic effects. The synergism depended on the order of enzyme addition. The digestion of fibers treated first with TrCel7A (exo) was accelerated by the addition of TrCel7B (endo). It was apparent from our direct observations that the fibers were preferentially digested from the top whereas the base of the fibers remained intact. In contrast, the addition of TrCel7A to the TrCel7B-pretreated fibers (endoexo case) significantly enhanced the reaction rates and increased the extent of hydrolysis relative to the exoendo case. Apparently, the exoendo synergy was not symmetric. As observed earlier directly in our work6,7 and by others,25,26 the endo activity of the enzymes removed amorphous cellulose, softened the fiber, and exposed single microfibrils, facilitating the attack by the exo enzymes. The single exo enzyme digested the endo enzyme-pretreated fiber, preferentially processing the fiber from one end (reducing or nonreducing). Synergism between the enzymes was most efficient for hydrolysis involving the three enzymes. The AFM images revealed that crystalline fibers pretreated with TrCel7B (endo) and TrCel6A (primarily exo enzyme digesting the nonreducing end) were processed from both ends (reducing and nonreducing) after the addition of TrCel7A (exo enzyme digesting the reducing end), leading to an almost 100% degradation of native bacterial cellulose fibers. The postulate of opposing directional processivities of the two CBHs to account for exoexo synergism was first proposed almost 30 years ago,40 but doubt was cast soon after when TrCel6A was also found to display some endo activity.19,20 Our observations following the sequential addition of the three enzymes suggest that the endo activity of TrCel6A does indeed contribute to the synergism, but to our knowledge, this is a unique report that directly demonstrates the bidirectional activity of the two CBHs and its importance in the complete hydrolysis of crystalline cellulose fibers. In this work, we have obtained the high-resolution, real-time nanoscale

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by a Collaborative Research and Development grant from the Natural Sciences and Engineering Research Council of Canada (NSERC), a research contract from Iogen Corporation, Ottawa, and NSERC Discovery grants to J.L., J.R.D., and A.J.C. J.L. and J.R.D. acknowledge Canada Research Chair Awards.

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