Biomacromolecules 2008, 9, 249–254
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The Physical Action of Cellulases Revealed by a Quartz Crystal Microbalance Study Using Ultrathin Cellulose Films and Pure Cellulases Peter Josefsson, Gunnar Henriksson, and Lars Wågberg* Royal Institute of Technology, Fibre and Polymer Technology, Teknikringen 56, 100 44 Stockholm, Sweden Received September 4, 2007; Revised Manuscript Received November 7, 2007
The effects of fungal cellulases on model cellulose films were studied using a high-resolution quartz crystal microbalance (QCM) sensitive to minute changes of the nanometer thick model cellulose films. It was found that endoglucanases not only produce new end groups but also cause a swelling of the cellulose film. The cellobiohydrolases degraded the films quickly, which was detected as a rapid decrease in the remaining amount of cellulose on the QCM crystal. However, changing viscoelastic properties of the films also indicated a softening of the film during the degradation. A defined mixture of selected cellulases caused a significantly higher rate of degradation than only cellobiohydrolases. Cellulase synergism is discussed with the endoglucanase swelling effects and film softening added.
Introduction Cellulose represents a reservoir of energy for bacteria and microorganisms, which have evolved highly specific and efficient enzyme systems to utilize this source of energy. Since the cellulose in plant cell walls is partly crystalline, of a high degree of polymerization, and also embedded in a matrix of wood polysaccharides and lignin, it is highly inaccessible to enzymatic attack. This situation has via evolution created a complex mechanism for enzymatic cellulose degradation by microorganisms. The existence of enzymes that are capable of degrading or modifying cellulose has been known since the beginning of the last century1,2 and has attracted a vast amount of research over the decades, due to the industrial potential in using enzymatic modifications.3,4 Thorough reviews of this topic have been published earlier and will not be given in the present work.5–8 Cellulases from aerobic fungi are the most commonly studied, since they are comparatively easy to produce and have many technical applications. These cellulases are typically modular, often consisting of a carbohydrate binding module (CBM), that gives the cellulase strong affinity for cellulose, a linker, and a catalytic core module,9 but there are also cellulases lacking linker and CBM. In the case of CBMs present in most aerobic fungal cellulases, the CBM brings the catalytic core module into close and prolonged contact with the carbohydrate through binding with three aromatic residues.10 This binding has been shown to be exothermic but is nevertheless mainly driven by a large positive change in entropy due to the release of bound water molecules.11 The binding of CBMs to cellulosic substrates has been reported to be both reversible and irreversible;12 however, despite irreversible binding a mobility of the CBM on the cellulose substrate has been reported.13 The CBMs themselves lack a catalytic site. However, indications have been reported that they can show nonhydrolytic breakdown14 or a “sloughing * To whom correspondence may be addressed. E-mail: wagberg@ polymer.kth.se. Telephone: + 46 8 790 82 94. Fax: +46 8 790 81 01.
off” effect15 on the cellulose. This has been shown to be the case for a CBM from chitinases.16 Efficient saccharification of cellulose requires a mixture of several functional types of cellulases, for example endoglucanases that can cause hydrolysis within the chain and cellobiohydrolases hydrolyzing from the reducing and nonreducing end. Different cellulases also show strong synergy.17–19 This has been explained mainly at the primary structure level of cellulose; endoglucanases create sites for cellobiohydrolases by cleaving cellulose chains, and cellobiohydrolases acting on the nonreducing end exposing new sites for cellobiohydrolases acting on the reducing end and vice versa.5,6 Synergistic effects on a higher morphological level have not been extensively studied, although it has been suggested that endoglucanases without CBM may have a function in separating cellulose fibrils, and thereby increasing the accessibility for other cellulases.4 Such effects are nevertheless probably of importance from both a biological and technical perspective, since changes in the higher level of organization of cellulose in cellulose fiber structures most likely will affect fiber properties such as water absorption, fiber/fiber interaction, and chemical reactivity. The technique of using model cellulose films allows online measurements of the effects of cellulases on the cellulose film properties, which has not been possible using conventional techniques. Previous investigations, employing model films, have been focused on studying the interaction between model cellulose films and cellulases using ellipsometry and amorphous cellulose films,20,21 concluding that an endoglucanase lacking CBM shows less activity than the corresponding endoglucanase with CBM. Also, the dependence of the degradation rate on pH was eliminated for the endoglucanase lacking CBM. A quartz crystal microbalance (QCM) has also been used, with crude mixtures of cellulases, stating that QCM and model cellulose films are a suitable model system for studying cellulase activity.22 In the present work, the action of purified cellulases as well as a defined mixture on model cellulose films has been studied in order to gain knowledge of their action as monocomponents and as synergistic components. To accomplish this, model
10.1021/bm700980b CCC: $40.75 2008 American Chemical Society Published on Web 12/29/2007
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Figure 1. Image of a QCM crystal (left) spin coated with a thin cellulose film, obtained with a digital camera, and phase images from an atomic force microscope. Table 1. Categorization and Names of Cellulases from Aerobic Fungi type cellobiohydrolase (EC 3.2.1.91) cellobiohydrolase endoglucanase with CBM (EC 3.2.1.4)
endoglucanase without CBM
mode
used in this study
acts progressively from reducing end acts progressively from nonreducing end attacks amorphous cellulose, adsorbs to and possibly nicks crystalline cellulose
cellobiohydrolase 58 (CBH 58) cellobiohydrolase 50 (CBH 50) endoglucanase I (EG I)
in comparison small adsorption affinity, and slow/no activity on crystalline cellulose
endoglucanase V (EG V) inactive endoglucanase V none
cellulose films have been developed that are smooth and nonporous. They also comply with known properties of fibers such as swelling, crystallinity, and fibrillar structure.23–25 Films produced according to this methodology have been shown to be 30 nm thick, being partly crystalline (cellulose II-type).23–25 However, the molecular weight of the cellulose in the films, 160 kDa, is lower than cellulose in wood and pulp which are around 5100 kDa and 400 kDa, respectively.25,26
Experimental Procedures Preparation of Model Cellulose Films. Model cellulose films, shown in Figure 1, were prepared similarly to those of Fält et al.24 Polyvinylamine (PVAm, Catiofast VFH, BASF, Germany) was precipitated in ethanol and vaccuum-dried overnight followed by dissolution in Milli-Q water to 0.1 g/L. The polymer was then adsorbed to QCM silicon oxide crystals (Q-Sense, Sweden) at 0.1 g/L and pH 8 for 20 min, washed with Milli-Q water, and then dried using nitrogen gas. Five grams of dissolving pulp (Domsjö fabriker, Sweden) was Soxhlet-extracted with acetone in order to remove traces of extractives in the pulp. The pulp was then dissolved in 25 mL of N-methylmorpholine N-oxide (NMMO) at 115–120 °C. When a clear solution of NMMO and cellulose was obtained, 75 mL of dimethyl sulfoxide was added and the temperature adjusted to 120 °C. Thereafter a film was spun on the PVAm precovered quartz QCM crystals using a spin coater (Chemat Technology, KW-4A, U.K.). The cellulose film was precipitated in Milli-Q water for at least 24 h and then dried in a desiccator at room temperature. Imaging with Atomic Force Microscopy. The cellulose films were imaged in tapping-mode using an atomic force microscope (Picoforce SPM; Veeco, Santa Barbara, CA) at 23 °C and 50% relative humidity using a TESP rectangular silica cantilever (Veeco, Santa Barbara, CA). Cellulases. Data of the cellulases used in this study can be found in Table 1. CBH 58 (P. c. Cel 7D) was purified from a cellulolytic culture filtrate of Phanerochaete chrysosporium K3 cultivated as described earlier,27–29 by chromatographic methods as described earlier.27–29 The EG I (T. r. Cel 17B) was purified from a cellulolytic culture filtrate of Trichoderma reesei strain QM9414 cultivated as described by Szabo et al.29 purified with chromatographic methods as described elsewhere.30
other names
organism
from G. Johansson30 G. Johansson30
Cel 7B
Phanerochaete chrysosporium Phanerochaete chrysosporium Trichoderma reseei
Cel 45A
Humicola insolens
Novozymes
D10N
Humicola insolens
Novozymes
Cel7D Cel6A
G. Johansson30
EG V (H.i. Cel45A) was obtained from Novozymes (Denmark) as the semicommercial product Novozym 476. All enzymes were homogeneous according to analysis with SDS PAGE.32 The cellobiohydrolases did not show any significant endoglucanase activity, according to the method of released reducing sugars from carboxymethyl cellulose.33 The concentrations used in the experiments were EG I and EG V (active and inactive) 10 µM, CBH 50 30 µM, CBH 58 50 µM, and the synthetic mixture 10 µM (50% CBH 50, 25% CBH 58 and 25% EG I). The QCM Technique. The QCM technique is based on the measurement of vibrations of a disk-shaped, piezoelectric quartz crystal, caused by a driving voltage with a known frequency. For a film that is flat, uniform, firmly attached to the crystal, and fairly rigid, the change in mass of the film is directly proportional to the change in frequency, as shown in the Sauerbrey equation34
∆mass ) -C
∆frequency n
(1)
where C ()0.177 mg m-2 Hz-1 at f ) 5 MHz) is the constant for the mass sensitivity and n, here 3, is the overtone number. When the film is viscoelastic, eq 1 underestimates the film mass. In order to determine the properties of a viscous and elastic film, a dissipation factor D
D)
Elost 1 ) 2πEStored πfτ
(2)
has been defined, where EStored is the energy stored and Elost is the energy dissipated during one oscillation period. The time constant, τ, can be determined from the exponential decay in the amplitude of the resonator when the driving voltage is turned off. The higher the dissipation, the more viscoelastic is the layer on the crystal; that is, a high dissipation can be interpreted as lower viscosity and lower elastic modulus of the film.35 QCM Measurements. For each measurement a model cellulose film on a QCM crystal was mounted in a QCM D300 instrument, Q-Sense, Sweden. A buffer, 50 mM acetate, pH 4.5, was injected, and the swelling of the film was monitored. When the instrument had stabilized, ∆f < 0.03 Hz min-1, ∆D < 0.01 min-1, 2 mL of cellulase in buffer solution was injected, followed by rinsing with more than 3 mL of buffer after 5 min of adsorption. The adsorbed amount of enzymes was calculated from the frequency
Physical Action of Cellulases
Figure 2. The adsorption process of endoglucanases on model cellulose films as determined with a QCM. The endoglucanases behave similarly during adsorption, regardless of type or activity, EG I or EG V, inactive or active catalytic core module. 50 mM sodium acetate buffer, pH 4.5, 23 °C was used. See Figure 4 for an expanded time frame.
difference of the third overtone prior to cellulase injection and after rinsing, and the film thickness change due to cellulase action was calculated using nonlinear modeling.35
Results The Adsorption Phase. The cellulases were injected into the QCM and allowed to interact with the cellulose film surface for 5 min which was followed by rigorous rinsing with a buffer solution. The adsorption processes for the endoglucanses were very similar regarding both frequency and dissipation as shown in Figure 2. The frequency and dissipation changes were between -62 and -83 Hz and 1.5 units, respectively, for the three different endoglucanases. For CBH 50 the change in frequency was –70 Hz, and for CBH 58 the frequency change was -140 Hz. The corresponding values for the dissipation changes were 1.6 and 1.2 units, respectively, as shown in Figure 3. The cellulase mixture changed the frequency by -160 Hz, while the dissipation increased 9.3 units during these 5 min. The Postrinsing Phase. As seen in Figure 4, the both active endoglucanases, EG V and EG I, performed in a similar way subsequent to rinsing, however with different kinetics. The frequency signals decreased by 100 and 150 Hz while the dissipations steadily increased by 7 and 12 units for EG I and EG V, respectively, during 10 h. The inactive EG V′s effect on the signals was small in comparison with the active endoglucanases, and the results show that the frequency increased with 20 Hz while the dissipation decreased with 1 unit over a 10 h period.
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Figure 3. The adsorption process for cellobiohydrolases and for a mixture consisting of 50% CBH 50, 25% CBH 58, and 25% EG I as determined with QCM. The frequency signals during adsorption were similar for the cellobiohydrolases, while the rapid action of the enzymes affected the dissipation signal significantly during the adsorption, making interpretations of the adsorption difficult to evaluate. 50 mM sodium acetate buffer, pH 4.5, 23 °C was used. See Figure 5 for an expanded time frame.
The cellobiohydrolases caused a continuous increase in frequency accompanied by an initial increase in dissipation that then decreased back to the starting value, Figure 5. The maxima in dissipation were typically seen at half the time it took the frequency signal to reach the plateau level. CBH 50 reached the dissipation maximum (28 units) after 90 min while CBH 58 reached the maximum after 110 min and the frequency plateau was approximately 640 and 1400 Hz (still increasing) for CBH 58 and CBH 50, respectively. The viscoelastic properties of films exposed to cellulase mixtures showed similar trends as for films exposed to cellobiohydrolases, yet with different kinetics. For the mixture consisting of 50% CBH 50, 25% CBH 58, and 25% EG I, the dissipation maximum of 40 units was reached after 35 min and after 100 min the dissipation level had decreased back to the starting level. During these 100 min, the frequency reached the plateau level of 1200 Hz.
Discussion Traditionally, cellulase action has been studied using indirect methods, for example by measuring the amount of released or reducing sugars. Also microscopy techniques, such as scanning electron microscopy and transmission electron microscopy, have been valuable tools. However, a methodology to study how cellulases physically affect the cellulose film properties has been needed in order to gain deeper understanding for the interaction between the cellulases and cellulose structures.
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Figure 4. The action of endoglucanase on model cellulose films was rather limited in the frequency signal as determined with the QCM; however, strong effects were seen in the dissipation signal, indicating swelling of the film. 50 mM sodium acetate buffer, pH 4.5, 23 °C was used. 50 mM sodium acetate buffer, pH 4.5, 23 °C was used.
In this study, nanometer thin cellulose films were employed in combination with QCM to determine cellulose film properties during exposure to cellulases. The technique measures the frequency and dissipation changes of the cellulose film which then can be related to properties such as film viscoelasticity and thickness. The films used in this study were 30 nm thick, with an estimated weight of 20 mg/m2 and show similarities to cellulose fibers in parameters such as swelling, crystallinity, and fibrillar structure as illustrated in Figure 1. With this methodology, both the adsorption and catalysis process can be monitored in situ, revealing information about the film properties during the degradation. This information can then support a deeper understanding for cellulase action. The cellulose films were placed in the QCM and allowed to swell until reaching equilibrium. Cellulases were then injected and allowed to interact with the film for 5 min. During these minutes, called the adsorption phase, adsorption and catalysis occurs. The flow cell was then rinsed in order to decouple adsorption from catalytic effects. This was possible since during rinsing the abundant enzymes in the bulk phase are rinsed away which will enable interpretation of the subrinsing phase as “pure” action of the enzymes without interference from adsorption processes. The frequency and dissipation changes during the adsorption phase were determined using the signals prior to enzyme injection and just after rinsing disturbances had leveled out. Since the cellulose films contain chains of lower degree of polymerization than those in delignified or wood fibers, there is a difference between fibers and cellulose films. This difference
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Figure 5. The action of cellobiohydrolases and a mixture consisting of 50% CBH 50, 25% CBH 58, and 25% as determined with the QCM. The frequency signal increased rapidly, while the dissipation signal initially increased to a maximum followed by a decrease back to the initial level. This was interpreted as degradation, which was accompanied by an initial phase in which the film became less dense. 50 mM sodium acetate buffer, pH 4.5, 23 °C was used.
will affect the relative importance of the effect of the endoglucanases. Most probably the importance of endoglucanase effects decreases with decreasing degree of polymerization, while the number of groups available for the cellobiohydrolases should increase as the molecular mass of the cellulose decreases. The Action of Endoglucanases. An inactive endoglucanase was investigated as a control experiment. The adsorption of the inactive endoglucanase was 3.7 mg m-2, as calculated with eq 1 and the dissipation decreased by 1 unit and increased the frequency by 20 Hz during the 10 h after adsorption. These changes would correspond to a 1.2 mg m-2 mass decrease or around one-third of the adsorbed endoglucanase. This mass decrease can be caused either by deswelling of the cellulose film or by enzyme desorption. Desorption can be driven by structural rearrangements at the interface or by changes in enzyme concentration due to the equilibrium process between being adsorbed and desorbed. In either case, this mass decrease shows that the CBM of the EG V lacks a significant “sloughing off” effect on this substrate, since then a significant increase in the dissipation of the film would be expected. As shown in Figure 2, the adsorption of the endoglucanases was similar. According to eq 1, the adsorption of both types of enzymes was 3.7 ( 0.3 mg m-2 (including solid enzyme and immobilized water). The adsorbed endoglucanases then caused an increase of 7–10 dissipation units and decreases in frequency by 100–150 Hz, as seen in Figure 4. The large increase in the
Physical Action of Cellulases
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Figure 6. Proposed model for the mechanism of enzymatic cellulose degradation by fungal cellulases. Please note that the figure is not drawn to scale.
dissipation signal indicates that the internal structure of the film changes and adopts a lower degree of integrity, which means that it becomes more loosely bound and/or more extended into the solution. With Voinova modeling35 these decreasing frequencies and dissipation increases were found to be equivalent to thickness increases of 5–10 nm. Considering that the only available medium to cause this mass increase was water, the endoglucanases must therefore cause swelling of the film. By use of the density of water, the thickness increase would correspond to a mass increase of 5–10 mg m-2, which would correspond to approximately 20–50% increase in the total film mass. The most probable reason for this swelling is a change in the balance between swelling and restraining forces within the film. The swelling forces can be traced back to charges and to hydrophilic hydroxyl groups within the film. These charges are most probably carboxyl groups23,24 emanating from small remaining fractions of hemicellulose and possibly also oxidized cellulose end groups. The swelling forces are counteracted by the interaction between the cellulose molecules within the film. As the cellulose degree of polymerization decreases due to endoglucanase action, these counteracting forces decrease resulting in an increased swelling of the film. As the swelling increases, the outer parts of the film becomes less dense, exposing the chains more to the water phase. Thus, the endoglucanases generates chain ends by decreasing the degree of polymerization but also causing swelling and an exposure of these chains to the environment. The Action of Cellobiohydrolases. Using the same methodology as for the endoglucanases to calculate the cellobiohydrolase adsorption resulted in 4.1 and 8.3 mg m-2 for CBH50 and CBH58, respectively. After the adsorption phase, the
cellobiohydrolases continuously increased the frequency signal until reaching a plateau level. The dissipation signal initially increased to a peak level followed by a decrease leveling out close to the starting value. This can be interpreted as continuous degradation in two phases: (1) Initially the catalysis causes the film to become less dense. (2) As the catalysis progress reaches the end of the cellulose film and as less and less cellulose remains, the film appear to become denser. However, what really happens is that more and more of the less viscoelastic PVAmcoated SiO2 film becomes exposed to the water phase. CBH 50 resulted in a larger change in frequency compared to CBH 58, 1400 and 640 Hz respectively. Furthermore, the dissipation maximum was higher and was reached faster for CBH 50 than for CBH 58. This can be interpreted as a higher rate of degradation for CBH 50 but also that CBH 58 did not completely degrade the film. The complete degradation cause of CBH 50 can be explained by the fact that CBH 50 possesses some endoglucanase activity36 whereas CBH 58 does not. On comparison of the cellobiohydrolase and cellulase mixtures, similarities are found in the increasing dissipation while the frequency change is negative for endoglucanases and positive for cellobiohydrolases. This clearly shows the different actions of these cellulase types. The Action of a Cellulase Mixture. The defined mixture of endoglucanases and cellobiohydrolases was an attempt to mimic the composition of cellulases occurring in nature during fungal cellulose degradation. The mixture consisted of 50% CBH 50, 25% CBH 58, and 25% EG I. Figure 5 shows that the signals of the cellulase mixture were similar to the cellobiohydrolase signals, yet with significantly higher rates. The adsorption during 5 min resulted in an increase of 9.3 dissipation units and a decrease in frequency by 160 Hz. The large dissipation change
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is most probably not mainly caused by adsorption but by catalysis. Therefore, since the catalysis contribution to the signals is significant, it is difficult to determine precise adsorption levels. When the time to reach the plateau level in frequency was considered, the mixture was around three times faster than only monocomponent cellobiohydrolases. The fact that endoglucanases and cellobiohydrolases degrade cellulose more rapidly together than as pure enzymes has been known since before 1980,19 and it has been shown that this effect stems from the process in which the endoglucanases increase the number of free chain ends for the cellobiohydrolases to act on. This is supported by the data in the present study. But also, the nondegrading swelling effect of the endoglucanases, revealed in the present study, is also of significant importance for efficient hydrolysis of cellulose by cellobiohydrolases. A Morphological Model for the Synergy of Cellulase Mixtures. The present data show that endoglucanases and cellobiohydrolases have physically different effects on the properties of cellulose model films. The endoglucanase causes the films to bind water and to swell, which results in weight increase and with that decreasing viscosity and elasticity of the film. The cellobiohydrolases also cause decreasing viscosity and elasticity, but this process is accompanied by significant weight losses of the film. Mixtures of endoglucanases and cellobiohydrolases showed similar trends as the cellobiohydrolases but with faster kinetics. The higher rate of degradation can be explained as synergy effects where endoglucanases increase the number of sites available for cellobiohydrolases to act on. This is achieved by decreasing the degree of polymerization increasing the number of available sites for attack but also by causing a swelling of the outer cellulose structure resulting in an increased availability of the new sites. From the results described above, the model in Figure 6 is suggested, showing that the endoglucanase opens the surface of the cellulose film by increasing the number of chain ends and by that also swelling it. This increases the number of available sites and the availability of those sites resulting in a higher apparent substrate concentration for cellobiohydrolases to act on and degrade the cellulose chains to cellobiose units. Thus, synergy effects between endoglucanase and cellobiohydrolases exist on a higher hierarchical level than the primary molecular level. Such effects of endoglucanases might also explain why even short treatments with endoglucanases increase the chemical reactivity of cellulose.3 Preliminary results in our group have also shown that the swelling of these films upon exposure to cellulose can be transferred to the action of cellulases on wood and delignified fibers.
Conclusions The action of the investigated endoglucanases caused softening and swelling of the cellulose film while the cellobiohydrolases caused film softening accompanied with degradation. The swelling caused by endoglucanases was probably a consequence of the endoglucanase action which reduces the restraining forces within the cellulose film. As the restraining forces are decreased, the remaining swelling forces will cause a swelling of the film. The swelling caused by the endoglucanases shows that synergy effects between endoglucanases and cellobiohydrolases also exist on a higher level than the primary molecular level. An inactive endoglucanase adsorbed in equal amounts as the active version yet caused desorption and no swelling. This suggests that the particular CBM does not possess properties other than binding.
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Acknowledgment. The Bo Rydin Foundation is gratefully acknowledged for financing of Peter Josefsson. We are also grateful to Gunnar Johansson and Novozymes for providing cellulases and Lars Ödberg for fruitful discussions.
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BM700980B
