Nanoscale dynamics of cellulose digestion by the cellobiohydrolase TrCel7A

Understanding the mechanism by which cellulases from bacteria, fungi, and protozoans catalyze the digestion of lignocellulose is important for developing cost-effective strategies for bioethanol production. Cel7A from the fungus Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. Here, we used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A molecules binding to and moving processively along immobilized cellulose. Individual enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzyme molecules bound to cellulose in a static state and dissociated without detectable movement, whereas a minority of molecules moved processively for an average distance of 39 nm at an average speed of 3.2 nm/s. These data were integrated into a three-state model in which TrCel7A molecules can bind from solution into either static or processive states and can reversibly switch between states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state, either by dissociation from the cellulose surface or by initiation of a processive run. Thus, accelerating the transition of Cel7A out of its static state is a potential avenue for improving cellulase efficiency.     

Modeling the activity burst in the initial phase of cellulose hydrolysis by the processive cellobiohydrolase Cel7A

The hydrolysis of cellulose by processive cellulases, such as exocellulase TrCel7A from Trichoderma reesei, is typically characterized by an initial burst of high activity followed by a slowdown, often leading to incomplete hydrolysis of the substrate. The origins of these limitations to cellulose hydrolysis are not yet fully understood. Here, we propose a new model for the initial phase of cellulose hydrolysis by processive cellulases, incorporating a bound but inactive enzyme state. The model, based on ordinary differential equations, accurately reproduces the activity burst and the subsequent slowdown of the cellulose hydrolysis and describes the experimental data equally well or better than the previously suggested model. We also derive steady-state expressions that can be used to describe the pseudo-steady state reached after the initial activity burst. Importantly, we show that the new model predicts the existence of an optimal enzyme-substrate affinity at which the pseudo-steady state hydrolysis rate is maximized. The model further allows the calculation of glucose production rate from the first cut in the processive run and reproduces the second activity burst commonly observed upon new enzyme addition. These results are expected to be applicable also to other processive enzymes.     

A kinetic model for the burst phase of processive cellulases

Cellobiohydrolases (exocellulases) hydrolyze cellulose processively, i.e. by sequential cleaving of soluble sugars from one end of a cellulose strand. Their activity generally shows an initial burst, followed by a pronounced slowdown, even when substrate is abundant and product accumulation is negligible. Here, we propose an explicit kinetic model for this behavior, which uses classical burst phase theory as the starting point. The model is tested against calorimetric measurements of the activity of the cellobiohydrolase Cel7A from Trichoderma reesei on amorphous cellulose. A simple version of the model, which can be solved analytically, shows that the burst and slowdown can be explained by the relative rates of the sequential reactions in the hydrolysis process and the occurrence of obstacles for the processive movement along the cellulose strand. More specifically, the maximum enzyme activity reflects a balance between a rapid processive movement, on the one hand, and a slow release of enzyme which is stalled by obstacles, on the other. This model only partially accounts for the experimental data, and we therefore also test a modified version that takes into account random enzyme inactivation. This approach generally accounts well for the initial time course (approximately 1 h) of the hydrolysis. We suggest that the models will be useful in attempts to rationalize the initial kinetics of processive cellulases, and demonstrate their application to some open questions, including the effect of repeated enzyme dosages and the 'double exponential decay' in the rate of cellulolysis.     

Single-molecule Imaging Analysis of Elementary Reaction Steps of Trichoderma reesei Cellobiohydrolase I (Cel7A) Hydrolyzing Crystalline Cellulose Iα and IIII

Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose I_α and III_I were previously reported as substrates with different crystalline forms and different susceptibilities to hydrolysis by TrCel7A. In this study, we observed that different susceptibilities of cellulose I_α and III_I are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose I_α and III_I. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose I_α and III_I. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low. Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose I_α and III_I and similar fractions of productive binding on cellulose I_α and III_I. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose I_α and III_I with similar translational rates. With structural models of cellulose I_α and III_I, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lanes.     

Mechanism of initial rapid rate retardation in cellobiohydrolase catalyzed cellulose hydrolysis

Despite intensive research, the mechanism of the rapid retardation in the rates of cellobiohydrolase (CBH) catalyzed cellulose hydrolysis is still not clear. Interpretation of the hydrolysis data has been complicated by the inability to measure the catalytic constants for CBH‐s acting on cellulose. We developed a method for measuring the observed catalytic constant (k^obs) for CBH catalyzed cellulose hydrolysis. It relies on in situ measurement of the concentration of CBH with the active site occupied by the cellulose chain. For that we followed the specific inhibition of the hydrolysis of para‐nitrophenyl‐β‐D ‐lactoside by cellulose. The method was applied to CBH‐s TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium and their isolated catalytic domains. Bacterial microcrystalline cellulose, Avicel, amorphous cellulose, and lignocellulose were used as substrates. A rapid decrease of k^obs in time was observed on all substrates. The k^obs values for PcCel7D were about 1.5 times higher than those for TrCel7A. In case of both TrCel7A and PcCel7D, the k^obs values for catalytic domains were similar to those for intact enzymes. A model where CBH action is limited by the average length of obstacle‐free way on cellulose chain is proposed. Once formed, productive CBH–cellulose complex proceeds with a constant rate determined by the true catalytic constant. After encountering an obstacle CBH will “get stuck” and the rate of further cellulose hydrolysis will be governed by the dissociation rate constant (k_off), which is low for processive CBH‐s. Biotechnol. Bioeng. 2010;106: 871–883. © 2010 Wiley Periodicals, Inc.     

Enzymatic transformations of cellulose assessed by quantitative high-throughput fourier transform infrared spectroscopy (QHT-FTIR)

Enzymatic hydrolysis of bacterial microcrystalline cellulose was performed with the thermophile enzyme system of Thermobifida fusca Cel5A (a classical endocellulase), Cel6B (a classical exocellulase), Cel9A (a processive endoglucanase), and a synergistic mixture of endo- and exocellulases. Different concentrations of enzymes were used to vary the extent of hydrolysis. Following standardization, the concentration of cellulose was directly correlated to the absorbance of the cellulose signals. Crystallinity indexes (Lateral Order Index (LOI), Total Crystallinity Index, Hydrogen Bonding Index), allomorphic composition, conversion of specific atomic bonds (including the β-glucosidic bonds) were extracted from the spectral data obtained by QHT-FTIR. By quantifying the disruption of the H-bonding in complement to the sugar production, a more dynamic and complex picture of the role of cellulases in the hydrolysis of cellulose was demonstrated. The disruption of the H-bonding within the cellulose matrix appears as a quantifiable activity of the enzymes which was not correlated with the production of sugars in solution. The results also demonstrate that Cel9A activities from the cellulose transformation standpoint were partially similar to the activities of the synergistic mixture. In addition, Cel9A preferentially degraded the I(α) fraction of the crystalline cellulose while the Cel5A and Cel6B synergistic mixture preferentially degraded the I(β) fraction.     

A kinetic study of Trichoderma reesei Cel7B catalyzed cellulose hydrolysis

One prominent feature of Trichoderma reesei (Tr) endoglucanases catalyzed cellulose hydrolysis is that the reaction slows down quickly after it starts (within minutes). But the mechanism of the slowdown is not well understood. A structural model of Tr- Cel7B catalytic domain bound to cellulose was built computationally and the potentially important binding residues were identified and tested experimentally. The 13 tested mutants show different binding properties in the adsorption to phosphoric acid swollen cellulose and filter paper. Though the partitioning parameter to filter paper is about 10 times smaller than that to phosphoric acid swollen cellulose, a positive correlation is shown for two substrates. The kinetic studies show that the reactions slow down quickly for both substrates. This slowdown is not correlated to the binding constant but anticorrelated to the enzyme initial activity. The amount of reducing sugars released after 24 h by Cel7B in phosphoric acid swollen cellulose, Avicel and filter paper cellulose hydrolysis is correlated with the enzyme activity against a soluble substrate p-nitrophenyl lactoside. Six of the 13 tested mutants, including N47A, N52D, S99A, N323D, S324A, and S346A, yield ∼15–35% more reducing sugars than the wild type (WT) Cel7B in phosphoric acid swollen cellulose and filter paper hydrolysis. This study reveals that the slowdown of the reaction is not due to the binding of the enzyme to cellulose. The activity of Tr- Cel7B against the insoluble substrate cellulose is determined by the enzyme’s capability in hydrolyzing the soluble substrate.     

Two noncellulosomal cellulases of Clostridium thermocellum, Cel9I and Cel48Y, hydrolyse crystalline cellulose synergistically

The genome of Clostridium thermocellum contains a number of genes for polysaccharide degradation-associated proteins that are not cellulosome bound. The list includes beta-glucanases, glycosidases, chitinases, amylases and a xylanase. One of these 'soluble'-enzyme genes codes for a second glycosyl hydrolase (GH)48 cellulase, Cel48Y, which was expressed in Escherichia coli and biochemically characterized. It is a cellobiohydrolyse with activity on native cellulose such as microcrystalline and bacterial cellulose, and low activity on carboxymethylcellulose. It is about 100 times as active on amorphic cellulose and mixed-linkage barley beta-glucan compared with cellulase Cel9I. The enzyme Cel48Y shows a distinct synergism of 2.1 times with the noncellulosomal processive endoglucanase Cel9I on highly crystalline bacterial cellulose at a 17-fold excess of Cel48Y over Cel9I. These data show that C. thermocellum has, besides the cellulosome, the genes for a second cellulase system for the hydrolysis of crystalline cellulose that is not particle bound.     

Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations.The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K(M) and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.     

Binding and Movement of Individual Cel7A Cellobiohydrolases on Crystalline Cellulose Surfaces Revealed by Single-molecule Fluorescence Imaging

The efficient catalytic conversion of biomass to bioenergy would meet a large portion of energy requirements in the near future. A crucial step in this process is the enzyme-catalyzed hydrolysis of cellulose to glucose that is then converted into fuel such as ethanol by fermentation. Here we use single-molecule fluorescence imaging to directly monitor the movement of individual Cel7A cellobiohydrolases from Trichoderma reesei (TrCel7A) on the surface of insoluble cellulose fibrils to elucidate molecular level details of cellulase activity. The motion of multiple, individual TrCel7A cellobiohydrolases was simultaneously recorded with ∼15-nm spatial resolution. Time-resolved localization microscopy provides insights on the activity of TrCel7A on cellulose and informs on nonproductive binding and diffusion. We measured single-molecule residency time distributions of TrCel7A bound to cellulose both in the presence of and absence of cellobiose the major product and a potent inhibitor of Cel7A activity. Combining these results with a kinetic model of TrCel7A binding provides microscopic insight into interactions between TrCel7A and the cellulose substrate.     

Enhanced microbial utilization of recalcitrant cellulose by an ex vivo cellulosome-microbe complex

A cellulosome-microbe complex was assembled ex vivo on the surface of Bacillus subtilis displaying a miniscaffoldin that can bind with three dockerin-containing cellulase components: the endoglucanase Cel5, the processive endoglucanase Cel9, and the cellobiohydrolase Cel48. The hydrolysis performances of the synthetic cellulosome bound to living cells, the synthetic cellulosome, a noncomplexed cellulase mixture with the same catalytic components, and a commercial fungal enzyme mixture were investigated on low-accessibility recalcitrant Avicel and high-accessibility regenerated amorphous cellulose (RAC). The cell-bound cellulosome exhibited 4.5- and 2.3-fold-higher hydrolysis ability than cell-free cellulosome on Avicel and RAC, respectively. The cellulosome-microbe synergy was not completely explained by the removal of hydrolysis products from the bulk fermentation broth by free-living cells and appeared to be due to substrate channeling of long-chain hydrolysis products assimilated by the adjacent cells located in the boundary layer. Our results implied that long-chain hydrolysis products in the boundary layer may inhibit cellulosome activity to a greater extent than the short-chain products in bulk phase. The findings that cell-bound cellulosome expedited the microbial cellulose utilization rate by 2.3- to 4.5-fold would help in the development of better consolidated bioprocessing microorganisms (e.g., B. subtilis) that can hydrolyze recalcitrant cellulose rapidly at low secretory cellulase levels.     

Free Energy Diagram for the Heterogeneous Enzymatic Hydrolysis of Glycosidic Bonds in Cellulose

Kinetic and thermodynamic data have been analyzed according to transition state theory and a simplified reaction scheme for the enzymatic hydrolysis of insoluble cellulose. For the cellobiohydrolase Cel7A from Hypocrea jecorina (Trichoderma reesei), we were able to measure or collect relevant values for all stable and activated complexes defined by the reaction scheme and hence propose a free energy diagram for the full heterogeneous process. For other Cel7A enzymes, including variants with and without carbohydrate binding module (CBM), we obtained activation parameters for the association and dissociation of the enzyme-substrate complex. The results showed that the kinetics of enzyme-substrate association (i.e. formation of the Michaelis complex) was almost entirely entropy-controlled and that the activation entropy corresponded approximately to the loss of translational and rotational degrees of freedom of the dissolved enzyme. This implied that the transition state occurred early in the path where the enzyme has lost these degrees of freedom but not yet established extensive contact interactions in the binding tunnel. For dissociation, a similar analysis suggested that the transition state was late in the path where most enzyme-substrate contacts were broken. Activation enthalpies revealed that the rate of dissociation was far more temperature-sensitive than the rates of both association and the inner catalytic cycle. Comparisons of one- and two-domain variants showed that the CBM had no influence on the transition state for association but increased the free energy barrier for dissociation. Hence, the CBM appeared to promote the stability of the complex by delaying dissociation rather than accelerating association.     

Imaging the enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolase Cel7A

Dispersed cellulose ribbons from bacterial cellulose were subjected to digestion with cloned Cel7A (cellobiohydrolase [CBH] I) and Cel6A (CBH II) from Humicola insolens either alone or in a mixture and in the presence of an excess of beta-glucosidase. Both Cel7A and Cel6A were effective in partially converting the ribbons into soluble sugars, Cel7A being more active than Cel6A. In combination, these enzymes showed substantial synergy culminating with a molar ratio of approximately two-thirds Cel6A and one-third Cel7A. Ultrastructural transmission electron microscopy (TEM) observations indicated that Cel7A induced a thinning of the cellulose ribbons, whereas Cel6A cut the ribbons into shorter elements, indicating an endo type of action. These observations, together with the examination of the digestion kinetics, indicate that Cel6A can be classified as an endo-processive enzyme, whereas Cel7A is essentially a processive enzyme. Thus, the synergy resulting from the mixing of Cel6A and Cel7A can be explained by the partial endo character of Cel6A. A preparation of bacterial cellulose ribbons appears to be an appropriate substrate, superior to Valonia or bacterial cellulose microcrystals, to visualize directly by TEM the endo-processivity of an enzyme such as Cel6A.     

Initial- and processive-cut products reveal cellobiohydrolase rate limitations and the role of companion enzymes

Efforts to improve the activity of cellulases, which catalyze the hydrolysis of insoluble cellulose, have been hindered by uncertainty surrounding the mechanistic origins of rate-limiting phenomena and by an incomplete understanding of complementary enzyme function. In particular, direct kinetic measurements of individual steps occurring after enzymes adsorb to the cellulose surface have proven to be experimentally elusive. This work describes an experimental and analytical approach, derived from a detailed mechanistic model of cellobiohydrolase action, for determining rates of initial- and processive-cut product generation by Trichoderma longibrachiatum cellobiohydrolase I (TlCel7A) as it catalyzes the hydrolysis of bacterial microcrystalline cellulose (BMCC) alone and in the presence of Talaromyces emersonii endoglucanase II (TemGH5). This analysis revealed that the rate of TlCel7A-catalyzed hydrolysis of crystalline cellulose is limited by the rate of enzyme complexation with glycan chains, which is shown to be equivalent to the rate of initial-cut product generation. This rate is enhanced in the presence of endoglucanase enzymes. The results confirm recent reports about the role of morphological obstacles in enzyme processivity and also provide the first direct evidence that processive length may be increased by the presence of companion enzymes, including small amounts of TemGH5. The findings of this work indicate that efforts to improve cellobiohydrolase activity should focus on enhancing the enzyme's ability to complex with cellulose chains, and the analysis employed provides a new technique for investigating the mechanism by which companion enzymes influence cellobiohydrolase activity.     

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

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

Effects of noncatalytic residue mutations on substrate specificity and ligand binding of Thermobifida fusca endocellulase cel6A.

The availability of a high-resolution structure of the Thermobifida fusca endocellulase Cel6A catalytic domain makes this enzyme ideal for structure-based efforts to engineer cellulases with high activity on native cellulose. In order to determine the role of conserved, noncatalytic residues in cellulose hydrolysis, 14 mutations of six conserved residues in or near the Cel6A active-site cleft were studied for their effects on catalytic activity, substrate specificity, processivity and ligand-binding affinity. Eleven mutations were generated by site-directed mutagenesis using PCR, while three were from previous studies. All the CD spectra of the mutant enzymes were indistinguishable from that of Cel6A indicating that the mutations did not dramatically change protein conformation. Seven mutations in four residues (H159, R237, K259 and E263) increased activity on carboxymethyl cellulose (CM-cellulose), with K259H (in glucosyl subsite -2) creating the highest activity (370%). Interestingly, the other mutations in these residues reduced CM-cellulose activity. Only the K259H enzyme retained more activity on acid-swollen cellulose than on filter paper, suggesting that this mutation affected the rate-limiting step in crystalline cellulose hydrolysis. All the mutations lowered activity on cellotriose and cellotetraose, but two mutations, both in subsite +1 (H159S and N190A), had higher kcat/Km values (6.6-fold and 5.0-fold, respectively) than Cel6A on 2,4-dinitrophenyl-beta-D-cellobioside. Measurement of enzyme : ligand dissociation constants for three methylumbelliferyl oligosaccharides and cellotriose showed that all mutant enzymes bound these ligands either to the same extent as or more weakly than Cel6A. These results show that conserved noncatalytic residues can profoundly affect Cel6A activity and specificity.     

ATP-dependent exonuclease V from Micrococcus luteus. The enzyme-DNA complex, the processive mechanism, and the role of ATP

Some kinetic predictions of the proposed processive mechanism for the hydrolysis of DNA by the ATP-dependent enzyme exonuclease V have been checked. The method is to trap enzyme molecules not attached to radioactive DNA substrate with an excess of nonradioactive DNA, so that enzyme molecules attached to the radioactive substrate contribute to the liberation of radioactive products only until they dissociate from it. The experiments show that enzyme molecules remain attached to a T7 double-stranded DNA molecule, while hydrolysing it, for about 2 min under our conditions, in agreement with the predictions of the processive mechanism. However, the mechanism of degradation of single-stranded DNA is not processive. Formation of an enzyme-DNA complex is largely dependent on the presence of ATP. This formation does not appear to be synchronous. ATP analogs do not stimulate formation of, nor stabilize, the enzyme-DNA complex. EDTA causes dissociation of enzyme molecules from the DNA complex.     

A Distinct Model of Synergism between a Processive Endocellulase (TfCel9A) and an Exocellulase (TfCel48A) from Thermobifida fusca

Lignocellulosic biomass is digested in nature by the synergistic activities of enzymes with complementary properties, and understanding synergistic interactions will improve the efficiency of industrial biomass use for sustainable fuels and chemicals. Cel9A and Cel48A from a model bacterium, Thermobifida fusca (TfCel9A and TfCel48A, respectively), are two cellulases with different properties and have previously been shown to synergize well with each other. TfCel9A is a processive endocellulase with relatively high activity on crystalline cellulose. TfCel48A is a reducing end-directed exocellulase with very low activity on crystalline cellulose. Neither enzyme fits its respective role in the classical synergism model of enzymatic cellulose digestion. Using the results of time course, endpoint, and sequential addition activity assays, we propose a model of synergistic cooperation between the two cellulases. TfCel9A is most effective on fresh bacterial cellulose with a presumably uniform surface at the molecular level. Its processive activity likely erodes the surface and thus reduces its own activity. TfCel48A is able to hydrolyze the TfCel9A-modified substrate efficiently and replenish the uniform surface required by TfCel9A, creating a feedback mechanism. The model of synergistic interactions is comparable to an earlier proposed model for Trichoderma reesei Cel7A and Cel7B, but the roles of endo- and exocellulases are reversed, a finding which suggests that bacteria and fungi may have evolved different approaches to efficient biomass degradation.     

Characterization of modular bifunctional processive endoglucanase Cel5 from Hahella chejuensis KCTC 2396

Cel5 from marine Hahella chejuensis is composed of glycoside hydrolase family-5 (GH5) catalytic domain (CD) and two carbohydrate binding modules (CBM6-2). The enzyme was expressed in Escherichia coli and purified to homogeneity. The optimum endoglucanase and xylanase activities of recombinant Cel5 were observed at 65 °C, pH 6.5 and 55 °C, pH 5.5, respectively. It exhibited K _m of 1.8 and 7.1 mg/ml for carboxymethyl cellulose and birchwood xylan, respectively. The addition of Ca²⁺ greatly improved thermostability and endoglucanase activity of Cel5. The Cel5 retained 90 % of its endoglucanase activity after 24 h incubation in presence of 5 M concentration of NaCl. Recombinant Cel5 showed production of cellobiose after hydrolysis of cellulosic substrates (soluble/insoluble) and methylglucuronic acid substituted xylooligosaccharides after hydrolysis of glucuronoxylans by endo-wise cleavage. These results indicated that Cel5 as bifunctional enzyme having both processive endoglucanase and xylanase activities. The multidomain structure of Cel5 is clearly distinguished from the GH5 bifunctional glycoside hydrolases characterized to date, which are single domain enzymes. Sequence analysis and homology modeling suggested presence of two conserved binding sites with different substrate specificities in CBM6-2 and a single catalytic site in CD. Residues Glu132 and Glu219 were identified as key catalytic amino acids by sequence alignment and further verified by using site directed mutagenesis. CBM6-2 plays vital role in catalytic activity and thermostability of Cel5. The bifunctional activities and multiple substrate specificities of Cel5 can be utilized for efficient hydrolysis of cellulose and hemicellulose into soluble sugars.     

Dynamic interaction of Trichoderma reesei cellobiohydrolases Cel6A and Cel7A and cellulose at equilibrium and during hydrolysis

The binding of cellobiohydrolases to cellulose is a crucial initial step in cellulose hydrolysis. In the search for a detailed understanding of the function of cellobiohydrolases, much information concerning how the enzymes and their constituent catalytic and cellulose-binding domains interact with cellulose and with each other and how binding changes during hydrolysis is still needed. In this study we used tritium labeling by reductive methylation to monitor binding of the two Trichoderma reesei cellobiohydrolases, Cel6A and Cel7A (formerly CBHII and CBHI), and their catalytic domains. Measuring hydrolysis by high-performance liquid chromatography and measuring binding by scintillation counting allowed us to correlate activity and binding as a function of the extent of degradation. These experiments showed that the density of bound protein increased with both Cel6A and Cel7A as hydrolysis proceeded, in such a way that the adsorption points moved off the initial binding isotherms. We also compared the affinities of the cellulose-binding domains and the catalytic domains to the affinities of the intact proteins and found that in each case the affinity of the enzyme was determined by the linkage between the catalytic and cellulose-binding domains. Desorption of Cel6A by dilution of the sample showed hysteresis (60 to 70% reversible); in contrast, desorption of Cel7A did not show hysteresis and was more than 90% reversible. These findings showed that the two enzymes differ with respect to the reversibility of binding.