Adsorption behaviors of two cellobio-hydrolases and their core proteins from Trichoderma reesei on Avicel PH 101

The cleavage of cellulose binding domain decreased the adsorption affinity and tightness of cellobiohydrolase I by 76.5 and 82.1%, as well as those of cellobiohydrolase II did by 20.7 and 68.3% at 25°C. The synergism of the two cellobiohydrolases can be explained by assuming the formation of a partial complex of between binding domain of cellobiohydrolases I and core protein of cellobiohydrolases II, which have higher adsorption affinity and tightness than those of individual components, and different specificities in their attack on cellulose.     

Specific quantification of trichoderma reesei cellulases in reconstituted mixtures and its application to cellulase-cellulose binding studies

Specific quantifications of the major cellulolytic components of the Trichoderma reesei enzyme complex, i.e., endoglucanases I and III and cellobiohydrolases I and II, are described and, employing a defined mixture of these four cellulases reconstituted according to the composition of the native Trichoderma cellulase complex, used to determine the binding of each individual component onto filter paper. During substrate degradation by this enzyme mixture, the specific adsorption of each individual cellulase gradually increases and no preferential binding of one enzyme component in any particular phase of cellulose hydrolysis is found. T. reesei cellobiohydrolases I and II admixed with endoglucanases I and III represent a "full-value" cellulase system that is capable of degrading semicrystalline cellulose efficiently. In comparison with the crude Trichoderma enzyme complex, almost identical adsorption properties and similar hydrolytic efficiency are found for the reconstituted mixture. (c) 1994 John Wiley & Sons, Inc.     

The mechanism of fungal cellulase action. Synergism between enzyme components of Penicillium pinophilum cellulase in solubilizing hydrogen bond-ordered cellulose.

Studies on reconstituted mixtures of extensively purified cellobiohydrolases I and II and the five major endoglucanases of the fungus Penicillium pinophilum have provided some new information on the mechanism by which crystalline cellulose in the form of the cotton fibre is rendered soluble. It was observed that there was little or no synergistic activity either between purified cellobiohydrolases I and II, or, contrary to previous findings, between the individual cellobiohydrolases and the endoglucanases. Cotton fibre was degraded to a significant degree only when three enzymes were present in the reconstituted enzyme mixture: these were cellobiohydrolases I and II and some specific endoglucanases. The optimum ratio of the cellobiohydrolases was 1:1. Only a trace of endoglucanase activity was required to make the mixture of cellobiohydrolases I and II effective. The addition of cellobiohydrolases I and II individually to endoglucanases from other cellulolytic fungi resulted in little synergistic activity; however, a mixture of endoglucanases and both cellobiohydrolases was effective. It is suggested that current concepts of the mechanism of cellulase action may be the result of incompletely resolved complexes between cellobiohydrolase and endoglucanase activities. It was found that such complexes in filtrates of P. pinophilium or Trichoderma reesei were easily resolved using affinity chromatography on a column of p-aminobenzyl-1-thio-beta-D-cellobioside.     

Structural basis for ligand binding and processivity in cellobiohydrolase Cel6A from Humicola insolens

The enzymatic digestion of cellulose entails intimate involvement of cellobiohydrolases, whose characteristic active-center tunnel contributes to a processive degradation of the polysaccharide. The cellobiohydrolase Cel6A displays an active site within a tunnel formed by two extended loops, which are known to open and close in response to ligand binding. Here we present five structures of wild-type and mutant forms of Cel6A from Humicola insolens in complex with nonhydrolyzable thio-oligosaccharides, at resolutions from 1.7-1.1 A, dissecting the structural accommodation of a processing substrate chain through the active center during hydrolysis. Movement of ligand is facilitated by extensive solvent-mediated interactions and through flexibility in the hydrophobic surfaces provided by a sheath of tryptophan residues.     

A binding-site-deficient, catalytically active, core protein of endoglucanase III from the culture filtrate of Trichoderma reesei

From the culture filtrate of Trichoderma reesei we have isolated a novel endoglucanase (38 kDa) which was shown to be identical to endoglucanase III (E III, 50 kDa), but lacking the first 61 N-terminal amino acids. This core protein, designated E III core, is fully active against soluble substrates, such as carboxymethylcellulose, whereas both activity against and adsorption to microcrystalline cellulose (Avicel) is markedly decreased. Sedimentation velocity experiments revealed that the intact E III enzyme has much higher asymmetry than the E III core protein, suggesting that the N-terminal region split off constitutes a protruding part of the native enzyme. These results lead to the proposal that native E III consists of two functionally separated domains: a catalytically active core and a protruding N-terminal domain which acts in the binding to insoluble cellulose. The N-terminal peptide missing in E III core corresponds to the heavily glycosylated common structural element found also in the N-terminus of cellobiohydrolase II and in the C-termini of cellobiohydrolase I and endoglucanase I. A similar bifunctional organization could thus be the rule for Trichoderma cellulases, endoglucanases as well as cellobiohydrolases.     

Hydrolysis of amorphous and crystalline cellulose by heterologously produced cellulases of Melanocarpus albomyces

Three thermostable neutral cellulases from Melanocarpus albomyces, a 20-kDa endoglucanase (Cel45A), a 50-kDa endoglucanase (Cel7A), and a 50-kDa cellobiohydrolase (Cel7B) heterologously produced in a recombinant Trichoderma reesei were purified and studied in hydrolysis (50 degrees C, pH 6.0) of crystalline and amorphous cellulose. To improve their efficiency, M. albomyces cellulases naturally harboring no cellulose-binding module (CBM) were genetically modified to carry the CBM of T. reesei CBHI/Cel7A, and were studied under similar experimental conditions. Hydrolysis performance and product profiles were used to evaluate hydrolytic features of the investigated enzymes. Each cellulase proved to be active against the tested substrates; the cellobiohydrolase Cel7B had greater activity than the endoglucanases Cel45A and Cel7A against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence of synergism was observed when mixtures of the novel enzymes were applied in a constant total protein dosage. Presence of the CBM improved the hydrolytic potential of each enzyme in all experimental configurations; it had a greater effect on the endoglucanases Cel45A and Cel7A than the cellobiohydrolase Cel7B, especially against crystalline substrate. The novel cellobiohydrolase performed comparably to the major cellobiohydrolase of T. reesei (CBHI/Cel7A) under the applied experimental conditions.     

Endo-exo Synergism in Cellulose Hydrolysis Revisited

Synergistic cooperation of different enzymes is a prerequisite for efficient degradation of cellulose. The conventional mechanistic interpretation of the synergism between randomly acting endoglucanases (EGs) and chain end-specific processive cellobiohydrolases (CBHs) is that EG-generated new chain ends on cellulose surface serve as starting points for CBHs. Here we studied the hydrolysis of bacterial cellulose (BC) by CBH TrCel7A and EG TrCel5A from Trichoderma reesei under both single-turnover and "steady state" conditions. Unaccountable by conventional interpretation, the presence of EG increased the rate constant of TrCel7A-catalyzed hydrolysis of BC in steady state. At optimal enzyme/substrate ratios, the "steady state" rate of synergistic hydrolysis became limited by the velocity of processive movement of TrCel7A on BC. A processivity value of 66 ± 7 cellobiose units measured for TrCel7A on (14)C-labeled BC was close to the leveling off degree of polymerization of BC, suggesting that TrCel7A cannot pass through the amorphous regions on BC and stalls. We propose a mechanism of endo-exo synergism whereby the degradation of amorphous regions by EG avoids the stalling of TrCel7A and leads to its accelerated recruitment. Hydrolysis of pretreated wheat straw suggested that this mechanism of synergism is operative also in the degradation of lignocellulose. Although both mechanisms of synergism are used in parallel, the contribution of conventional mechanism is significant only at high enzyme/substrate ratios.     

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.     

Description of cellobiohydrolases Ce16A and Ce17A fromTrichoderma reesei using Langmuir-type models

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 changes during hydrolysis is still needed. The adsorption of purffied two cellobiohydrolases (Ce17A and Ce16A) fromTrichoderma reesei cellulase to microcrystalline cellulose has been studied. Cellobiohydrolase II (Ce16A) does not affect the adsorption of cellobiohydrolase I (Ce17A) significantly, and there are specific binding sites for both Ce17A and Ce16A. The adsorption affinity and tightness of the cellulase binding domain (CBD) for Ce17A are larger that those of the CBD for Ce16A. The CBD for Ce17A binds more rapidly and tightly to Avicel than the CBD for Ce16A. The decrease in adsorption observed when the two cellobihydrolases are studied together would appear to be the result of competition for binding sites on the cellulose. Ce17A competes more efficiently for binding sites than Ce16A. Competition for binding sites is the dominating factor when the two enzymes are acting together, furthermore adsorption to sites specific for Ce17A and Ce16A, also contributes to the total adsorption.     

Analysis of Molecular Size Distributions of Cellulose Molecules during Hydrolysis of Cellulose by Recombinant Cellulomonas fimi β-1,4-Glucanases

Four β-1,4-glucanases (cellulases) of the cellulolytic bacterium Cellulomonas fimi were purified from Escherichia coli cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of β-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.Cellulose hydrolysis by aerobic fungi, such as Trichoderma reesei, is usually explained in terms of the synergistic activities of endo-β-1,4-glucanases and exocellobiohydrolases. Models that describe the attack of cellulose at susceptible regions by endoglucanases, followed by cellobiohydrolase attack at the newly formed chain ends, continue to form the basis of most discussions of enzymatic cellulose hydrolysis (2, 24).Although the occurrence of endoglucanases and cellobiohydrolases in fungi is firmly established, the extent to which the cellulase systems of aerobic bacteria resemble those from fungi was unclear until recently, because there was little evidence for the presence of cellobiohydrolases in bacteria. However, it now appears that at least some cellulolytic bacteria produce enzymes similar to the fungal cellobiohydrolases. For example, Cellulomonas fimi produces at least six β-1,4-glucanases, of which four (CenA, CenB, CenC, and CenD) are endoglucanases and two (CbhA and CbhB) appear to be cellobiohydrolases that are the functional equivalents of T. reesei CBHI and CBHII (6, 15, 21, 22). Similar cellobiohydrolases have been described for the actinomycete Thermomonospora fusca (9).

C. fimi cellobiohydrolases.

The preferential attack of cellulose at the ends of glucan chains by C. fimi cellobiohydrolases CbhA and CbhB is strongly suggested by hydrolysis experiments using cellooligosaccharides or carboxymethylcellulose (CMC) (14, 15, 21, 22). However, we lack direct evidence for exohydrolytic activity on cellulose itself. Accordingly, we have examined the activities of CbhA and CbhB on cellulose by measurement of molecular size distributions during hydrolysis. Analysis of CenA was also included to allow comparison of cellobiohydrolase and endoglucanase activities.

C. fimi CenC.

Previous studies have indicated that CenA attacks susceptible linkages in soluble CMC in a relatively nonprocessive manner (7, 14); i.e., the enzyme dissociates from the substrate after each hydrolytic event. While CenB and CenD attack CMC in a similar way (14, 23), C. fimi CenC seems to act in a more processive fashion (16, 23). Therefore, CenC activity was analyzed in order to determine if the enzyme behaves in a similarly processive manner on cellulose.

Cellulose substrates.

Previous determinations of molecular size distribution during hydrolysis have shown that the choice of substrate is an important consideration (10). In this study we used two forms of cellulose: bacterial microcrystalline cellulose (BMCC) and phosphoric acid-swollen cellulose (PASC). These celluloses were chosen in order to simplify the interpretation of data by avoiding complications due to low surface/volume ratios and substrate heterogeneity, which are associated with the use of substrates like cotton or pulp fibers (24). Both BMCC and PASC have a high surface/volume ratio (17). BMCC is a highly crystalline form of cellulose I prepared from cellulose produced by Acetobacter xylinum. PASC is produced by swelling microcrystalline cellulose in concentrated phosphoric acid; although often described as amorphous, it is probably a low-crystallinity form of cellulose II (1). Recent data suggest that cellulose I and cellulose II contain glucan chains arranged in parallel orientation (12).     

Analysis of Molecular Size Distributions of Cellulose Molecules during Hydrolysis of Cellulose by Recombinant Cellulomonas fimi β-1,4-Glucanases

Four β-1,4-glucanases (cellulases) of the cellulolytic bacterium Cellulomonas fimi were purified from Escherichia coli cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of β-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.     

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.     

Detection and characterization of the specific and nonspecific endoglucanases of Trichoderma reesei: Evidence demonstrating endoglucanase activity by cellobiohydrolase II

The cellulase enzyme system of Trichoderma reesei RUT C-30 has been separated by DEAE ion exchange chromatography into four fractions. Their specificity towards substituted cellulose and cellooligosaccharides was revealed by analytical IEF and activity stains. Fraction EGI (26% of the total protein) exhibited mainly endoglucanase activity on carboxymethylcellulose (CMC) whereas endoglucanases EGII and EGIII (15% of the total protein) showed high activity towards CMC as well as xylan, 4-methylumbelliferyl cellobioside [MeUmb(Glc)₂] and p-nitrophenyl lactoside (pNPL). A subfraction of EGI (pI 5.9) which has been described in the literature as a cellobiohydrolase (CBHII) was isolated by preparative isoelectric focusing, and was shown to have only 3 U CMCase activity per milligram. Turbidimetric measurements and phase contrast microscopy demonstrated differences between endoglucanase and cellobiohydrolase behaviour during the hydrolysis of purified cellulose (Solka Floc BW-40). Treatment of the purified cellulose with endoglucanases resulted in fibre breakdown into small particles. This was contrasted with no morphological change to the fibres when contacted with the cellobiohydrolase. By this technique it was revealed that the EGI subfraction (pI 5.9) behaves as an endoglucanase and not as a cellobiohydrolase. Incubation of this enzyme with acid-swollen cellulose resulted in cellotriose production, as it did with other endoglucanases which exhibited CMCase activities >; 100 U mg⁻¹. Cellotriose was not present during the hydrolysis of acid-swollen cellulose with the CBHI fraction.     

The cellulase of Penicillium pinophilum. Synergism between enzyme components in solubilizing cellulose with special reference to the involvement of two immunologically distinct cellobiohydrolases.

Two immunologically unrelated cellobiohydrolases (I and II), isolated from the extracellular cellulase system elaborated by the fungus Penicillum pinophilum, acted in synergism to solubilize the microcrystalline cellulose Avicel; the ratio of the two enzymes for maximum rate of attack was approx. 1:1. A hypothesis to explain the phenomenon of synergism between two endwise-acting cellobiohydrolases is presented. It is suggested that the cellobiohydrolases may be two stereospecific enzymes concerned with the hydrolysis of the two different configurations of non-reducing end groups that would exist in cellulose. Only one type of cellobiohydrolase has been isolated so far from the cellulases of the fungi Fusarium solani and Trichoderma koningii. Only cellobiohydrolase II of P. pinophilum acted synergistically with the cellobiohydrolase of the fungi T. koningii or F. solani to solubilize Avicel. Cellobiohydrolase II showed no capacity for co-operating with the endo-1,4-beta-glucanase of T. koningii or F. solani to solubilize crystalline cellulose, but cellobiohydrolase I did. These results are discussed in the context of the hypothesis presented.     

High Speed Atomic Force Microscopy Visualizes Processive Movement of Trichoderma reesei Cellobiohydrolase I on Crystalline Cellulose

Fungal cellobiohydrolases act at liquid-solid interfaces. They have the ability to hydrolyze cellulose chains of a crystalline substrate because of their two-domain structure, i.e. cellulose-binding domain and catalytic domain, and unique active site architecture. However, the details of the action of the two domains on crystalline cellulose are still unclear. Here, we present real time observations of Trichoderma reesei (Tr) cellobiohydrolase I (Cel7A) molecules sliding on crystalline cellulose, obtained with a high speed atomic force microscope. The average velocity of the sliding movement on crystalline cellulose was 3.5 nm/s, and interestingly, the catalytic domain without the cellulose-binding domain moved with a velocity similar to that of the intact TrCel7A enzyme. However, no sliding of a catalytically inactive enzyme (mutant E212Q) or a variant lacking tryptophan at the entrance of the active site tunnel (mutant W40A) could be detected. This indicates that, besides the hydrolysis of glycosidic bonds, the loading of a cellulose chain into the active site tunnel is also essential for the enzyme movement.     

Mechanism of cellobiose inhibition in cellulose hydrolysis by cellobiohydrolase

An experimental study of cellobiose inhibition in cellulose hydrolysis by synergism of cellobiohydrolyse I and endoglucanase I is presented. Cellobiose is the structural unit of cellulose molecules and also the main product in enzymatic hydrolysis of cellulose. It has been identified that cellobiose can strongly inhibit hydrolysis reaction of cellulase, whereas it has no effect on the adsorption of cellulase on cellulose surface. The experimental data of FT-IR spectra, fluorescence spectrum and circular dichroism suggested that cellobiose can be combined with tryptophan residue located near the active site of cellobiohydrolase and then form steric hindrance, which prevents cellulose molecule chains from diffusing into active site of cellulase. In addition, the molecular conformation of cellobiohydrolase changes after cellobiose binding, which also causes most of the non-productive adsorption. Under these conditions, microfibrils cannot be separated from cellulose chains, thus further hydrolysis of cellulose can hardly proceed.     

Surface hydrophobic amino acid residues in cellulase molecules as a structural factor responsible for their high denim-washing performance

The denim-washing performance of six purified fungal cellulases (four endo-1,4-beta-D-glucanases and two cellobiohydrolases) was compared using a model microassay. The performance of cellobiohydrolases per mg of protein was much lower than that of endoglucanases. For endoglucanases, it varied up to 5 times between the best and the worst enzyme. Experiments with amino acids immobilized on cross-linked agarose showed that their side chains may bind indigo owing to hydrophobic interactions and formation of hydrogen bonds. The best binding effects provided Tyr and Phe. Analysis of three-dimensional structures of cellulase molecules showed that a certain correlation exists between the washing performance of enzyme and (i) quantity (percentage) of aromatic residues exposed to solvent on the surface of protein globule or (ii) overall percentage of the surface hydrophobic residues. Data presented provide an evidence that the molecules of certain cellulases, which have hydrophobic domains (clusters of closely located non-polar residues) on their surface, may bind indigo and thus act as emulsifiers helping the dye to float out of cellulose fibers to the bulk solution.     

Mechanism of cellobiose inhibition in cellulose hydrolysis by cellobiohydrolase

An experimental study of cellobiose inhibition in cellulose hydrolysis by synergism of cellobiohydrolyse I and endoglucanase I is presented. Cellobiose is the structural unit of cellulose molecules and also the main product in enzymatic hydrolysis of cellulose. It has been identified that cellobiose can strongly inhibit hydrolysis reaction of cellulase, whereas it has no effect on the adsorption of cellulase on cellulose surface. The experimental data of FT-IR spectra, fluorescence spectrum and circular dichroism suggested that cellobiose can be combined with trypto-phan residue located near the active site of cellobiohydrolase and then form steric hindrance, which prevents cellulose molecule chains from diffusing into active site of cellulase. In addition, the molecular conformation of cellobiohydrolase changes after cellobiose binding, which also causes most of the non-productive adsorption. Under these conditions, microfibrils cannot be separated from cellulose chains, thus further hydrolysis of cell     

The structure and mechanism of action of cellulolytic enzymes

The modern structural classification of polysaccharases comprising cellulase–hemicellulase enzyme systems is dis cussed. Their catalytic domains are currently grouped into 15 of more than 80 known glycosyl hydrolase families, whereas substrate binding domains fall into 13 families. The structures of catalytic and substrate binding domains, as well as linker sequences, are briefly considered. A hypothetical mechanism of concerted action of catalytic and substrate binding domains of cellobiohydrolases on the surface of highly ordered cellulose is suggested.