A single molecule study of cellulase hydrolysis of crystalline cellulose (original) (raw)
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Journal of Biological Chemistry, 2014
The catalytic mechanism of Trichoderma reesei cellobiohydrolase I (TrCel7A) is still unclear. Results: TrCel7A exhibited similar reaction kinetics during crystalline cellulose I ␣ and III I hydrolysis. Conclusion: Not differences in kinetic parameters but surface properties of the crystalline cellulose influence the susceptibilities of cellulose I ␣ and III I to hydrolysis by TrCel7A. Significance: Single-molecule measurements further our understanding of TrCel7A mechanism. . 2 The abbreviations used are: HS-AFM, high speed atomic force microscope (microscopy); Cel7A, cellobiohydrolase I; Cy3-TrCel7A, Cy3-labeled Trichoderma reesei cellobiohydrolase I; pNPG, p-nitrophenyl--D-glucoside; pNPL, p-nitrophenyl lactoside; TrCel7A, Trichoderma reesei cellobiohydrolase I; TIRFM, total internal reflection fluorescence microscope (microscopy).
The Journal of biological chemistry, 2016
Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-Linker) on crystalline cellulose Iα The CBM-Linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high-affinity site than to the low-affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cell...
Langmuir, 2012
The biodegradation of cellulose involves the enzymatic action of cellulases (endoglucanases), cellobiohydrolases (exoglucanases), and β-glucosidases that act synergistically. The rate and efficiency of enzymatic hydrolysis of crystalline cellulose in vitro decline markedly with time, limiting the large-scale, cost-effective production of cellulosic biofuels. Several factors have been suggested to contribute to this phenomenon, but there is considerable disagreement regarding the relative importance of each. These earlier investigations were hampered by the inability to observe the disruption of crystalline cellulose and its subsequent hydrolysis directly. Here, we show the application of high-resolution atomic force microscopy to observe the swelling of a single crystalline cellulose fiber and its-hydrolysis in real time directly as catalyzed by a single cellulase, the industrially important cellulase 7B from Trichoderma reesei. Volume changes, the root-mean-square roughness, and rates of hydrolysis of the surfaces of single fibers were determined directly from the images acquired over time. Hydrolysis dominated the early stage of the experiment, and swelling dominated the later stage. The high-resolution images revealed that the combined action of initial hydrolysis followed by swelling exposed individual microfibrils and bundles of microfibrils, resulting in the loosening of the fiber structure and the exposure of microfibrils at the fiber surface. Both the hydrolysis and swelling were catalyzed by the native cellulase; under the same conditions, its isolated carbohydrate-binding module did not cause changes to crystalline cellulose. We anticipate that the application of our AFM-based analysis on other cellulolytic enzymes, alone and in combination, will provide significant insight into the process of cellulose biodegradation and greatly facilitate its application for the efficient and economical production of cellulosic ethanol.
Journal of the American Chemical Society, 2014
Analysis of heterogeneous catalysis at an interface is difficult because of the variety of reaction sites and the difficulty of observing the reaction. Enzymatic hydrolysis of cellulose by cellulases is a typical heterogeneous reaction at a solid/liquid interface, and a key parameter of such reactions on polymeric substrates is the processivity, i.e., the number of catalytic cycles that can occur without detachment of the enzyme from the substrate. In this study, we evaluated the reactions of three closely related glycoside hydrolase family 7 cellobiohydrolases from filamentous fungi at the molecular level by means of high-speed atomic force microscopy to investigate the structure−function relationship of the cellobiohydrolases on crystalline cellulose. We found that high moving velocity of enzyme molecules on the surface is associated with a high dissociation rate constant from the substrate, which means weak interaction between enzyme and substrate. Moreover, higher values of processivity were associated with more loop regions covering the subsite cleft, which may imply higher binding affinity. Loop regions covering the subsites result in stronger interaction, which decreases the velocity but increases the processivity. These results indicate that there is a trade-off between processivity and hydrolytic velocity among processive cellulases.
Molecular-Scale Investigations of Cellulose Microstructure during Enzymatic Hydrolysis
Biomacromolecules, 2010
Changes in cellulose microstructure have been proposed to occur throughout hydrolysis that impact enzyme access and hydrolysis rates. However, there are very few direct observations of such changes in ongoing reactions. In this study, changes in the microstructure of cellulose are measured by simultaneous confocal and atomic force microscopy and are correlated to hydrolysis extents and quantities of bound enzyme in the reaction. Minimally processed and never-dried cellulose I was hydrolyzed by a purified cellobiohydrolase, Trichoderma reesei Cel7A. Early in the reaction (∼30% hydrolysis), at high hydrolysis rates and high bound cellulase quantities, untwisting of cellulose microfibrils was observed. As the hydrolysis reaction neared completion (>80% hydrolysis), extensively thinned microfibrils (diameters of 3-5 nm) and channels (0.3-0.6 nm deep) along the lengths of the microfibrils were observed. The prominent microstructural changes in cellulose due to cellobiohydrolase action are discussed in the context of the overall hydrolysis reaction.
Biotechnology for biofuels, 2016
The main challenges of large-scale biochemical conversion involve the high costs of cellulolytic enzymes and the inefficiency in enzymatic deconstruction of polysaccharides embedded in the complex structure of the plant cell wall, leading to ongoing interests in studying the predominant mode of enzymatic hydrolysis. In this study, complete enzymatic hydrolysis of pretreated biomass substrates was visualized in situ and in real time by atomic force microscopy (AFM) topography and recognition imaging. Throughout the entire hydrolytic process, a hydrolysis mode for exoglucanase (CBH I) consisting of a peeling action, wherein cellulose microfibrils are peeled from sites on the pretreated cellulose substrate that have cracks sufficiently large for CBH I to immobilize. We quantitatively monitored the complete hydrolytic process on pretreated cellulose. The synergetic effect among the different enzymes can accelerate the cellulose hydrolysis rate dramatically. However, the combination of C...
Journal of Biological Chemistry, 2009
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.