Mechanochemical Coupling of Catalysis and Motion in a Cellulose-Degrading Multienzyme Nanomachine.

The cellulosome is a megadalton-size protein complex that functions as a biological nanomachine of cellulosic fiber degradation. We show that the cellulosome behaves as a Brownian ratchet that rectifies protein motions on the cellulose surface into a propulsion mechanism by coupling to the hydrolysis of cellulose chains. Movement on cellulose fibrils is unidirectional and results from "macromolecular crawl" composed of dynamic switches between elongated and compact spatial arrangements of enzyme subunits. Deletion of the main exocellulase Cel48S eliminates conformational bias for aligning the subunits to the long fibril axis, which we reveal as crucial for optimum coupling between directional movement and substrate degradation. Implications of the cellulosome acting as a mechanochemical motor suggest a distinct mechanism of enzymatic machinery in the deconstruction of cellulose assemblies.

Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase.

BACKGROUND: In whole-cell bio-catalysis, the biosystems engineering paradigm shifts from the global reconfiguration of cellular metabolism as in fermentation to a more focused, and more easily modularized, optimization of comparably short cascade reactions. Human milk oligosaccharides (HMO) constitute an important field for the synthetic application of cascade bio-catalysis in resting or non-living cells. Here, we analyzed the central catalytic module for synthesis of HMO-type sialo-oligosaccharides, comprised of CMP-sialic acid synthetase (CSS) and sialyltransferase (SiaT), with the specific aim of coordinated enzyme co-expression in E. coli for reaction flux optimization in whole cell conversions producing 3'-sialyllactose (3SL). RESULTS: Difference in enzyme specific activity (CSS from Neisseria meningitidis: 36 U/mg; α2,3-SiaT from Pasteurella dagmatis: 5.7 U/mg) was compensated by differential protein co-expression from tailored plasmid constructs, giving balance between the individual activities at a high level of both (α2,3-SiaT: 9.4 × 102 U/g cell dry mass; CSS: 3.4 × 102 U/g cell dry mass). Finally, plasmid selection was guided by kinetic modeling of the coupled CSS-SiaT reactions in combination with comprehensive analytical tracking of the multistep conversion (lactose, N-acetyl neuraminic acid (Neu5Ac), cytidine 5'-triphosphate; each up to 100 mM). The half-life of SiaT in permeabilized cells (≤ 4 h) determined the efficiency of 3SL production at 37 °C. Reaction at 25 °C gave 3SL (40 ± 4 g/L) in ∼ 70% yield within 3 h, reaching a cell dry mass-specific productivity of ∼ 3 g/(g h) and avoiding intermediary CMP-Neu5Ac accumulation. CONCLUSIONS: Collectively, balanced co-expression of CSS and SiaT yields an efficient (high-flux) sialylation module to support flexible development of E. coli whole-cell catalysts for sialo-oligosaccharide production.

Fabrication and characterization of a novel zein/pectin/pumpkin seed oil Pickering emulsion and the effects of myricetin on oxidation stability.

In this study, zein/pectin/pumpkin seed oil (PSO) Pickering emulsions (ZPPEs) were fabricated loading with myricetin (MYT), and the quality control methods of oxidation stability were innovatively investigated. The microstructure and particle properties of zein-pectin particles were determined. The zein to pectin ratio of 5:3 and oil phase fraction (φ = 50 %) turned out as the most optimal conditions for the stabilization of myricetin-loaded ZPPEs. The expected oil-in-water emulsion-type structure was confirmed by confocal laser scanning microscopy (CLSM). The internal 3D structure of Pickering emulsions (Lugol's solution improved the water-phase contrast) was imaged by micro-computed tomography (Micro-CT) for the first time. Results showed a sponge like structure of water phase in emulsion with 42 µm as mean droplet size. Light-induced oxidation was evaluated with the PetroOxy method and malondialdehyde (MDA) assays. Encapsuling ZPPEs with MYT could prevent the light induced oxidation, especially, loading of MYT at the core of the emulsion. The analysis of Electronic nose (E-nose) was used to analyze the odor before and after UV-induced oxidation, and showed a good discrimination. This study provided a new approach to prepare ZPPEs with high oxidation stability. Micro-CT, PetroOxy and E-nose could be new methods for characterization and quality assessment of Pickering emulsions.

Enzyme Synergy in Transient Clusters of Endo- and Exocellulase Enables a Multilayer Mode of Processive Depolymerization of Cellulose.

Biological degradation of cellulosic materials relies on the molecular-mechanistic principle that internally chain-cleaving endocellulases work synergistically with chain end-cleaving exocellulases in polysaccharide chain depolymerization. How endo-exo synergy becomes effective in the deconstruction of a solid substrate that presents cellulose chains assembled into crystalline material is an open question of the mechanism, with immediate implications on the bioconversion efficiency of cellulases. Here, based on single-molecule evidence from real-time atomic force microscopy, we discover that endo- and exocellulases engage in the formation of transient clusters of typically three to four enzymes at the cellulose surface. The clusters form specifically at regular domains of crystalline cellulose microfibrils that feature molecular defects in the polysaccharide chain organization. The dynamics of cluster formation correlates with substrate degradation through a multilayer-processive mode of chain depolymerization, overall leading to the directed ablation of single microfibrils from the cellulose surface. Each multilayer-processive step involves the spatiotemporally coordinated and mechanistically concerted activity of the endo- and exocellulases in close proximity. Mechanistically, the cooperativity with the endocellulase enables the exocellulase to pass through its processive cycles ∼100-fold faster than when acting alone. Our results suggest an advanced paradigm of efficient multienzymatic degradation of structurally organized polymer materials by endo-exo synergetic chain depolymerization.

Processive Enzymes Kept on a Leash: How Cellulase Activity in Multienzyme Complexes Directs Nanoscale Deconstruction of Cellulose.

Biological deconstruction of polymer materials gains efficiency from the spatiotemporally coordinated action of enzymes with synergetic function in polymer chain depolymerization. To perpetuate enzyme synergy on a solid substrate undergoing deconstruction, the overall attack must alternate between focusing the individual enzymes locally and dissipating them again to other surface sites. Natural cellulases working as multienzyme complexes assembled on a scaffold protein (the cellulosome) maximize the effect of local concentration yet restrain the dispersion of individual enzymes. Here, with evidence from real-time atomic force microscopy to track nanoscale deconstruction of single cellulose fibers, we show that the cellulosome forces the fiber degradation into the transversal direction, to produce smaller fragments from multiple local attacks ("cuts"). Noncomplexed enzymes, as in fungal cellulases or obtained by dissociating the cellulosome, release the confining force so that fiber degradation proceeds laterally, observed as directed ablation of surface fibrils and leading to whole fiber "thinning". Processive cellulases that are enabled to freely disperse evoke the lateral degradation and determine its efficiency. Our results suggest that among natural cellulases, the dispersed enzymes are more generally and globally effective in depolymerization, while the cellulosome represents a specialized, fiber-fragmenting machinery.

Engineering analysis of multienzyme cascade reactions for 3'-sialyllactose synthesis.

Sialo-oligosaccharides are important products of emerging biotechnology for complex carbohydrates as nutritional ingredients. Cascade bio-catalysis is central to the development of sialo-oligosaccharide production systems, based on isolated enzymes or whole cells. Multienzyme transformations have been established for sialo-oligosaccharide synthesis from expedient substrates, but systematic engineering analysis for the optimization of such transformations is lacking. Here, we show a mathematical modeling-guided approach to 3'-sialyllactose (3SL) synthesis from N-acetyl- d-neuraminic acid (Neu5Ac) and lactose in the presence of cytidine 5'-triphosphate, via the reactions of cytidine 5'-monophosphate-Neu5Ac synthetase and α2,3-sialyltransferase. The Neu5Ac was synthesized in situ from N-acetyl- d-mannosamine using the reversible reaction with pyruvate by Neu5Ac lyase or the effectively irreversible reaction with phosphoenolpyruvate by Neu5Ac synthase. We show through comprehensive time-course study by experiment and modeling that, due to kinetic rather than thermodynamic advantages of the synthase reaction, the 3SL yield was increased (up to 75%; 10.4 g/L) and the initial productivity doubled (15 g/L/h), compared with synthesis based on the lyase reaction. We further show model-based optimization to minimize the total loading of protein (saving: up to 43%) while maintaining a suitable ratio of the individual enzyme activities to achieve 3SL target yield (61%-75%; 7-10 g/L) and overall productivity (3-5 g/L/h). Collectively, our results reveal the principal factors of enzyme cascade efficiency for 3SL synthesis and highlight the important role of engineering analysis to make multienzyme-catalyzed transformations fit for oligosaccharide production.

A Biological Nanomachine at Work: Watching the Cellulosome Degrade Crystalline Cellulose.

The cellulosome is a supramolecular multienzymatic protein complex that functions as a biological nanomachine of cellulosic biomass degradation. How the megadalton-size cellulosome adapts to a solid substrate is central to its mechanism of action and is also key for its efficient use in bioconversion applications. We report time-lapse visualization of crystalline cellulose degradation by individual cellulosomes from Clostridium thermocellum by atomic force microscopy. Upon binding to cellulose, the cellulosomes switch to elongated, even filamentous shapes and morph these dynamically at below 1 min time scale according to requirements of the substrate surface under attack. Compared with noncomplexed cellulases that peel off material while sliding along crystalline cellulose surfaces, the cellulosomes remain bound locally for minutes and remove the material lying underneath. The consequent roughening up of the surface leads to an efficient deconstruction of cellulose nanocrystals both from the ends and through fissions within. Distinct modes of cellulose nanocrystal deconstruction by nature's major cellulase systems are thus revealed.

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.

Single-molecule study of oxidative enzymatic deconstruction of cellulose.

LPMO (lytic polysaccharide monooxygenase) represents a unique paradigm of cellulosic biomass degradation by an oxidative mechanism. Understanding the role of LPMO in deconstructing crystalline cellulose is fundamental to the enzyme's biological function and will help to specify the use of LPMO in biorefinery applications. Here we show with real-time atomic force microscopy that C1 and C4 oxidizing types of LPMO from Neurospora crassa (NcLPMO9F, NcLPMO9C) bind to nanocrystalline cellulose with high preference for the very same substrate surfaces that are also used by a processive cellulase (Trichoderma reesei CBH I) to move along during hydrolytic cellulose degradation. The bound LPMOs, however, are immobile during their adsorbed residence time ( ~ 1.0 min for NcLPMO9F) on cellulose. Treatment with LPMO resulted in fibrillation of crystalline cellulose and strongly ( ≥ 2-fold) enhanced the cellulase adsorption. It also increased enzyme turnover on the cellulose surface, thus boosting the hydrolytic conversion.Understanding the role of enzymes in biomass depolymerization is essential for the development of more efficient biorefineries. Here, the authors show by atomic force microscopy the real-time mechanism of cellulose deconstruction by lytic polysaccharide monooxygenases.

Active-Site His85 of Pasteurella dagmatis Sialyltransferase Facilitates Productive Sialyl Transfer and So Prevents Futile Hydrolysis of CMP-Neu5Ac.

Sialyltransferases of the GT-80 glycosyltransferase family are considered multifunctional because of the array of activities detected. They exhibit glycosyl transfer, trans-sialylation, and hydrolysis activities. How these enzymes utilize their active-site residues in balancing the different enzymatic activities is not well understood. In this study of Pasteurella dagmatis α2,3sialyltransferase, we show that the conserved His85 controls efficiency and selectivity of the sialyl transfer. A His85→Asn variant was 200 times less efficient than wild-type for sialylation of lactose, and exhibited relaxed site selectivity to form not only the α2,3- but also the α2,6-sialylated product (21 %). The H85N variant was virtually inactive in trans-sialylation but showed almost the same CMP-Neu5Ac hydrolase activity as wild-type. The competition between sialyl transfer and hydrolysis in the conversion of CMP-Neu5Ac was dependent on the lactose concentration; this was characterized by a kinetic partition ratio of 85 m-1 for the H85N variant, compared to 17 000 m-1 for the wild-type enzyme. His85 promotes the productive sialyl transfer to lactose and so prevents hydrolysis of CMP-Neu5Ac in the reaction.

Direct-Write Fabrication of Cellulose Nano-Structures via Focused Electron Beam Induced Nanosynthesis.

In many areas of science and technology, patterned films and surfaces play a key role in engineering and development of advanced materials. Here, we introduce a new generic technique for the fabrication of polysaccharide nano-structures via focused electron beam induced conversion (FEBIC). For the proof of principle, organosoluble trimethylsilyl-cellulose (TMSC) thin films have been deposited by spin coating on SiO2 / Si and exposed to a nano-sized electron beam. It turns out that in the exposed areas an electron induced desilylation reaction takes place converting soluble TMSC to rather insoluble cellulose. After removal of the unexposed TMSC areas, structured cellulose patterns remain on the surface with FWHM line widths down to 70 nm. Systematic FEBIC parameter sweeps reveal a generally electron dose dependent behavior with three working regimes: incomplete conversion, ideal doses and over exposure. Direct (FT-IR) and indirect chemical analyses (enzymatic degradation) confirmed the cellulosic character of ideally converted areas. These investigations are complemented by a theoretical model which suggests a two-step reaction process by means of TMSC → cellulose and cellulose → non-cellulose material conversion in excellent agreement with experimental data. The extracted, individual reaction rates allowed the derivation of design rules for FEBIC parameters towards highest conversion efficiencies and highest lateral resolution.

Functional characterization of the native swollenin from Trichoderma reesei: study of its possible role as C1 factor of enzymatic lignocellulose conversion.

BACKGROUND: Through binding to cellulose, expansin-like proteins are thought to loosen the structural order of crystalline surface material, thus making it more accessible for degradation by hydrolytic enzymes. Swollenin SWO1 is the major expansin-like protein from the fungus Trichoderma reesei. Here, we have performed a detailed characterization of a recombinant native form of SWO1 with respect to its possible auxiliary role in the enzymatic saccharification of lignocellulosic substrates. RESULTS: The swo1 gene was overexpressed in T. reesei QM9414 Δxyr1 mutant, featuring downregulated cellulase production, and the protein was purified from culture supernatant. SWO1 was N-glycosylated and its circular dichroism spectrum suggested a folded protein. Adsorption isotherms (25 °C, pH 5.0, 1.0 mg substrate/mL) revealed SWO1 to be 120- and 20-fold more specific for binding to birchwood xylan and kraft lignin, respectively, than for binding to Avicel PH-101. The SWO1 binding capacity on lignin (25 µmol/g) exceeded 12-fold that on Avicel PH-101 (2.1 µmol/g). On xylan, not only the binding capacity (22 µmol/g) but also the affinity of SWO1 (K d = 0.08 µM) was enhanced compared to Avicel PH-101 (K d = 0.89 µM). SWO1 caused rapid release of a tiny amount of reducing sugars (<1 % of total) from different substrates (Avicel PH-101, nanocrystalline cellulose, steam-pretreated wheat straw, barley ß-glucan, cellotetraose) but did not promote continued saccharification. Atomic force microscopy revealed that amorphous cellulose films were not affected by SWO1. Also with AFM, binding of SWO1 to cellulose nanocrystallites was demonstrated at the single-molecule level, but adsorption did not affect this cellulose. SWO1 exhibited no synergy with T. reesei cellulases in the hydrolysis of the different celluloses. However, SWO1 boosted slightly (1.5-fold) the reducing sugar release from a native grass substrate. CONCLUSIONS: SWO1 is a strongly glycosylated protein, which has implications for producing it in heterologous hosts. Although SWO1 binds to crystalline cellulose, its adsorption to xylan is much stronger. SWO1 is not an auxiliary factor of the enzymatic degradation of a variety of cellulosic substrates. Effect of SWO1 on sugar release from intact plant cell walls might be exploitable with certain (e.g., mildly pretreated) lignocellulosic feedstocks.

The micromorphology of Trichoderma reesei analyzed in cultivations on lactose and solid lignocellulosic substrate, and its relationship with cellulase production.

BACKGROUND: Trichoderma reesei is the principal producer of cellulolytic enzymes. Because of the strong influence on the enzyme production, the morphology of the filamentous fungi is a key parameter for process optimization. For cost-effective production of cellulolytic enzymes, the cultivation of T. reesei is performed on lignocellulosic waste streams. These insoluble substrates prevent the application of the conventional light microscopy for the analysis of fungal morphology. Here, we present a novel method for the micromorphological analysis based on confocal laser-scanning microscopy (CLSM) and the computer-aided image analysis. This method enabled the quantification of the dimensions of the single cell (intercalary length and cell width) and the degree of branching in cultivations on the industrially relevant substrates wheat straw and lactose. The micromorphology of two T. reesei strains, QM9414 and a carbon catabolite derepressed cre1 knockout mutant (Δcre1), was analyzed in dependence of substrate, inoculation method, and agitation velocity. RESULTS: Trichoderma reesei strain Δcre1 formed shorter cells (10.09 µm) on average and developed more ramified mycelia (0.36 branches/cell) than strain QM9414 (12.03 µm, 0.22 branches/cell). Cultivated on wheat straw, the average cell length of QM9414 (10.87 µm) and Δcre1 (9.74 µm) was 10 and 21 % shorter as compared to reference cultivations on lactose. When inoculation was done with spores as compared to hyphal biomass, cell lengths of QM9414 (10.97 µm) and Δcre1 (9.10 µm) were on average about 20 % shorter. Strain performance was evaluated in protein concentration and total cellulase activity, which varied between 0.69 and 2.31 FPU/mL for Δcre1 and between 0.84 and 1.64 FPU/mL for QM9414. The cell length exhibited slightly negative correlation with the protein (regression coefficient -0.04 g/(L µm), R (2) 0.33) and the cellulase (-0.30 FPU/(mL µm), R (2) 0.53) production. CONCLUSIONS: The dimensions of the single cell of T. reesei were dependent on strain background, substrate used and process conditions applied. Micromorphological changes were correlated semi-quantitatively with the efficiency of enzyme production. In providing a process analytical tool for enzyme production by T. reesei on lignocellulosic substrate, this study has relevance for the characterization and optimization of a critical step in the overall saccharification process.

Cellular automata modeling depicts degradation of cellulosic material by a cellulase system with single-molecule resolution.

BACKGROUND: Enzymatic hydrolysis of cellulose involves the spatiotemporally correlated action of distinct polysaccharide chain cleaving activities confined to the surface of an insoluble substrate. Because cellulases differ in preference for attacking crystalline compared to amorphous cellulose, the spatial distribution of structural order across the cellulose surface imposes additional constraints on the dynamic interplay between the enzymes. Reconstruction of total system behavior from single-molecule activity parameters is a longstanding key goal in the field. RESULTS: We have developed a stochastic, cellular automata-based modeling approach to describe degradation of cellulosic material by a cellulase system at single-molecule resolution. Substrate morphology was modeled to represent the amorphous and crystalline phases as well as the different spatial orientations of the polysaccharide chains. The enzyme system model consisted of an internally chain-cleaving endoglucanase (EG) as well as two processively acting, reducing and non-reducing chain end-cleaving cellobiohydrolases (CBHs). Substrate preference (amorphous: EG, CBH II; crystalline: CBH I) and characteristic frequencies for chain cleavage, processive movement, and dissociation were assigned from biochemical data. Once adsorbed, enzymes were allowed to reach surface-exposed substrate sites through "random-walk" lateral diffusion or processive motion. Simulations revealed that slow dissociation of processive enzymes at obstacles obstructing further movement resulted in local jamming of the cellulases, with consequent delay in the degradation of the surface area affected. Exploiting validation against evidence from atomic force microscopy imaging as a unique opportunity opened up by the modeling approach, we show that spatiotemporal characteristics of cellulose surface degradation by the system of synergizing cellulases were reproduced quantitatively at the nanometer resolution of the experimental data. This in turn gave useful prediction of the soluble sugar release rate. CONCLUSIONS: Salient dynamic features of cellulose surface degradation by different cellulases acting in synergy were reproduced in simulations in good agreement with evidence from high-resolution visualization experiments. Due to the single-molecule resolution of the modeling approach, the utility of the presented model lies not only in predicting system behavior but also in elucidating inherently complex (e.g., stochastic) phenomena involved in enzymatic cellulose degradation. Thus, it creates synergy with experiment to advance the mechanistic understanding for improved application.

Tunable Semicrystalline Thin Film Cellulose Substrate for High-Resolution, In-Situ AFM Characterization of Enzymatic Cellulose Degradation.

In the field of enzymatic cellulose degradation, fundamental interactions between different enzymes and polymorphic cellulose materials are of essential importance but still not understood in full detail. One technology with the potential of direct visualization of such bioprocesses is atomic force microscopy (AFM) due to its capability of real-time in situ investigations with spatial resolutions down to the molecular scale. To exploit the full capabilities of this technology and unravel fundamental enzyme-cellulose bioprocesses, appropriate cellulose substrates are decisive. In this study, we introduce a semicrystalline-thin-film-cellulose (SCFTC) substrate which fulfills the strong demands on such ideal cellulose substrates by means of (1) tunable polymorphism via variable contents of homogeneously sized cellulose nanocrystals embedded in an amorphous cellulose matrix; (2) nanoflat surface topology for high-resolution and high-speed AFM; and (3) fast, simple, and reproducible fabrication. The study starts with a detailed description of SCTFC preparation protocols including an in-depth material characterization. In the second part, we demonstrate the suitability of SCTFC substrates for enzymatic degradation studies by combined, individual, and sequential exposure to TrCel6A/TrCel7A cellulases (Trichoderma reesei) to visualize synergistic effects down to the nanoscale.

Cellulose surface degradation by a lytic polysaccharide monooxygenase and its effect on cellulase hydrolytic efficiency.

Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization.

Surface structural dynamics of enzymatic cellulose degradation, revealed by combined kinetic and atomic force microscopy studies.

Highly heterogeneous and usually weakly defined substrate morphologies complicate the study of enzymatic cellulose hydrolysis. The cellulose surface has a non-uniform shape in particular, with consequent impacts on cellulase adsorption and activity. We have therefore prepared a cellulosic model substrate which is shown by atomic force microscopy (AFM) to display a completely smooth surface, the residual squared mean roughness being 10 nm or lower, and applied it for kinetic analysis of cellulase action. The substrate consists of an amorphous cellulose matrix into which variably sized crystalline fibers are distributed in apparently irregular fashion. Its conversion into soluble sugars by Trichoderma sp. cellulase at 50 °C proceeded without apparent limitation up to 70% completion and was paralleled by a steady increase in cellulase adsorption to the cellulose. Individual cellulase components (CBH I, CBH II, EG) also showed strongly enhanced adsorption with progressing cellulose conversion, irrespective of their preference for degrading the amorphous or crystalline substrate parts as revealed by AFM. The specific activity of the adsorbed cellulases, however, decreased concomitantly. Cellulose surface morphologies evolving as a consequence of cellulase action were visualized by AFM. Three-dimensional surface degradation by the cellulases resulted in a large increase in cellulose surface area for enzyme adsorption. However, the decline in enzyme specific activity during conversion was caused by factors other than surface ablation and disruption. Based on kinetic evidence for enzymatic hydrolyses of the smooth-surface model substrate and microcrystalline cellulose (Avicel), we hypothesize that, due to gradual loss of productive dynamics in their interactions with the cellulose surface, individual cellulases get progressively confined to substrate parts where they are no longer optimally active. This eventually leads to an overall slow-down of hydrolysis.

Dissecting and reconstructing synergism: in situ visualization of cooperativity among cellulases.

Cellulose is the most abundant biopolymer and a major reservoir of fixed carbon on earth. Comprehension of the elusive mechanism of its enzymatic degradation represents a fundamental problem at the interface of biology, biotechnology, and materials science. The interdependence of cellulose disintegration and hydrolysis and the synergistic interplay among cellulases is yet poorly understood. Here we report evidence from in situ atomic force microscopy (AFM) that delineates degradation of a polymorphic cellulose substrate as a dynamic cycle of alternating exposure and removal of crystalline fibers. Direct observation shows that chain-end-cleaving cellobiohydrolases (CBH I, CBH II) and an internally chain-cleaving endoglucanase (EG), the major components of cellulase systems, take on distinct roles: EG and CBH II make the cellulose surface accessible for CBH I by removing amorphous-unordered substrate areas, thus exposing otherwise embedded crystalline-ordered nanofibrils of the cellulose. Subsequently, these fibrils are degraded efficiently by CBH I, thereby uncovering new amorphous areas. Without prior action of EG and CBH II, CBH I was poorly active on the cellulosic substrate. This leads to the conclusion that synergism among cellulases is morphology-dependent and governed by the cooperativity between enzymes degrading amorphous regions and those targeting primarily crystalline regions. The surface-disrupting activity of cellulases therefore strongly depends on mesoscopic structural features of the substrate: size and packing of crystalline fibers are key determinants of the overall efficiency of cellulose degradation.