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.

Effect of ionic liquid on the enzymatic synthesis of cello-oligosaccharides and their assembly into cellulose materials.

Imidazolium-based ionic liquids are important solvents for the processing of natural cellulose. Little is known about their use in synthesizing cellulose via bottom-up polymerization of ß-1,4-d-glucosyl chains in solution. Here, we analyzed cellodextrin phosphorylase-catalyzed synthesis of cello-oligosaccharides, and the subsequent spontaneous self-assembly of the chains, in the presence of cellulose-dissolving ionic liquid, 1,3-dimethylimidazolium dimethyl phosphate ([Dmim]DMP) or 1-ethyl-3-methylimidazolium acetate ([Emim]OAc). The average chain length dropped from ~7.4 in buffer to ~6.4 in ionic liquid (30 vol%). The synthetic cellulose exhibited allomorph II crystal structure and showed nanosheet morphology of 4-5 nm thickness and several µm length. Its suspensions were hydrogels with viscoelastic properties dependent on solvent conditions used. Reactions in 10 vol% [Dmim]DMP or [Emim]OAc gave a hydrogel with elastic modulus of ~13 kPa and loss factor of ~0.18. Collectively, interactions of the ionic liquid with enzyme and cello-oligosaccharides delimit the polymerization and tune the assembly into cellulose networks.

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.

Reducing end thiol-modified nanocellulose: Bottom-up enzymatic synthesis and use for templated assembly of silver nanoparticles into biocidal composite material.

Nanoparticle-polymer composites are important functional materials but structural control of their assembly is challenging. Owing to its crystalline internal structure and tunable nanoscale morphology, cellulose is promising polymer scaffold for templating such composite materials. Here, we show bottom-up synthesis of reducing end thiol-modified cellulose chains by iterative bi-enzymatic ß-1,4-glycosylation of 1-thio-ß-d-glucose (10 mM), to a degree of polymerization of ∼8 and in a yield of ∼41% on the donor substrate (α-d-glucose 1-phosphate, 100 mM). Synthetic cellulose oligomers self-assemble into highly ordered crystalline (cellulose allomorph II) material showing long (micrometers) and thin nanosheet-like morphologies, with thickness of 5-7 nm. Silver nanoparticles were attached selectively and well dispersed on the surface of the thiol-modified cellulose, in excellent yield (≥ 95%) and high loading efficiency (∼2.2 g silver/g thiol-cellulose). Examined against Escherichia coli and Staphylococcus aureus, surface-patterned nanoparticles show excellent biocidal activity. Bottom-up approach by chemical design to a functional cellulose nanocomposite is presented. Synthetic thiol-containing nanocellulose can expand the scope of top-down produced cellulose materials.

Deposition of Cellulose-Based Thin Films on Flexible Substrates.

This study investigates flexible (polyamide 6.6 PA-6.6, polyethylene terephthalate PET, Cu, Al, and Ni foils) and, for comparison, stiff substrates (silicon wafers and glass) differing in, for example, in surface free energy and surface roughness and their ability to host cellulose-based thin films. Trimethylsilyl cellulose (TMSC), a hydrophobic acid-labile cellulose derivative, was deposited on these substrates and subjected to spin coating. For all the synthetic polymer and metal substrates, rather homogenous films were obtained, where the thickness and the roughness of the films correlated with the substrate roughness and its surface free energy. A particular case was the TMSC layer on the copper foil, which exhibited superhydrophobicity caused by the microstructuring of the copper substrate. After the investigation of TMSC film formation, the conversion to cellulose using acidic vapors of HCl was attempted. While for the polymer foils, as well as for glass and silicon, rather homogenous and smooth cellulose films were obtained, for the metal foils, there is a competing reaction between the formation of metal chlorides and the generation of cellulose. We observed particles corresponding to the metal chlorides, while we could not detect any cellulose thin films after HCl treatment of the metal foils as proven by cross-section imaging using scanning electron microscopy (SEM).