The enzymatic degradation of recalcitrant plant biomass is one of the key industrial challenges of the 21st century. Accordingly, there is a continuing drive to discover new routes to promote polysaccharide degradation. Perhaps the most promising approach involves the application of "cellulase-enhancing factors," such as those from the glycoside hydrolase (CAZy) GH61 family. Here we show that GH61 enzymes are a unique family of copper-dependent oxidases. We demonstrate that copper is needed for GH61 maximal activity and that the formation of cellodextrin and oxidized cellodextrin products by GH61 is enhanced in the presence of small molecule redox-active cofactors such as ascorbate and gallate. By using electron paramagnetic resonance spectroscopy and single-crystal X-ray diffraction, the active site of GH61 is revealed to contain a type II copper and, uniquely, a methylated histidine in the copper's coordination sphere, thus providing an innovative paradigm in bioinorganic enzymatic catalysis.ellulose is Earth's most abundant biopolymer. Its exploitation as an energy source plays a critical role in the global ecology and carbon cycle. Industrial production of fuels and chemicals from this plentiful and renewable resource holds the potential to displace petroleum-based sources, thus reducing the associated economic and environmental costs of oil and gas production (1, 2) and promoting energy security as part of a balanced energy portfolio. However, despite the burgeoning potential of cellulose as a biofuel source, its remarkable recalcitrance to depolymerization has so far hindered the economical use of any form of lignocellulosic biomass as a feedstock for biofuel production (3, 4).In addressing the issue of cellulose recalcitrance, much effort has been directed toward harnessing the known cellulosedegrading enzymatic pathways found in fungi. The consensus model of enzymatic degradation involves the concerted action of a consortium of different endoglucanases and "exo"-acting cellobiohydrolases (collectively termed "cellulases"); both enzyme classes perform classical glycoside hydrolysis through attack of water at the anomeric center of oligo/polysaccharide substrates (5-9). Necessarily as part of the overall enzymatic degradation of cellulose, the initial enzymatic step must overcome cellulose's inertness by disrupting the cellulosic structure, thus allowing attack by traditional cellulases. Originally, Reese et al. (10) suggested that undefined enzymes could play a major role in this step. This notion remained a hypothesis until very recently when, in a key paper, Harris et al. (11) demonstrated that inclusion of a novel enzyme class, currently termed GH61 glycoside hydrolases in the CAZy database of carbohydrate-active enzymes (12), greatly increases the performance of Hypocrea jecorina (Trichoderma reesei) cellulases in lignocellulose hydrolysis. From this work, it was suggested that GH61s act directly on cellulose rendering it more accessible to traditional cellulase action (11). Moreover, recent genomi...

Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulose hydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewable transportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families of glycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than family 61 (GH61), originally classified based on measurement of very weak endo-1,4-beta-d-glucanase activity in one family member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselves but in the presence of various divalent metal ions can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and find that it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donor and nucleophile/base in a canonical hydrolytic reaction, and we conclude that the GH61 proteins are unlikely to be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residues for GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loading (and hence the cost) required to hydrolyze lignocellulosic biomass.

The remarkable progress in efforts to prepare conductive self-healing hydrogels mimicking human skin's functions has been witnessed in recent years. However, it remains a great challenge to develop an integrated conductive gel combining excellent self-healing and mechanical properties, which is derived from their inherent compromise between the dynamic cross-links for healing and steady cross-links for mechanical strength. In this work, we design a tough, self-healing, and self-adhesive ionic gel by constructing synergistic multiple coordination bonds among tannic acid-coated cellulose nanocrystals (TA@CNCs), poly(acrylic acid) chains, and metal ions in a covalent polymer network. The incorporated TA@CNC acts as a dynamic connected bridge in the hierarchically porous network mediated by multiple coordination bonds, endowing the ionic gels the superior mechanical performance. Reversible nature of dynamic coordination interactions leads to excellent recovery property as well as reliable mechanical and electrical self-healing property without any assistance of external stimuli. Intriguingly, the ionic gels display durable and repeatable adhesiveness ascribed to the presence of catechol groups from the incorporated tannic acid, which can be adhered directly on human skin without inflammatory response and residual. Additionally, the ionic gels with a great strain sensitivity can be employed as flexible strain sensors to monitor and distinguish both large motions (e.g., joints bending) and subtle motions (e.g., pulse and breath), which enable us to analyze the data on the user interface of smart phone via programmable wireless transmission. This work provides a new prospect for the design of the biocompatible cellulose-based hydrogels with stretchable, self-adhesive, self-healing, and strain-sensitive properties for potential applications in wearable electronic sensors and healthcare monitoring.

No abstract

A comparative study has been performed with several fungal laccases for the oxidation of a series of phenols, anilines, and benzenethiols and for the inhibition by halides. The observed K(m) and kcat were correlated to the structure of substrate. The change in log (kcat/K(m)) was found to be proportional to the one-electron redox potential difference between laccase's type 1 copper site and substrate. This correlation indicated that the first electron transfer from substrate to laccase was governed by the "outersphere" mechanism. Compared to the electronic factor, the steric effect of small o-substituents (such as methyl and methoxy groups) was found to be unimportant. The depth of the laccase's type 1 copper site was estimated as approximately 10 A by comparing the steric effect among five 2-methoxyphenols whose 4-substituents ranged from 0.1 to 14 kDa in mass. The observed inhibition potency order of F- > Cl- > Br- was attributed to limited accessibility of laccase's type 2/type 3 trinuclear copper cluster site. Although the enzymes studied have homologous primary sequences and predicted similar backbone structures, the difference exhibited by each enzyme (in interacting with individual substrate or inhibitor) suggested the structural variation in their functional domains.