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A deeper mechanistic understanding of the saccharification of cellulosic biomass could enhance the efficiency of biofuels development. We report here the real-time visualization of crystalline cellulose degradation by individual cellulase enzymes through use of an advanced version of high-speed atomic force microscopy. Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules were observed to slide unidirectionally along the crystalline cellulose surface but at one point exhibited collective halting analogous to a traffic jam. Changing the crystalline polymorphic form of cellulose by means of an ammonia treatment increased the apparent number of accessible lanes on the crystalline surface and consequently the number of moving cellulase molecules. Treatment of this bulky crystalline cellulose simultaneously or separately with T. reesei cellobiohydrolase II (TrCel6A) resulted in a remarkable increase in the proportion of mobile enzyme molecules on the surface. Cellulose was completely degraded by the synergistic action between the two enzymes.

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A series of alkylimidazolium salts containing dimethyl phosphate, methyl methylphosphonate, or methyl phosphonate prepared by a facile, one-pot procedure were obtained as room temperature ionic liquids, which have the potential to solubilize cellulose under mild conditions. Especially, N-ethyl-N9-methylimidazolium methylphosphonate enables the preparation of 10 wt% cellulose solution by keeping it at 45 uC for 30 min with stirring and rendered soluble 2-4 wt% cellulose without pretreatments and heating.

Conversion of 1,2-dihydroxyl groups to dialdehyde by periodate oxidation is a useful method of derivatizing polysaccharides but has not been extensively utilized in derivatization of cellulose because of complicacy due to the crystalline nature of cellulose. To understand the influence of cellulose crystallinity on this reaction, we investigated how the periodate oxidation proceeds with a highly crystalline cellulose of the marine alga Cladophora sp. The crystallinity of the oxidized cellulose, determined by X-ray diffraction, decreased according to the oxidation level. The half-height widths of equatorial diffraction peaks were nearly unchanged. The solid-state 13C NMR spectra did not show peaks corresponding to aldehyde groups, but solution 13C NMR spectra showed the presence of dicarboxylic groups after subsequent oxidation by sodium chlorite. Transmission electron microscopy showed that microfibrils of Cladophora tended to be bent and more flexible than the original sample. Gold labeling of the aldehyde groups, mediated by thiosemicarbazide derivatization, revealed a highly uneven distribution of dialdehyde groups. When treated by 50% (w/v) sulfuric acid, partially oxidized Cladophora cellulose gave many short fragments of microfibril. These features indicate that the periodate oxidation proceeds by forming dialdehyde groups in longitudinally spaced, bandlike domains.

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.

The crystal and molecular structure, together with the hydrogen-bonding system in ammonia-mercerized cellulose III I, has been determined using synchrotron X-ray and neutron fiber diffraction data. The structure has a one-chain monoclinic unit cell with an asymmetric unit that contains only one glucosyl residue and with the hydroxymethyl group in the gt conformation. The hydrogen-bonding system is well-defined with no evidence of disorder. A bifurcated hydrogen bond links a donating secondary alcohol O3 atom to a ring O5 atom (major) and a primary alcohol O6 atom (minor) of an adjacent residue in the same chain. Two hydrogen bonds are present between neighboring chains, perpendicular to the chain direction. A detailed comparison of the crystal structure and hydrogen-bonding system reported here for cellulose III I and those reported previously for the other cellulose polymorphs is given. The conformation of the chain in cellulose IIII has features similar to that of the center chain in the highly stable cellulose II allomorph. However, unlike in cellulose II, the chains are parallel, as in cellulose IR and cellulose Iβ.

Aerogels with their low density (0.004-0.500 g cm À3 ), large internal surface area, and large open pores are promising candidates for various advanced applications.[1] The utilization of inorganic aerogels, however, has been hampered by their poor mechanical properties. A prominent example is silica aerogel, which is prepared by an organic sol-gel process, [2] and has unique features, such as ultralow density (the lightest silica aerogel has a density that is similar to the density of air, which is 0.00129 g cm À3 ), near transparency, and low thermal conductivity. However, the extreme fragility of this aerogel necessitates its reinforcement for practical uses. A typical method is hybridization with organic polymers, such as polyurea, polyurethane, poly(methyl methacrylate), polyacrylonitrile, and polystyrene. [3] Other candidates for the reinforcement of inorganic aerogels are insoluble polysaccharides, which are abundantly available and show wide varieties in structure and properties.[4] The useful features of these compounds are hydrophilicity, biocompatibility, hydroxy reactivity, and reasonable thermal and mechanical stabilities.[5] For example, nanofibrillar bacterial cellulose and microfibrillated cellulose gel have been proposed as templates for cobalt ferrite nanoparticles and titanium dioxide. [6] While in the above-mentioned work native cellulose with cellulose I crystallinity was used, cellulose can be prepared as a hydrogel with cellulose II crystallinity through dissolution and coagulation. Some of the resulting aerogels have remarkable mechanical strength and light transmittance.[7] They have high porosity with open structures and thus provide an effective substrate for the synthesis of metallic nanoparticles.[8] To further utilize the regenerated cellulose gel, we herein attempted in situ synthesis of silica in cellulose gels.While a similar attempt has been reported, in which the cellulose gel was obtained from solution in N-methylmorpholine-N-oxide monohydrate, [9] the development of the nanostructure (nitrogen BET surface area of 220-290 m 2 g À1 ) and the level of silica loading (less than 13 % w/w) were rather limited. By using the aqueous alkali-based solvent, we obtained the cellulose aerogel with a surface area of 356 m 2 g À1 , and a silica loading of more than 60 % w/w resulted in surface areas that exceeded 600 m 2 g À1 . We used the sol-gel synthesis method toward nanostructured silica, which typically starts from tetraethyl orthosilicate (TEOS). The resulting composite gels were dried with supercritical CO 2 to give cellulose-silica aerogels with low density, moderate light transmittance, a large surface area, high mechanical integrity, and excellent heat insulation. This method can also lead to fabrication of silica-only aerogels through the removal of cellulose by calcination, that is, the use of cellulose aerogel as sacrificial template. Figure 1 shows the preparation of the aerogel. The cellulose hydrogel is a transparent material that has a water content of 92 % and a poro...