Elucidation of the Specific Ion Effects and Intermediate Structures of Cellulose Fibers Swollen in Inorganic Salt Solutions via In Situ X-ray Diffraction.

The solubilities of many substances are significantly affected by specific ions, as demonstrated by the Hofmeister series of proteins. Cellulose has a resistant fibrillar structure that hinders its swelling and dissolution. Certain inorganic salt solutions are effective swelling agents and solvents for cellulose. However, the precise effects of these ions on cellulose are not fully understood. In this study, we studied the intermediate structures of cellulose fibers during their swelling process in ZnCl2 and LiBr solutions via in situ X-ray diffraction. Two swollen phases with characteristic morphologies were observed for both salt treatments. Only the surfaces of the fibers were swollen in ZnCl2, whereas the ions penetrated the fibers and formed complexes with cellulose while the morphology of the fibers was maintained in LiBr. Our findings clarify the reasons that ZnCl2 has been used as an excellent swelling agent, whereas LiBr has been used as a good solvent for cellulose.

X-ray crystal structure of anhydrous chitosan at atomic resolution.

We determined the crystal structure of anhydrous chitosan at atomic resolution, using X-ray fiber diffraction data extending to 1.17 Å resolution. The unit cell [a = 8.129(7) Å, b = 8.347(6) Å, c = 10.311(7) Å, space group P21 21 21 ] of anhydrous chitosan contains two chains having one glucosamine residue in the asymmetric unit with the primary hydroxyl group in the gt conformation, that could be directly located in the Fourier omit map. The molecular arrangement of chitosan is very similar to the corner chains of cellulose II implying similar intermolecular hydrogen bonding between O6 and the amine nitrogen atom, and an intramolecular bifurcated hydrogen bond from O3 to O5 and O6. In addition to the classical hydrogen bonds, all the aliphatic hydrogens were involved in one or two weak hydrogen bonds, mostly helping to stabilize cohesion between antiparallel chains. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 361-368, 2016.

Cellulose is not degraded in the tunic of the edible ascidian Halocynthia roretzi contracting soft tunic syndrome.

Soft tunic syndrome is a fatal disease in the edible ascidian Halocynthia roretzi, causing serious damage to ascidian aquaculture in Korea and Japan. In diseased individuals, the tunic, an integumentary extracellular matrix of ascidians, softens and eventually tears. This is an infectious disease caused by the kinetoplastid flagellate Azumiobodo hoyamushi. However, the mechanism of tunic softening remains unknown. Because cellulose fibrils are the main component of the tunic, we compared the contents and structures of cellulose in healthy and diseased tunics by means of biochemical quantification and X-ray diffractometry. Unexpectedly, the cellulose contents and structures of cellulose microfibrils were almost the same regardless of the presence or absence of the disease. Therefore, it is unlikely that thinning of the microfibrils occurred in the softened tunic, because digestion should have resulted in decreases in crystallinity index and crystallite size. Moreover, cellulase was not detected in pure cultures of A. hoyamushi in biochemical and expressed sequence tag analyses. These results indicate that cellulose degradation does not occur in the softened tunic.

The tryptophan residue at the active site tunnel entrance of Trichoderma reesei cellobiohydrolase Cel7A is important for initiation of degradation of crystalline cellulose.

BACKGROUND: Mutation of Trp-40 in the Cel7A cellobiohydrolase from Trichoderma reesei (TrCel7A) causes a loss of crystalline cellulose-degrading ability. RESULTS: Mutant W40A showed reduced specific activity for crystalline cellulose and diffused the cellulose chain from the entrance of the active site tunnel. CONCLUSION: Trp-40 is essential for chain end loading to initiate processive hydrolysis of TrCel7A. SIGNIFICANCE: The mechanisms of crystalline polysaccharide degradation are clarified. The glycoside hydrolase family 7 cellobiohydrolase Cel7A from Trichoderma reesei is one of the best studied cellulases with the ability to degrade highly crystalline cellulose. The catalytic domain and the cellulose-binding domain (CBD) are both necessary for full activity on crystalline substrates. Our previous high-speed atomic force microscopy studies showed that mutation of Trp-40 at the entrance of the catalytic tunnel drastically decreases the ability to degrade crystalline cellulose. Here, we examined the activities of the WT enzyme and mutant W40A (with and without the CBD) for various substrates. Evaluation and comparison of the specific activities of the enzymes (WT, W40A, and the corresponding catalytic subunits (WTcat and W40Acat)) adsorbed on crystalline cellulose indicated that Trp-40 is involved in recruiting individual substrate chains into the active site tunnel to initiate processive hydrolysis. This was supported by molecular dynamics simulation study, i.e. the reducing end glucose unit was effectively loaded into the active site of WTcat, but not into that of W40Acat, when the simulation was started from subsite -7. However, when similar simulations were carried out starting from subsite -5, both enzymes held the substrate for 50 ns, indicating that the major difference between WTcat and W40Acat is the length of the free chain end of the substrate required to allow initiation of processive movements; this also reflects the difference between crystalline and amorphous celluloses. The CBD is important for enhancing the enzyme population on crystalline substrate, but it also decreases the specific activity of the adsorbed enzyme, possibly by attaching the enzyme to non-optimal places on the cellulose surface and/or hindering processive hydrolysis.

Trade-off between processivity and hydrolytic velocity of cellobiohydrolases at the surface of crystalline cellulose.

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.

Catalytic activity of Cu2O nanoparticles supported on cellulose beads prepared by emulsion-gelation using cellulose/LiBr solution and vegetable oil.

Nanocatalysts tend to aggregate and are difficult to recycle, limiting their practical applications. In this study, an environmentally friendly method was developed to produce cellulose beads for use as supporting materials for Cu-based nanocatalysts. Cellulose beads were synthesized from a water-in-oil emulsion using cellulose dissolved in an LiBr solution as the water phase and vegetable oil as the oil phase. Upon cooling, the gelation of the cellulose solution produced spherical cellulose beads, which were then oxidized to introduce surface carboxyl groups. These beads (diameter: 95-105 µm; specific surface area: 165-225 m2 g-1) have a three-dimensional network of nanofibers (width: 20-30 nm). Furthermore, the Cu2O nanoparticles were loaded onto oxidized cellulose beads before testing their catalytic activity in the reduction of 4-nitrophenol using NaBH4. The apparent reaction rate constant increased with increasing loading of Cu2O nanoparticles and the conversion efficiency was >90 %. The turnover frequency was 376.2 h-1 for the oxidized cellulose beads with the lowest Cu2O loading, indicating a higher catalytic activity compared to those of other Cu-based nanoparticle-loaded materials. In addition to their high catalytic activity, the cellulose beads are reusable and exhibit excellent stability.

Hydrolytic activities of crystalline cellulose nanofibers.

Cellulose is commonly believed to be inactive to organic substances; this inertness is an essential requirement for raw materials in industrial products. Here we demonstrate the contradictory but promising properties, which are the hydrolytic activities of crystalline cellulose nanofibers for the ester, monophosphate, and even amide bonds of small organic substrates under extremely mild conditions (neutral pH, moderate temperature, and atmospheric pressure). The hydrolytic activities were significantly extended to decompose the coat proteins of model viruses, followed by a drastic decrease in their infection capabilities to the host cells.

Adsorption characteristics of fungal family 1 cellulose-binding domain from Trichoderma reesei cellobiohydrolase I on crystalline cellulose: negative cooperative adsorption via a steric exclusion effect.

Cellobiohydrolases (CBHs) hydrolyzing crystalline cellulose share a two-domain structure of catalytic domain (CD) and cellulose-binding domain (CBD). To focus on the binding characteristics of CBD, we analyzed the adsorption of fusion protein of fungal family 1 CBD from Trichoderma reesei CBH I and red-fluorescent protein on crystalline and amorphous celluloses. Binding data were better fitted by Hill's model with negative cooperativity than by other adsorption models, suggesting the occurrence of a steric exclusion effect among the fusion molecules on the cellulose surfaces. The degree of negative cooperativity depended on the nature of the cellulose. The significance of this phenomenon for catalysis by intact CBHI is discussed.

Untangling the threads of cellulose mercerization.

Naturally occurring plant cellulose, our most abundant renewable resource, consists of fibers of long polymer chains that are tightly packed in parallel arrays in either of two crystal phases collectively referred to as cellulose I. During mercerization, a process that involves treatment with sodium hydroxide, cellulose goes through a conversion to another crystal form called cellulose II, within which every other chain has remarkably changed direction. We designed a neutron diffraction experiment with deuterium labelling in order to understand how this change of cellulose chain direction is possible. Here we show that during mercerization of bacterial cellulose, chains fold back on themselves in a zigzag pattern to form crystalline anti-parallel domains. This result provides a molecular level understanding of one of the most widely used industrial processes for improving cellulosic materials.

High speed atomic force microscopy visualizes processive movement of Trichoderma reesei cellobiohydrolase I on crystalline cellulose.

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.

Looking at hydrogen bonds in cellulose.

A series of cellulose crystal allomorphs has been studied using high-resolution X-ray and neutron fibre diffraction to locate the positions of H atoms involved in hydrogen bonding. One type of position was always clearly observed in the Fourier difference map (F(d)-F(h)), while the positions of other H atoms appeared to be less well established. Despite the high crystallinity of the chosen samples, neutron diffraction data favoured some hydrogen-bonding disorder in native cellulose. The presence of disorder and a comparison of hydrogen-bond geometries in different allomorphs suggests that although hydrogen bonding may not be the most important factor in the stabilization of cellulose I, it is essential for stabilizing cellulose III, which is the activated form, and preventing it from collapsing back to the more stable cellulose I.

Highly swellable hydrogel of regioselectively aminated (1→3)-α-d-glucan crosslinked with ethylene glycol diglycidyl ether.

(1→3)-α-d-glucan synthesized by glucosyltransferase J (GtfJ) cloned from Streptococcus salivarius was regioselectively aminated as 6-amino-6-deoxy-(1→3)-α-d-glucan (aminoglucan) through three steps: bromination, azidation, and reduction. The degree of substitution of the amino group was determined by elemental analysis to be 0.97 and the molecular weight was 3.74×104 as measured by size exclusion chromatography. The regioselective amination at the C6 position of every pyranose ring was confirmed by 1H/13C NMR and solid state 15N cross polarization/magic angle spinning NMR spectroscopy. Aminoglucan was characterized by FT-IR, X-ray diffraction and thermogravimetric analysis. Solubility of aminoglucan in various solvents was investigated and confirmed in aqueous solution at pH ≤ 11. Therefore, aminoglucan was crosslinked with ethylene glycol diglycidyl ether (EGDE) by an epoxy-ring opening reaction under alkaline conditions. The obtained EGDE-crosslinked aminoglucan hydrogels were highly swellable in water owing to a strong water-holding ability and no water was released on compression and breaking of the gels.

Nanoporous cellulose as metal nanoparticles support.

Despite considerable progress in the field of metal nanoparticles synthesis, major challenges remain in many practical applications of nanoparticles which require their immobilization on solid substrates, presenting additional difficulty in separation and processing. Here, transparent nanoporous cellulose gel obtained from aqueous alkali hydroxide-urea solution was examined as supporting medium for noble metal nanoparticles. Silver, gold, and platinum nanoparticles were synthesized in the gel by hydrothermal reduction by cellulose or by added reductant. Both methods gave nanoparticles embedded with high dispersion in cellulose gels. Supercritical CO2 drying of the metal-carrying gel gave corresponding aerogels with high transmittance, porosity, surface area, moderate thermal stability, and good mechanical strength. The cellulose and metal-cellulose gels were characterized by UV/vis spectroscopy, optical microscopy, SEM, TEM, XRD, nitrogen physisorption, TGA, and tensile testing, systematically.

X-ray crystallographic, scanning microprobe X-ray diffraction, and cross-polarized/magic angle spinning 13C NMR studies of the structure of cellulose III(II).

The X-ray crystallographic structure of cellulose III(II) is characterized by disorder; the unit cell (space group P2(1); a = 4.45 A, b = 7.64 A, c = 10.36 A, alpha = beta = 90 degrees, gamma = 106.96 degrees) is occupied by one chain that is the average of statistically disordered antiparallel chains. 13C CP/MAS NMR studies reveal the presence of three distinct molecular conformations that can be interpreted as a mixture of two different crystal forms, one equivalent to cellulose III(I), and another with two independent glucosyl conformations in the asymmetric unit. Both X-ray crystallographic and 13C NMR spectroscopic results are consistent with an aggregated microdomain structure for cellulose III(II). This structure can be generated from a new crystal form (space group P2(1); a = 4.45 A, b = 14.64 A, c = 10.36 A, alpha = beta = 90 degrees, gamma = 90.05 degrees; two crystallographically independent and antiparallel chains; gt hydroxymethyl groups) by multiple dislocation defects. These defects produce microdomains of the new crystal form and cellulose III(I) that scanning microprobe diffraction studies show are distributed consistently through the cellulose III(II) fiber.

Bone tissue engineering with premineralized silk scaffolds.

Silk fibroin biomaterials are being explored as novel protein-based systems for cell and tissue culture. In the present study, biomimetic growth of calcium phosphate on porous silk fibroin polymeric scaffolds was explored to generate organic/inorganic composites as scaffolds for bone tissue engineering. Aqueous-derived silk fibroin scaffolds were prepared with the addition of polyaspartic acid during processing, followed by the controlled deposition of calcium phosphate by exposure to CaCl(2) and Na(2)HPO(4). These mineralized protein-composite scaffolds were subsequently seeded with human bone marrow stem cells (hMSC) and cultured in vitro for 6 weeks under osteogenic conditions with or without BMP-2. The extent of osteoconductivity was assessed by cell numbers, alkaline phosphatase and calcium deposition, along with immunohistochemistry for bone-related outcomes. The results suggest increased osteoconductive outcomes with an increase in initial content of apatite and BMP-2 in the silk fibroin porous scaffolds. The premineralization of these highly porous silk fibroin protein scaffolds provided enhanced outcomes for the bone tissue engineering.

Structure and thermal behavior of a cellulose I-ethylenediamine complex.

We prepared highly crystalline samples of a cellulose I-ethylenediamine (EDA) complex by immersing oriented films of algal (Cladophora) cellulose microcrystals in EDA at room temperature for a few days. The unit-cell parameters were determined to be a = 0.455, b = 1.133, and c = 1.037 nm (fiber repeat) and gamma = 94.02 degrees. The space group was P2(1). On the basis of unit cell, density, and thermogravimetry analyses, the asymmetric unit is composed of one anhydrous glucose residue and one EDA molecule. The chemical and thermal stabilities of the cellulose I-EDA complex were also investigated by the use of X-ray diffraction. When the cellulose I-EDA complex was immersed in methanol or water at room temperature, cellulose III I or I beta was obtained, respectively. However, immersion in a nonpolar solvent such as toluene did not affect the crystal structure of the complex. The cellulose I-EDA complex was stable up to a temperature of approximately 130 degrees C, whereas the boiling point of EDA is 117 degrees C. This thermal stability of the complex is probably caused by intermolecular hydrogen bonds between EDA molecules and cellulose. When heated above 150 degrees C, the cellulose I-EDA complex decomposed into cellulose I beta.

Cellulose hydrogel with tunable shape and mechanical properties: From rigid cylinder to soft scaffold.

Cellulose hydrogel from aqueous solution of lithium bromide demonstrated excellent tunability of mechanical property and shape. A series of compression tests showed that cellulose hydrogel covered a wide range of mechanical property, where the compressive Young's modulus was controllable from 30 kPa to 1.3 MPa by changing the initial concentration of cellulose solution. Meanwhile, the diameter of the building block of gel, namely nano-fibrous cellulose, was constant at 15-20 nm irrelevant of the initial concentration of cellulose solution. Moreover, thanks to the biocompatibility of cellulose, the cultivation of cartilage tissue was successful in the micro-porous sponge-like cellulose hydrogel prepared by salt-leaching process. These findings show that this environmentally-benign versatile gel offers a new substrate for the biomaterial-based nanomaterial in biomedical applications.

Activation of crystalline cellulose to cellulose III(I) results in efficient hydrolysis by cellobiohydrolase.

The crystalline polymorphic form of cellulose (cellulose I(alpha)-rich) of the green alga, Cladophora, was converted into cellulose III(I) and I(beta) by supercritical ammonium and hydrothermal treatments, respectively, and the hydrolytic rate and the adsorption of Trichoderma viride cellobiohydrolase I (Cel7A) on these products were evaluated by a novel analysis based on the surface density of the enzyme. Cellobiose production from cellulose III(I) was more than 5 times higher than that from cellulose I. However, the amount of enzyme adsorbed on cellulose III(I) was less than twice that on cellulose I, and the specific activity of the adsorbed enzyme for cellulose III(I) was more than 3 times higher than that for cellulose I. When cellulose III(I) was converted into cellulose I(beta) by hydrothermal treatment, cellobiose production was dramatically decreased, although no significant change was observed in enzyme adsorption. This clearly indicates that the enhanced hydrolysis of cellulose III(I) is related to the structure of the crystalline polymorph. Thus, supercritical ammonium treatment activates crystalline cellulose for hydrolysis by cellobiohydrolase.

Protein adsorption of dialdehyde cellulose-crosslinked chitosan with high amino group contents.

Crosslinked chitosan was prepared by Schiff base formation between the aldehyde groups of dialdehyde cellulose (DAC) and the amino groups of chitosan and a subsequent reduction. DAC was obtained through periodate oxidation of cellulose and solubilization in hot water at 100°C for 1h. Three grades of DAC-crosslinked chitosan were prepared by adding various amounts DAC. The degrees of crosslinking as determined by amino group content were 3.8, 8.3, and 12.1%, respectively. DAC-crosslinked chitosan showed higher stability in the pH 2-9 range and no cytotoxicity was identified over the course of a 21-day long-term stability test. Also, DAC-crosslinked chitosan showed remarkably high bovine serum albumin (BSA) adsorption capacity at pH 5.5 as a result of the increased amino group content, due to the reaction between DAC and chitosan molecular chains occurring at multiple points even though DAC-crosslinked chitosan showed a lower degree of crosslinking.