Solid-state 13C NMR study of na-cellulose complexes.

The interaction of microcrystalline cellulose from cotton and aqueous sodium hydroxide was investigated by 13C NMR solid-state spectroscopy as a function of temperature and sodium hydroxide concentration. When the concentration of NaOH was increased, the initial cellulose spectrum was replaced successively by that of Na-cellulose I followed by that of Na-cellulose II. In Na-cellulose I, each carbon atom occurred as a singlet, thus implying that one glucosyl moiety was the independent magnetic residue in the structure of this allomorph. In addition, the occurrence of the C6 near 62 ppm is an indication of a gt conformation for the hydroxymethyl group of Na-cellulose I. In Na-cellulose II, the analysis of the resonances of C1 and C6 points toward a structure based on a cellotriosyl moiety as the independent magnetic residue, in agreement with the established X-ray analysis that has shown that for this allomorph, the fiber repeat was also that of a cellotriosyl residue. For Na-cellulose II, the occurrence of the C6 in the 60 ppm region indicates an overall gg conformation for the hydroxymethyl groups. A comparison of the spectra recorded at 268 K and at room temperature confirms the stronger interaction of NaOH with cellulose when the temperature is lowered. In the Q region, corresponding to NaOH concentrations of around 9% and temperatures below 277 K, most of the sample was dissolved and no specific solid-state 13C NMR spectrum could be recorded, except for that of a small fraction of undissolved cellulose I. The same experiment run on a wood pulp sample leads to a new spectrum, with spectral characteristics different from those of Na-cellulose I and Na-cellulose II. This new spectrum is assigned to the Q phase, which appears to result from topological constraints that are present in whole wood pulp fibers but not in microcrystalline cellulose. A spectrum recorded for samples in the Na-cellulose III conditions resembled that of Na-cellulose II but of lower resolution. Similarly, a spectrum of a sample of Na-cellulose IV was identical to that of hydrated cellulose II. These observations have allowed us to propose a simplified phase diagram of the cellulose/NaOH system in terms of temperature and NaOH concentration. This diagram, which is simpler than the one deduced from X-ray analysis, consists of only four different regions partially overlapping.

Single crystals of V-amylose complexed with alpha-naphthol.

Lamellar square single crystals of V-amylose were obtained by adding alpha-naphthol to metastable dilute aqueous solutions of synthetic amylose chains with an average degree of polymerization of 100. The morphology and structure of the crystals were studied using low-dose transmission electron microscopy including high-resolution imaging, as well as electron and X-ray diffraction. The crystals are crystallized in a tetragonal P4(1)2(1)2 or P4(3)2(1)2 space group with unit cell parameters, calculated from X-ray diffraction data, a = b = 2.2844 nm (+/-0.0005) and c = 0.7806 nm (+/-0.001), implying the presence of two amylose chains per unit cell. High-resolution lattice images of the crystals confirmed that the amylose chains were crystallized as 8-fold helices corresponding to the repeat of four maltosyl units.

Morphological and structural aspects of the giant starch granules from Phajus grandifolius.

The morphology and structure of giant starch granules from the pseudo-bulbs of Phajus grandifolius were investigated, using a number of microscopy techniques together with synchrotron radiation microdiffration analysis. Most of the granules, which had sizes between 100 and 200 microm, occurred as ogival particles with the hilum or proximal end located at the apex of the granules. A small percentage of granules held a protuberance extending orthogonally to the underlying parent granule. Growth rings were observed in all granules: strongly curved close to the hilum, but planar toward the distal end of the granules or in the protuberances. Specific mechanical disruption followed by enzymatic digestion revealed the susceptibility of the disorganized parts of the growth rings, which were preferentially carved away during the digestion, leaving behind the better-organized domains. Microdiffraction analysis achieved with synchrotron radiation revealed the crystalline features of the granules and provided orientation maps of the amylopectin molecules in the various parts of the granules. In simple ogival granules the amylopectin molecules were uniformly oriented with their axes running from the hilum toward the distal end of the granule. In granules with a protuberance, the axes of the amylopectin molecules kept their direction in the parent granule, but took an orthogonal direction in the protuberance. The occurrence of these morphological and structural features is tentatively correlated with the mode of growth of these granules.

Crystal structure and hydrogen bonding system in cellulose I(alpha) from synchrotron X-ray and neutron fiber diffraction.

The crystal and molecular structure, together with the hydrogen-bonding system in cellulose I(alpha), has been determined using atomic-resolution synchrotron and neutron diffraction data recorded from oriented fibrous samples prepared by aligning cellulose microcrystals from the cell wall of the freshwater alga Glaucocystis nostochinearum. The X-ray data were used to determine the C and O atom positions. The resulting structure is a one-chain triclinic unit cell with all glucosyl linkages and hydroxymethyl groups (tg) identical. However, adjacent sugar rings alternate in conformation giving the chain a cellobiosyl repeat. The chains organize in sheets packed in a "parallel-up" fashion. The positions of hydrogen atoms involved in hydrogen-bonding were determined from a Fourier-difference analysis using neutron diffraction data collected from hydrogenated and deuterated samples. The differences between the structure and hydrogen-bonding reported here for cellulose I(alpha) and previously for cellulose I(beta) provide potential explanations for the solid-state conversion of I(alpha) --> I(beta) and for the occurrence of two crystal phases in naturally occurring cellulose.

Fast intracrystalline hydration of beta-chitin revealed by combined microdrop generation and on-line synchrotron radiation microdiffraction.

Water microdrops of about 50 microm in diameter, generated by an ink-jet system, have been used to hydrate fragments of Pogonophora tubes. In situ X-ray microdiffraction with a beam size of 10 microm was used to follow the structural transformations that affected the crystalline beta-chitin part of the specimens. Starting from anhydrous chitin, the formation of a full beta-chitin dihydrate was observed within about 90 s. A disordered intermediate phase with variable d-spacing that could be due to a mixture of anhydrous and hydrated beta-chitin layers was also detected.

Structural and morphological diversity of (1-->3)-beta-D-glucans synthesized in vitro by enzymes from Saprolegnia monoïca. Comparison with a corresponding in vitro product from blackberry (Rubus fruticosus).

Detergent extracts of microsomal fractions from Saprolegnia monoïca and blackberry (Rubus fruticosus) cells were incubated with UDP-glucose to yield in vitro (1-->3)-beta-d-glucans. The insoluble products were analyzed by conventional and cryo transmission electron microscopy, X-ray diffraction, and (13)C CP/MAS NMR, and their molecular weights were determined by light scattering experiments. All the products were microfibrillar, but for the detergent extracts from S. monoïca, important morphological differences were observed when the pH of the synthesizing medium was modified. At pH 6, the product had a weight average degree of polymerization () exceeding 20 000 and consisted of endless ribbon-like microfibrils. The microfibrils obtained at pH 9 had a length of only 200-300 nm, and their was approximately 5000. Of all the in vitro (1-->3)-beta-d-glucans, the one from R. fruticosus had the shortest length and the smallest. Crystallographic and spectroscopic data showed that the three in vitro samples consisted of triple helices of (1-->3)-beta-d-glucans and contained substantial amounts of water molecules in their structure, the shortest microfibrils being more hydrated. In addition, the long microfibrils from S. monoïca synthesized at pH 6 were more resistant toward the action of an endo-(1-->3)-beta-d-glucanase than the shorter ones obtained at pH 9. These results are discussed in terms of molecular biosynthetic mechanisms of fungal and plant (1-->3)-beta-d-glucans, and in relation with the possible existence of several (1-->3)-beta-d-glucan synthases in a given organism. The interpretation and discussion of these observations integrate the current knowledge of the structure and function of (1-->3)-beta-d-glucans.

Fluorescent cellulose microfibrils as substrate for the detection of cellulase activity.

To devise a sensitive cellulase assay based on substrates having most of the physical characteristics of native cellulose, 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) was used as a grafting agent to prepare suspensions of fluorescent microfibrils from bacterial cellulose. These suspensions were digested by a series of commercially relevant cellulases from Humicola insolens origin: cloned Cel6B and Cel 45A as well as crude H. insolens complex. The digestion induced the release of fluorescent cellodextrins as well as reducing sugars. After adequate centrifugation, these soluble products were analyzed as a function of grafting content, digestion time, and cellulase characteristics. The resulting data allowed the grafting conditions to be optimized in order to maximize the quantity of soluble products and therefore to increase the sensitivity of the detection. A comparison between the amount of released fluorescence and that of released reducing sugar allowed the differentiation between processive exo and endo cellulase activities. The casting of films of DTAF-grafted microfibrils at the bottom of the microwell titer plates also led to sensitive cellulase detection. As these films kept their integrity and remained firmly glued to the well bottom during the digestion time, they are tailored made for a full automation of the cellulases testing.

Crystal structure and hydrogen-bonding system in cellulose Ibeta from synchrotron X-ray and neutron fiber diffraction.

The crystal and molecular structure together with the hydrogen-bonding system in cellulose Ibeta has been determined using synchrotron and neutron diffraction data recorded from oriented fibrous samples prepared by aligning cellulose microcrystals from tunicin. These samples diffracted both synchrotron X-rays and neutrons to better than 1A resolution (>300 unique reflections; P2(1)). The X-ray data were used to determine the C and O atom positions. The resulting structure consisted of two parallel chains having slightly different conformations and organized in sheets packed in a "parallel-up" fashion, with all hydroxymethyl groups adopting the tg conformation. The positions of hydrogen atoms involved in hydrogen-bonding were determined from a Fourier-difference analysis using neutron diffraction data collected from hydrogenated and deuterated samples. The hydrogen atoms involved in the intramolecular O3...O5 hydrogen bonds have well-defined positions, whereas those corresponding to O2 and O6 covered a wider volume, indicative of multiple geometry with partial occupation. The observation of this disorder substantiates a recent infrared analysis and indicates that, despite their high crystallinity, crystals of cellulose Ibeta have an inherent disorganization of the intermolecular H-bond network that maintains the cellulose chains in sheets.

In vitro versus in vivo cellulose microfibrils from plant primary wall synthases: structural differences.

Detergent extracts of microsomal fractions from suspension cultured cells of Rubus fruticosus (blackberry) were tested for their ability to synthesize in vitro sizable quantities of cellulose from UDP-glucose. Both Brij 58 and taurocholate were effective and yielded a substantial percentage of cellulose microfibrils together with (1-->3)-beta-d-glucan (callose). The taurocholate extracts, which did not require the addition of Mg(2+), were the most efficient, yielding roughly 20% of cellulose. This cellulose was characterized after callose removal by methylation analysis, electron microscopy, and electron and x-ray synchrotron diffractions; its resistance toward the acid Updegraff reagent was also evaluated. The cellulose microfibrils synthesized in vitro had the same diameter as the endogenous microfibrils isolated from primary cell walls. Both polymers diffracted as cellulose IV(I), a disorganized form of cellulose I. Besides these similarities, the in vitro microfibrils had a higher perfection and crystallinity as well as a better resistance toward the Updegraff reagent. These differences can be attributed to the mode of synthesis of the in vitro microfibrils that are able to grow independently in a neighbor-free environment, as opposed to the cellulose in the parent cell walls where new microfibrils have to interweave with the already laid polymers, with the result of a number of structural defects.

The unique ribbon morphology of the major ampullate silk of spiders from the genus Loxosceles (recluse spiders).

The morphology of silk produced by recluse spiders (Loxosceles arizonica) was investigated by scanning electron microscopy, atomic force microscopy, and transmission electron microscopy. This silk consisted entirely of very long, thin ribbons of width 2-4 microm and thicknesses of no more than 40 nm. The correspondence in shape and dimension between the silk ribbons and the elongated aperture of the major ampullate spigot indicated that these ribbons were major ampullate silk. Selected area electron diffraction patterns from single ribbons were indexed with an orthorhombic unit cell (a = 9.43(2) A, b = 8.96(3) A, c = 6.96(1) A). This unit cell is in good agreement with that previously reported for synthetic poly(L-alanylglycine). Thus it is likely that the crystalline regions of the major ampullate silk of L. arizonica consist of an alternating glycine-L-alanine motif that has adopted a beta-sheet structure. The amino acid composition achieved with the silk of L. arizonica as well as that of L. laeta confirmed that the major amino acid constituents of this silk were glycine and L-alanine in nearly equal amounts. As it was noticed that the dry ribbons were highly electrostatic, it is suggested that the electrostatic interaction plays an important role in prey capture for Loxoseles.