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Biomedical subjects

H Chanzy

Publications and source records attributed to H Chanzy.

At least 19 recordsLinked to original sources
PubMed2001

Optimized mixtures of recombinant Humicola insolens cellulases for the biodegradation of crystalline cellulose.

The digestion of bacterial cellulose ribbons by ternary mixtures of enzymes consisting of recombinant cellulases (two cellobiohydrolases, Cel6A and Cel7A, and the endoglucanase Cel45A) from Humicola insolens was investigated over a wide range of mixture composition. The extent of digestion was followed by soluble sugar release (saccharification) analysis together with transmission electron microscopy (TEM) observations. It was found that the addition of minute quantities of Cel45A induced a spectacular increase in saccharification of the substrate with either Cel7A or the mixture of Cel6A and Cel7A. Conversely, only a moderate saccharification resulted from the mixing of Cel45A and Cel6A. This difference is believed to originate from (1) the occasional endo character of Cel6A and (2) the competition of Cel6A and Cel45A for the substrate sites that are sensitive to endo activity. Interestingly, the mixture of enzymes giving rise to the highest saccharification rate did not always correspond to mixtures of enzymes generating the highest synergy. TEM images revealed that the bacterial cellulose ribbons became at the same time cut and narrowed down under the action of an optimized mixture of the three enzymes.

Ascomycota↗
PubMed2001

X-ray structure of mercerized cellulose II at 1 a resolution.

A revised crystal structure for mercerized cellulose based on high-resolution synchrotron X-ray data collected from ramie fibers is reported (space group P2(1), a = 8.10(3) A, b = 9.03(3) A, c = 10.31(5) A, gamma = 117.10(5) degrees; 751 reflections in 304 composite spots; theta < 21.11 degrees; lambda = 0.7208 A; LALS refinement with d > 1.5 A, R' ' = 0.16; SHELX97 refinement with d > 1 A, R = 0.21). As with regenerated cellulose the crystal structure consists of antiparallel chains with different conformations but with the hydroxymethyl groups of both chains near the gt position. However, the conformation of the hydroxymethyl group of the center chain in the structure reported here differs significantly from the conformation in regenerated cellulose. This may be related to a large observed difference in the amount of hydroxymethyl group disorder: approximately 30% for regenerated cellulose and approximately 10% for mercerized cellulose.

Boehmeria↗
PubMed2001

Degradation of mannan I and II crystals by fungal endo-beta-1,4-mannanases and a beta-1,4-mannosidase studied with transmission electron microscopy.

We have used the endo-beta-1,4-mannanase from Trichoderma reesei (Tr Man5A), the endo-beta-1,4-mannanase from Aspergillus niger (An Man5A) and the exo-beta-1,4-mannosidase from A. niger (An Mnd2A) to follow the enzymatic degradation of mannan I and II crystals. The degradation process was studied by transmission electron microscopy and also followed by analysis of the released soluble reducing sugars. The mannan crystals were degraded by the endo-beta-1,4-mannanases and to a lesser extent by the exo-beta-1,4-mannosidase. The observed hydrolysis pattern on mannan I crystals is fully consistent with the current view of the molecular structure of these crystals. The molecular organization of the mannan chains in mannan II crystals is less clear and the digestion results give some further information about the ultrastructure of mannan II. In addition, insight is provided into the mode of the enzymatic attack on the crystals of mannan I and mannan II.

Aspergillus niger↗
PubMed2001

Biosynthesis of (1-->3)-beta-D-glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana.

The aim of this work was to develop a biochemical approach to study (1-->3)-beta-D-glucan (callose) biosynthesis using suspension cultures of Arabidopsis thaliana. Optimal conditions for in vitro synthesis of callose corresponded to an assay mixture containing 50 mM Mops buffer, pH 6.8, 1 mM UDP-glucose, 8 mM Ca2+ and 20 mM cellobiose. The enzyme was Ca2+-dependent, and addition of Mg2+ to the reaction mixture did not favour cellulose biosynthesis. Enzyme kinetics suggested the existence of positive homotropic cooperativity of (1-->3)-beta-D-glucan synthase for the substrate UDP-glucose, in agreement with the hypothesis that callose synthase consists of a multimeric complex containing several catalytic subunits. Detergents belonging to different families were tested for their ability to extract and preserve membrane-bound (1-->3)-beta-D-glucan synthase activity. Cryo-transmission electron microscopy experiments showed that n-octyl-beta-D-glucopyranoside allowed the production of micelle-like structures, whereas vesicles were obtained with Chaps and Zwittergent 3-12. The morphology and size of the (1-->3)-beta-D-glucans synthesized in vitro by fractions obtained with different detergents were affected by the nature of the detergent tested. These data suggest that the general organization of the glucan synthase complexes and the properties of the in vitro products are influenced by the detergent used for protein extraction. The reaction products synthesized by different detergent extracts were characterized by infrared spectroscopy, methylation analysis, 13C-NMR spectroscopy, electron microscopy and X-ray diffraction. These products were identified as linear (1-->3)-beta-D-glucans having a degree of polymerization higher than 100, a microfibrillar structure, and a low degree of crystallinity.

Arabidopsis↗
PubMed2000

Structural data on the intra-crystalline swelling of beta-chitin.

The intra-crystalline swelling of the highly crystalline beta-chitin from Tevnia jerichonana was investigated by X-ray crystallography and Fourier transform infrared (FTIR) spectroscopy, using hydrogenated and deuterated hydrochloric acids as swelling agents. Three levels of swelling were identified that could be defined as inter- and intra-sheet swelling. A moderate and reversible swelling in water and methanol gave crystalline beta-chitin cystallosolvates, namely dihydrate and methanolate, respectively. In these, an inter-sheet swelling was observed, corresponding to an expansion of only the b parameter of the unit cell of beta-chitin. Under these swelling conditions, the use of deuterated reagents had no effect on the amide N&z.sbnd;H&ctdot;O&z.dbnd6;C hydrogen bonds that hold the structure of beta-chitin together, but only induced a partial and reversible deuteration of the chitin hydroxymethyl groups. A more severe swelling - but still reversible - occurred with 6 N HCl or DCl, which converted the crystals of beta-chitin into a paracrystalline gel-like product resulting from inter-sheet+intra-sheet swelling. With this acid strength, the deuteration pattern indicated that a fraction of the amide hydrogen bonds was broken and became susceptible to an irreversible deuteration. A very severe and irreversible swelling occurred with 8 N HCl or DCl. In that case, the inter- and intra-sheet swelling was extensive to the point where all memory of the parallel-chain beta-chitin was lost. In addition, this swelling was accompanied by a drastic and rapid depolymerization. The treatment with 8 N HCl led invariably to crystalline alpha-chitin when the samples were neutralized.

Animals↗
PubMed2000

Digestion of single crystals of mannan I by an endo-mannanase from Trichoderma reesei.

The enzymatic degradation of single crystals of mannan I with the catalytic core domain of a beta-mannanase (EC 3.2.1.78 or Man5A) from Trichoderma reesei was investigated by transmission electron microscopy and electron diffraction. The enzyme attack took place at the edge of the crystals and progressed towards their centres. Quite remarkably the crystalline integrity of the crystals was preserved almost to the end of the digestion process. This behaviour is consistent with an endo-mechanism, where the enzyme interacts with the accessible mannan chains located at the crystal periphery and cleaves one mannan molecule at a time. The endo mode of digestion of the crystals was confirmed by an analysis of the soluble degradation products.

Chromatography, Gel↗
PubMed2000

Imaging the enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolase Cel7A.

Dispersed cellulose ribbons from bacterial cellulose were subjected to digestion with cloned Cel7A (cellobiohydrolase [CBH] I) and Cel6A (CBH II) from Humicola insolens either alone or in a mixture and in the presence of an excess of beta-glucosidase. Both Cel7A and Cel6A were effective in partially converting the ribbons into soluble sugars, Cel7A being more active than Cel6A. In combination, these enzymes showed substantial synergy culminating with a molar ratio of approximately two-thirds Cel6A and one-third Cel7A. Ultrastructural transmission electron microscopy (TEM) observations indicated that Cel7A induced a thinning of the cellulose ribbons, whereas Cel6A cut the ribbons into shorter elements, indicating an endo type of action. These observations, together with the examination of the digestion kinetics, indicate that Cel6A can be classified as an endo-processive enzyme, whereas Cel7A is essentially a processive enzyme. Thus, the synergy resulting from the mixing of Cel6A and Cel7A can be explained by the partial endo character of Cel6A. A preparation of bacterial cellulose ribbons appears to be an appropriate substrate, superior to Valonia or bacterial cellulose microcrystals, to visualize directly by TEM the endo-processivity of an enzyme such as Cel6A.

Aspergillus niger↗
PubMed1999

High resolution neutron fibre diffraction data on hydrogenated and deuterated cellulose.

High-resolution fibre neutron diffraction data were recorded from cellulose samples on a D19 diffractometer at the Institut Laue-Langevin (Grenoble). Highly crystalline cellulose I samples from Cladophora (cellulose I alpha + I beta) or Halocynthia (cellulose I beta) origin were prepared in the form of oriented films. Samples were studied in a hydrogenated form and in a hydrogen-deuterium exchanged deuterated form corresponding to all OH moieties being replaced by ODs. These samples, which diffracted to a resolution of around 0.9 A, gave diffraction diagrams consisting of several hundred independent diffraction spots. Crystalline cellulose II fibres resulting from the mercerization of flax were also studied in a hydrogenated form using NaOH/H2O as mercerizing medium and in a deuterated form using NaOD/D2O. Both of these samples diffracted to around 1.2 A, giving fibre diffraction diagrams slightly less resolved than those of cellulose I, but still consisting of more than one hundred independent diffraction spots. For cellulose I as well as for cellulose II, significant differences between the hydrogenated and deuterated patterns were observed and recorded. These new data should lead to improved structures for cellulose and direct identification of the position of hydrogen atoms involved in hydrogen bonding.

Cellulose↗
PubMed1999

Digestion of crystalline cellulose substrates by the clostridium thermocellum cellulosome: structural and morphological aspects.

The action of cellulosomes from Clostridium thermocellum on model cellulose microfibrils from Acetobacter xylinum and cellulose microcrystals from Valonia ventricosa was investigated. The biodegradation of these substrates was followed by transmission electron microscopy, Fourier-transform IR spectroscopy and X-ray diffraction analysis, as a function of the extent of degradation. The cellulosomes were very effective in catalysing the complete digestion of bacterial cellulose, but the total degradation of Valonia microcrystals was achieved more slowly. Ultrastructural observations during the digestion process suggested that the rapid degradation of bacterial cellulose was the result of a very efficient synergistic action of the various enzymic components that are attached to the scaffolding protein of the cellulosomes. The degraded Valonia sample assumed various shapes, ranging from thinned-down microcrystals to crystals where one end was pointed and the other intact. This complexity may be correlated with the multi-enzyme content of the cellulosomes and possibly to a diversity of the cellulosome composition within a given batch. Another aspect of the digestion of model celluloses by cellulosomes is the relative invariability of their crystallinity, together with their Ialpha/Ibeta composition throughout the degradation process. Comparison of the action of cellulosomes with that of fungal enzymes indicated that the degradation of cellulose crystals by cellulosomes occurred with only limited levels of processivity, in contrast with the observations reported for fungal enzymes. The findings were consistent with a mechanism whereby initial attack by a cellulosome of an individual cellulose crystal results in its 'commitment' towards complete degradation.

Acetobacter↗
PubMed1999

Ultrastructural aspects of phytoglycogen from cryo-transmission electron microscopy and quasi-elastic light scattering data.

Phytoglycogen particles extracted from the sugary maize mutant su 1 and dispersed in water were studied using transmission electron microscopy (TEM) and light scattering. Dried specimens were either negatively stained with uranyl acetate or shadowed with W/Ta. Frozen-hydrated unstained particles embedded in a thin film of vitreous ice were also observed using cryo-TEM. The particles exhibited a spheroidal shape, with a diameter ranging from 30 to 100 nm. Some of them presented a multilobular morphology and appeared to be formed by smaller subunits, 20-30 nm in diameter, resembling the described beta-particles for animal glycogen. The diameter of stained and ice-embedded particles was measured from electron micrographs. The corresponding size distribution histograms showed that the average weight diameter of ice-embedded particles was higher than that of stained ones. In the latter case, a shrinkage of the particle was believed to occur during the drying process. Light scattering experiments confirmed the diameter of ice-embedded particles and indicated that they could be considered as uniformly dense spheroidal objects.

Cryoelectron Microscopy↗
PubMed1998

Cassia spectabilis DC seed galactomannan: structural, crystallographical and rheological studies.

The seeds of Cassia spectabilis DC (family: Leguminoseae), an Indian fast growing spreading tree, contain about 40% of endosperm and possess the characteristics of becoming a potential source of commercial gum. The purified galactomannan shows Mw 1.1 x 10(6), intrinsic viscosity [eta] 615ml/g with k' = 1.706 x 10(-1), and a mannose to galactose ration of 2.65. The hydrolysis of the fully methylated polysaccharide reveals clearly the expected structure of legume galactomannans. The orthorhombic lattice constants of the hydrated gums are as follows: a = 9.12 A, b = 25.63 A and c = 10.28 A. The results of X-ray fiber studies show that the b dimension of the unit cell is very sensitive to relative humidity (RH), galactose substitution and orientation of the films. The probable space group symmetry of the unit cell is P2(1)2(1)2. Rheological studies of the galactomannan have shown that the transition from semi-dilute to dilute regime occurs at a critical concentration Cc* = 2.75. The slope of the log-log plot of specific viscosity versus C at zero shear rate is 5.87 in the more concentrated regime. The viscoelastic and critical shear rate behavior indicate the characteristics of a coil polymer. The large dependence of the viscosity on the coil overlap parameter is probably due to polymer-polymer interactions and peculiarity of the galactose distribution along the chain. Above 20 g/L concentration, the rheological behavior of the gum is like the one of a weak-gel.

Cassia↗
PubMed1998

Cellulose, cellulases and cellulosomes.

The structural complexity and rigidity of cellulosic substrates have given rise to a phenomenal diversity of degradative enzymes--the cellulases. Cellulolytic microorganisms produce a wide variety of different catalytic and noncatalytic enzyme modules, which form the cellulases and act synergistically on their substrate. In some microbes, several types of cellulases are organized into an elaborate multifunctional supramolecular complex, known as the cellulosome. A combination of molecular genetic, biochemical, chemical, crystallographic and microscopic techniques are paving the way for new insights into both the structure of cellulose and the mechanisms of its hydrolysis.

Bacteria↗
PubMed1996

The Cellulose System in the Cell Wall of Micrasterias

The cellulose system of the cell wall of Micrasterias denticulata and Micrasterias rotata was analyzed by diffraction contrast transmission electron microscopy, electron diffraction, and X-ray analysis. The studies, achieved on disencrusted cell ghosts, confirmed that the cellulose microfibrils occurred in crisscrossed bands consisting of a number of parallel ribbon-like microfibrils. The individual microfibrils had thicknesses of 5 nm for a width of around 20 nm, but in some instances, two or three microfibrils merged into one another to yield larger monocrystalline domains reaching up to 60 nm in lateral size. The orientation of the cellulose of Micrasterias is very unusual, as it was found that in the cell wall, the equatorial crystallographic planes of cellulose having a d-spacing of 0.60 nm [(11;0) in the Ibeta cellulose unit cell defined by Sugiyama et al., 1991, Macromolecules 24, 4168-4175] were oriented perpendicular to the cell wall surface. Up to now, such orientation has been found only in Spirogyra, another member of the Zygnemataceae group. The unusual structure of the secondary wall cellulose of Micrasterias may be tentatively correlated with the unique organization of the terminal complexes, which in this alga occur as hexagonal arrays of rosettes.

Journal Article↗
PubMed1996

Single crystals of V amylose complexed with glycerol.

Lamellar single crystals of amylose V glycerol were grown at 100 degrees C by evaporating water from solutions of amylose in aqueous glycerol. The crystals which were square, with lateral dimensions of several micrometers, gave sharp electron diffraction patterns presenting an orthorhombic symmetry with a probable space group P2(1)2(1)2(1) and unit cell parameters: a = 1.93 +/- 0.01 nm, b = 1.86 +/- 0.01 nm and c (fiber axis) = 0.83 +/- 0.03 nm. The amylose Vglycerol crystal structure which is isomorphous to that of VDMSO consists of an antiparallel pair of left-handed six-fold amylose helices centered on the two-fold screw axes of the cell and probably separated by glycerol molecules. This packing mode is confirmed by de-solvation experiments where the Vglycerol amylose crystals, annealed in ethanol, could be converted into VH amylose without losing their external appearance. The Vglycerol amylose crystals could be seeded by cellulose microfibrils to yield a shish-kebab structure where the amylose crystalline lamellae grew perpendicular to the microfibril directions.

Amylases↗
PubMed1996

Single crystals of inulin.

Lamellar crystals of inulin were grown by crystallizing sharp fractions of low molecular weight inulin from dilute aqueous ethanol solutions. The crystals were analyzed using three-dimensional electron diffraction and X-ray powder diagrams. Two crystalline polymorphs were observed, depending on the hydration conditions: a hydrated form which indexed on an orthorhombic unit cell, with space group P2(1)2(1)2(1) and with cell dimensions of a = 1.670 nm, b = 0.980 nm and c (chain axis) = 1.47 nm, together with a pseudo-hexagonal semi-hydrated form with unit cell parameters a = 1.670 nm, b = 0.965 nm and c (chain axis) = 1.44 nm. These parameters, together with the density data, indicate that inulin crystallizes along a pseudo-hexagonal six-fold symmetry with an advance per monomer of 0.24 nm. The difference between the hydrated and the semi-hydrated unit cells does not seem to correspond to any change in the conformation of inulin, but rather to a variation in water content.

Carbohydrate Conformation↗
PubMed1995

The crystal structure of methyl beta-cellotrioside monohydrate 0.25 ethanolate and its relationship to cellulose II.

The crystal structure of methyl beta-cellotrioside (methyl O-beta-D-glucopyranosyl-(1-->4)-O-beta-d_guycopyranosyl-(1-->4)-be ta-D- glucopyranoside) complexed with water and ethanol, C19H34O16. H2O.0.25[C2H6O] was determined by combining Cu K alpha X-ray and synchrotron data collected at room temperature. The crystals have the monoclinic space group P21 with Z = 8 and unit cell parameters a = 7.9978(11), b = 76.38(4), c = 8.9908(6) A and beta = 116.40(1) degree. The structure, which was solved by direct methods and refined to a final R-factor of 0.067, contains four independent molecules of methyl beta-cellotrioside with an extended conformation. They are arranged parallel to the long b axis of the unit cell, and organized in two pairs of antiparallel molecules. Each beta-D-glucopyranosyl residue of the four independent molecules is in the 4C1 pyranose conformation, and each (O-6) primary hydroxyl group has the gt conformation. The crystal structure of methyl beta-cellotrioside has many points in common with that of cellotetraose hemihydrate as well as with the structure of cellulose II. Thus, it is likely that the precise atomic coordinates obtained in this study can be directly transposed to give an improved structure for cellulose II where, in particular, only the gt conformation would be present at the primary hydroxyl groups of both polysaccharide chains.

Carbohydrate Conformation↗
PubMed1994

An electron microscope and electron diffraction study of the effect of calcofluor and congo red on the biosynthesis of chitin in vitro.

The structure of chitin made in vitro by chitin synthetase was studied by electron microscopy and electron diffraction. Two different forms of chitin synthetase from the fungus Mucor rouxii were tested: chitosomes and 16 S particles. The long chitin fibrils produced by chitosomes had a high degree of crystallinity as revealed by electron diffraction. Specimen regions made of largely parallel microfibril bundles produced distinct fiber diagrams, an indication that the chitin chains were aligned along the fibril axis. Microdiffractometry of the shorter chitin crystals synthesized by 16 S particles also showed a similar alignment of chitin chains. Calcofluor and Congo red were powerful inhibitors of chitin synthetase and had profound effects on the color, macroscopic texture, electron microscopic morphology, and crystal structure of the biosynthesized chitin. Chitin made in the presence of Congo red had a bright red color; the one made in the presence of Calcofluor was strongly fluorescent and had a distinctly blue hue when illuminated by daylight. The dyes were tightly bound to the chitin and could not be removed by washing with water or ethanol. At low dye concentration, a mixture of two kinds of crystals was produced by 16 S particles: some were of the same dimensions as those made in the absence of dyes, but others were much thinner. At high dye concentration, there were only thin crystals. With increasing concentrations of Calcofluor or Congo red, the typical electron diffraction reflections of alpha-chitin, particularly the strong equatorial band at 0.466 nm became fainter and a new additional reflection centered at 0.40 nm arose as the dominant feature of the patterns. We regard the gel-like material, synthesized at highly inhibitory dye concentrations, as an apposition complex where the dye does not form part of the crystal structure of chitin and the bulk of the complex consists of molecular stacks of dye associated with nascent chitin chains or narrow chitin microfibrils.

Benzenesulfonates↗