Polysaccharides, tightly bound to cellulose in cell wall of flax bast fibre: Isolation and identification

Part of matrix polymers of flax bast fibre cell wall is tightly bound to cellulose and can not be extracted by conventional methods. To analyze these polymers, the residue, remaining after cell wall treatment with chelators and alkali, was dissolved in solution of lithium chloride in N,N-dimethylacetamide. Cellulose was precipitated by water and completely degraded by cellulase, giving the possibility to separate matrix polysaccharides, which remained in polymeric form. The obtained polymers were fractionated by gel permeation chromatography and characterized by monosaccharide analysis, staining with LM5 antibody and Yariv reagent, ¹H and ¹³C NMR. The total yield of the polysaccharides that are tightly bound to cellulose in flax fibre, was 4.6%. The major fractions (molecular mass 100–400 kDa) were composed of galactose, accompanied by two other significant monomers, GalA and Rha, with the ratio 1.1–1.4. Composition and structure of the cellulose bound galactan permit to consider it as fragment of the high-molecular mass (2000 kDa) galactan, synthesized by the developing fibres, while forming the secondary cell wall of gelatinous type.     

Biosynthesis of C-labeled branched polysaccharides by pestalotiopsis from C-labeled glucoses and the mechanism of formation

Biosynthesis of branched glucan by Pestalotiopsis from media containing D-(1-¹³C)glucose, D-(2-¹³C)glucose, D-(4-¹³C)glucose, D-(6-¹³C)glucose or a mixture of D-(1-¹³C)glucose and D-(2-¹³C)glucose was carried out to elucidate biosynthetic mechanism of branched polysaccharides. ¹³C NMR spectra of the labeled polysaccharides were determined and assigned. Analysis of ¹³C NMR spectra of glucitol acetates obtained from hydrolysates of the labeled branched polysaccharides indicated that transfer of labeling from C-1 to C-3 and C-6 carbons, from C-2 to C-1, C-3 and C-5 carbons, and from C-6 to C-1 carbon. From the results the percentages of routes via which the polysaccharide is biosynthesized are estimated. They show that the biosynthesis of the polysaccharide via the Embden-Meyerhof pathway and that from lipids and proteins are more active, and the pentose cycle is less active, than in the biosynthesis of cellulose and curdlan. As for the results, labeling at C-6 carbon in the branched polysaccharide cultured from D-(6-¹³C)glucose was low, compared to that of cellulose and curdlan.     

Analysis of the biosynthetic process of cellulose and curdlan using C-labeled glucoses

In order to elucidate the biosynthetic process of cellulose and curdlan, ¹³C-labeled polysaccharides were biosynthesized by Acetobacter xylinum (IFO 13693) and Agrobacterium sp. (ATCC 31749), from culture media containing -(1-¹³C)glucose, -(2-¹³C)glucose, -(4-¹³C)glucose, or -(6-¹³C)glucose as the carbon source, and their structures were determined by ¹³C NMR spectroscopy. The labeling was mainly found in the original position, indicating direct polymerization of introduced glucoses. In addition, the transfer of labeling from C-2 to C-1, C-3 and C-5, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 was found in celluloses. In curdlan, the transfer of labeling from C-1 to C-3, from C-2 to C-1 and C-3, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 and C-3 was observed. From analysis of this labeling, the biosynthetic process of cellulose and curdlan was explained as involving six routes. The percentages of each route via which cellulose or curdlan is biosynthesized were estimated for upper (C-1 to C-3) and lower portions (C-4 to C-6) of glucosidic units in the polysaccharides. It is noted that very few polysaccharides are formed via the Embden-Meyerhof pathway. The lower half (C-4 to C-6) structure of introduced glucoses is well preserved in the polysaccharides.     

Structural studies of the glucuronic acid oligomers produced by Gluconacetobacter hansenii strain

Gluconacetobacter hansenii PJK, a cellulose producing bacterium recently isolated from the rotten apples, produced fair amounts of the water-soluble polysaccharides (WSPS). WSPS were studied for their monosaccharides composition after acid hydrolysis, which revealed that the hydrolysates consist only of one sugar, glucuronic acid. The structure of the WSPS was investigated using various spectroscopic techniques including FT-IR, MALDI-TOF MS and ¹H, and ¹³C NMR. These studies revealed that the product is a mixture of oligomers with the -glucuronic acid as building blocks. The possible structure of the major oligosaccharide in the mixture has been deduced.     

Distribution of carboxylate groups introduced into cotton linters by the TEMPO-mediated oxidation

The 2,2,6,6-tetramethylpiperidine-1-oxy radial (TEMPO)-mediated oxidation was applied to aqueous slurries of cotton linters. The water-insoluble fibrous fractions thus obtained in the yields of more than 78% were characterized by solid-state ¹³C-NMR, X-ray diffraction and scanning electron microscopic analyses for evaluation of distribution of carboxylate groups formed in the TEMPO-oxidized celluloses. The patterns of solid-state ¹³C-NMR spectra revealed that the oxidation occurred at the C6 primary hydroxyl groups of cellulose. X-ray diffraction and scanning electron microscopic analyses showed that such C6 oxidation took place at the surfaces of cellulose I crystallites without any oxidation at the C6 of inside cellulose I crystallites. Thus, carboxylate and aldehyde groups introduced into the TEMPO-oxidized celluloses are densely present on the surfaces of cellulose I crystallites. In addition, the obtained results revealed that the shoulder signal due to non-crystalline C6 carbons at about 63 ppm in solid-state ¹³C-NMR spectra of native celluloses is ascribed to those of surfaces of cellulose I crystallites or those of cellulose microfibrils.     

Behavior of cellulose production of Acetobacter xylinum in C-enriched cultivation media including movements on nematic ordered cellulose templates

Acetobacter xylinum NQ-5 (ATCC 53582) and AY-201 (ATCC 23769) cultivated in the Hestrin–Schramm medium containing d-glucose with a natural ¹³C-abundance of 1.1% were investigated regarding their cell division rates and the bacterial movements. Comparative studies were carried out in the presence of d-glucose-U-¹³C₆ with a uniform ¹³C-labeling of 99% as the sole carbon source. The bacterial growth rates in numbers were found to increase in the ¹³C-enriched media by about 13% for NQ-5 and 26% for AY-201, respectively. The movements of single cells caused by the inverse force of the secretion and deposition of cellulose nanofibers on nematic ordered cellulose (NOC) templates were investigated by real-time video analyzes using light microscopy. As a result, d-glucose-U-¹³C₆ reduced the speed of motion for both strains, which was an opposite trend to the above growth rates in the cell division. The deposited cellulose fibers were proved to be in a cellulose crystalline form by CP/MAS ¹³C NMR and NIR FT Raman spectroscopic measurements, although the biosynthesized and thereafter-deposited cellulose has a strong interaction with the surface of NOC.     

Solid-state CP/MAS C-NMR analysis of cellulose and tri-O-substituted cellulose ethers

Highly crystalline tri-O-substituted cellulose ethers having ethyl, n-propyl, n-butyl, allyl, and methallyl substituents were prepared from low-molecular weight cellulose (DP = 15). Influences of conformational and packing effects on solid-state ¹³C-NMR spectra were studied by using X-ray diffraction and solid- and solution-state ¹³C-NMR analyses of the cellulose derivatives. Unit-cell sizes tentatively obtained from X-ray diffraction patterns of the cellulose derivatives indicated that conformations and packing states of cellulose chains and alkyl chains of substituents were different between the derivatives. Solid- and solution-state ¹³C-NMR spectra of cellulose allomorphs, and effects of hydrogen bonds present in celluloses I, II, and III on chemical shifts of their solid-state ¹³C-NMR spectra were proposed.     

Isolation and characterisation of cellulose obtained by a two-stage treatment with organosolv and cyanamide activated hydrogen peroxide from wheat straw

Seven wheat straw cellulose preparations were isolated by a two-stage acidic organosolv treatment followed by cyanamide activated hydrogen peroxide bleaching. The effects of concentration of acetic and formic acids on the yield of cellulose and degradation of lignin and non-cellulose polysaccharides were investigated. Organic acids were more effective than alcohols on the degradation of lignin and hemicelluloses. Formic acid/acetic acid/water (30/60/10, v/v/v) system was found to be the most effective in delignification and removal of non-cellulose polysaccharides from the straw and did not have any undesirable effects on cellulose properties such as its intrinsic viscosity. In this case, the treatment removed 94.1% of the original lignin and 76.5% of the original hemicelluloses using 0.1% HCl as a catalyst at 85 °C for 4 h. Cyanamide activated hydrogen peroxide bleaching degraded substantial amounts of residual hemicelluloses and lignin, produced the cellulose samples having a relatively high purity. Under a best condition, a cellulose relatively free of lignin (0.7%) and with intrinsic viscosity of 393 ml g⁻¹ and favourable molar mass (213,940 g mol⁻¹) was obtained. Both unbleached and bleached cellulose preparations were further characterised by FT-IR and CP/MAS ¹³C NMR spectroscopy, and thermal stability.     

Synthesis of starch derivatives with labile cationic groups

A new route to starch derivatives bearing hydrolyzable cationic groups was developed. This was based on reacting starch compounds with betaine derivatives in the presence of diisopropylcarbodiimide and 4-dimethylaminopyridine as coupling reagents in an aprotic polar solvent. Water-soluble starches with a perfectly controlled degree of substitution were thus obtained which were fully characterized by infrared, ¹H and ¹³C spectroscopy and viscosity measurements. The cationic groups grafted on the polysaccharides are shown to hydrolyze slowly upon storage at room temperature.     

NMR study of paper

High quality antique sheets of paper have been characterized by ¹H NMR relaxations and ¹³C CP MAS spectra. Paper can be regarded as a bicomponent material made of cellulose and water plus a small amount of organic and inorganic impurities. Semicrystalline fibrous cellulose, rich in water, is present in the I and I_β forms. The amorphous cellulose, with a low water content, contains a higher amount of paramagnetic impurities and it is characterized by quite short ¹H spin-lattice relaxations and by ¹¹³C resonances with noticeable chemical shifts. Ad hoc tailored pulse sequences are able to produce ¹³C CP MAS spectra in which only the amorphous content of paper is clearly observed. It is shown that water is fully bound to the cellulose lattice. It also seems reasonable to formulate the hypothesis that a larger concentration of paramagnetic ions is located in the amorphous fraction of highly degraded paper compared with paper in good condition.     

红壤和风沙土添加不同化学结构有机碳对外源碳去向及累积贡献的影响

植物凋落物碳输入显著影响陆地生态系统土壤CO₂排放和有机碳(SOC)形成,然而,针对不同质地土壤添加不同化学结构外源碳去向依然不清楚。本研究将¹³C标记的葡萄糖、淀粉和纤维素添加至红壤和风沙土,比较2种质地土壤添加不同化学结构外源碳在土壤释放的CO₂、SOC、可溶性有机碳(DOC)和微生物生物量碳(MBC)库的净累积量、回收率及贡献比例上的差异。结果表明: 添加外源有机碳显著提高了CO₂、SOC、DOC和MBC的δ¹³C值,且随着外源有机碳化学结构复杂性的增加,CO₂的δ¹³C峰值依次延迟出现;外源有机碳种类、土壤类型和培养时间均显著改变外源碳去向及其在各碳库的贡献比例;在风沙土中,外源有机碳更多被矿化为CO₂,且CO₂库的外源碳净累积量和回收率大小依次为葡萄糖>淀粉>纤维素;红壤添加外源碳转变为SOC的累积量和回收率显著高于风沙土,且红壤SOC库的外源碳净累积量和回收率大小顺序也为葡萄糖>淀粉>纤维素。可见,外源有机碳化学结构和土壤质地共同调控外源碳去向及累积贡献。     

Acetone-soluble cellulose acetates prepared by one-step homogeneous acetylation of cornhusk cellulose in an ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl)

Cellulose samples extracted from cornhusk have been successfully acetylated in an ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl). Without using any catalyst, cornhusk cellulose acetates (CCAs) with the degree of substitution (DS) in a range from 2.16 to 2.63 were prepared in one-step. Under the homogeneous state, the DS value of CCAs was easily controlled by the acetylation time. The obtained CCAs were characterized by means of FT-IR, ¹³C NMR, DSC, TGA, and a mechanical test. The NMR results showed that the distribution of the acetyl moiety among the three OH groups of the anhydroglucose unit shows a preference at the C₆ position. The CCAs exhibited good solubility in some organic solvents, such as acetone and DMSO. The cast CCA films from their acetone solutions had good mechanical properties. At the end of each acetylation of cornhusk cellulose, the ionic liquid AmimCl could be effectively recovered. Therefore, this study presents a promising approach and “green process” to make use of crop by-products.     

Application of CP-MAS and liquid-like solid-state NMR experiments for the study of the ripening-associated cell wall changes in tomato

¹³C and ¹H NMR spectra of an ethanol insoluble material (EIM) prepared from the pericarp of mature-green (MG) and red-ripe (RR) tomato fruits were acquired in ‘liquid-like’ and cross-polarisation with dipolar decoupling and magic angle spinning (CPMAS) conditions using the same triple resonance probe. Such a strategy allowed acquisitions of various NMR experiments aimed at detecting compositional differences as well as distinguishing differences in molecular mobility for various constituent polysaccharides related with the two ripening stages. Increase of the proton dipolar decoupling power levels from 3 to 50–55 kHz during single pulse ¹³C acquisition, led to more intense signals for pectic and hemicellulosic polysaccharides. This behaviour was interpreted as reflecting motional restrictions of these polysaccharides inside the porous cell wall network. Measurements of the proton rotating frame relaxation times T_1ρ in the ‘liquid-like’ conditions and of the proton transverse relaxation times T₂ from CPMAS spectra, revealed changes in mobilities for some pectic polysaccharides in relation with ripening, particularly for the H1 and H5 protons of -1,5 arabinan (Ara) side chains of rhamnogalacturonans. These data are discussed in relation with known pectic modifications occurring during ripening and associated with the tomato fruit softening.     

Free radical grafting onto cellulose in homogeneous conditions 1. Modified cellulose–acrylonitrile system

Synthesis of cellulose–polyacrylonitrile copolymers has been studied in a homogeneous solution of N,N-dimethylacetamide/LiCl. The method is based on the preliminary reaction of a portion of OH cellulosic groups with acryloyl chloride to give cellulose with a certain number of pendant double bonds. Successively, acrylonitrile is grafted onto the unsaturated groups by free radical polymerization using azobisisobutyronitrile as initiator. The optimum grafting conditions are evaluated by changing reaction temperature, as well as monomer/initiator and monomer/unsaturated group molar ratios. The products are characterized by size exclusion chromatography, i.r. and ¹³C n.m.r. spectroscopy and a possible reaction mechanism is deduced.     

Pullulans produced by strains of Cryphonectria parasitica—II. Nuclear magnetic resonance evidence

The structure of pullullan-like polysaccharides produced as exocellular material by different strains of Cryphonectria parasitica, the fungus responsible for chestnut tree cankers, was investigated with nuclear magnetic resonance (NMR) techniques. ¹³C, mono- and bidimensional ¹H, and ¹H–¹³C heteronuclear correlated NMR spectra (HSQC and HMBC) were recorded. Advanced analysis of the NMR spectra allowed the main resonance of the atoms in the maltotriose and in the maltotetraose repeat units of pullulan-like polysaccharides from C. parasitica to be recognised with confidence. In all cases investigated, the presence of large amounts of -(1→6) maltotetraose subunits was evidenced, in addition to the -(1→6) maltotriose subunits, corresponding to the repeating unit of pullulan produced by Aureobasidium pullulans and other fungi. The results were in agreement with other data from this laboratory, obtained with independent techniques. The belief that in ‘pullulans’ the maximum amount of -(1→6) maltotetraose subunits is about 7% can thus be considered as definitely outdated.     

Amphiphilic derivatives of sodium alginate and hyaluronate: synthesis and physico-chemical properties of aqueous dilute solutions

This paper reports on the synthesis and the physico-chemical characterisation of various amphiphilic derivatives of two natural polysaccharides, sodium alginate and sodium hyaluronate, in which a rather small proportion of the carboxylic groups (≤10% mol) was esterified by long alkyl chains (C₁₂H₂₅ or C₁₈H₃₇).

The derivatives thus prepared were characterised by gas chromatography, ¹H and ¹³C n.m.r. spectroscopy and size exclusion chromatography coupled to a multi-angle laser light scattering detection. The tendency of these water-soluble compounds to hydrophobic association in aqueous solutions was evidenced firstly in dilute regime using capillary viscometry as well as fluorescence spectroscopy in the presence of a molecular probe, 1,1-dicyano-(4′-N,N-dimethylaminophenyl)-1,3-butadiene.     

Sudies of O-specific polysaccharide chains of Pseudomonas solanacearum lipopolysaccharides consisting of structually different repeating units.

The structure of the O-antigenic ploysaccharide chains of lipopolysaccharides of a number of Pseudomonas solanacearum strains were elucidated mainly with the help of methylation analysis and ¹³CNMR spectroscopy, including a computer-assisted ¹³CNMR-based analysis. Six structually distinct but related polysaccharides were identified. They have a backbone which is built up of three -rhammoyranosyl resides and one 2-acetamido-2-deoxy- -glucopyranosyl residue, and is unsubtituted or substituted with a residue of -xylopyranose or -rhamnopyranose as a monosaccharide side chain. The lipopolysaccharides of most of the strains contain polysaccharide chains consisting of at least two structually different types of repeating units. Three of the polysaccharides are common to more than one strain.     

Mannitol metabolism in Eimeria tenella.

, and 1992. Mannitol metabolism in Eimeria tenella. International Journal for Parasitology 22: 1157–1163. Unsporulated oocysts of Eimeria tenella contain large quantities of carbohydrates, namely amylopectin, mannitol and glucose. Analysis of the carbohydrate content of sporulating oocysts revealed that mannitol content increased markedly during early stages of sporogony (first 4–6 h) but slowly diminished during the next 40 h of sporulation. Accumulation of mannitol was accompanied by a rapid decrease in amylopectin and free glucose, suggesting that mannitol might be synthesized from glucose released from amylopectin. Mannitol was also detected in sporozoite and merozoite extracts. All four mannitol cycle enzymes were detected in oocysts. Sporozoites excysted in vitro had lower activities of all four enzymes. Mannitol-1 -phosphatase and mannitol dehydrogenase activity was also detected in merozoites obtained from the second stage schizonts. Sporozoites incubated with ¹⁴C-glucose accumulated radioactively labelled precursor continuously for over 12 h and some of the ¹⁴C-glucose was converted into ¹⁴C-mannitol. These results indicate that mannitol plays an important role in the metabolism and development of the intracellular stages of the parasite.     

Surface properties and synthesis of the cellulose-based amphoteric polymeric surfactant

A new family of amphoteric surfactant – 2-hydroxy-3-(N,N-dimethyl-N-dodecyl-ammonium)-propyloxy cellulose sulfate (GDCS) was synthesized by etherification of cellulose sulfate with glycidyl dodecyl dimethylammonium chloride (GDDMAC) in this paper. GDCS was characterized by FT-IR spectroscopy, ¹³C NMR, elemental analysis and TG. The surface-active properties such as surface tension, critical aggregation concentration were determined by the Wilhelmy plate method. The micellar conformation of GDCS in aqueous solution at different concentration was investigated by environmental scanning electro microscopy (ESEM). Rheological measurements of GDCS indicated that the solution first behaved like a pseudoplastic property, the apparent viscosity decreased sharply, and then exhibited a Newtonian property, the apparent viscosity did not change obviously anymore.     

A general method for assaying homo-and hetero-transglycanase activities that act on plant cell-wall polysaccharides

Transglycanases(endotransglycosylases) cleave a polysaccharide(donor-substrate) in mid-chain, and then transfer a portion onto another poly-or oligosaccharide(acceptor-substrate). Such enzymes contribute to plant cellwall assembly and/or re-structuring. We sought a general method for revealing novel homo- and hetero-transglycanases, applicable to diverse polysaccharides and oligosaccharides, separating transglycanase-generated3 Hpolysaccharides from unreacted3H-oligosaccharides—the former immobilized(on filter-paper, silica-gel or glassfiber),the latter eluted. On filter-paper, certain polysaccharides [e.g.(1!3, 1!4)-b-D-glucans] remained satisfactorily adsorbed when water-washed; others(e.g. pectins) were partially lost. Many oligosaccharides(e.g. arabinan-, galactan-, xyloglucan-based) were successfully eluted in appropriate solvents, but others(e.g. [3H]xylohexaitol, [3H]mannohexaitol[3H]cellohexaitol) remained immobile. On silica-gel, all3 Holigosaccharides left an immobile ‘ghost' spot(contaminating any3H-polysaccharides), which was diminished but not prevented by additives e.g. sucrose or Triton X-100. The best stratum was glassfiber(GF), onto which the reactionmixture was dried then washed in 75% ethanol. Washing led to minimal loss or lateral migration of3H-polysaccharides if conducted by slow percolation of acidified ethanol. The effectiveness of GF-blotting was well demonstrated for Chara vulgaris transb-mannanase. In conclusion, our novel GF-blotting technique ef ficiently frees transglycanase-generated3H-polysaccharides from unreacted3H-oligosaccharides,enabling high-throughput screening of multiple postulated transglycanase activities utilising chemically diverse donorand acceptor-substrates.