Characterisation of the polysaccharide produced by Acetobacter xylinum strain CR1/4 by light scattering and atomic force microscopy

The molecular weight of the extracellular polysaccharide (CR1/4) produced by Acetobacter xylinum strain CR1/4 has been shown to be dependent upon growth conditions. Under normal growth conditions a high molecular weight polysaccharide (>1×10⁶ Da) is produced. Maintaining the pH at 5 results in an order of magnitude increase in the total yield of polysaccharide, but also an order of magnitude decrease in molecular weight. Analysis of the CR1/4 polysaccharides by the techniques of atomic force microscopy and static light scattering suggests that they are double helices. In solution the molecules behave as stiff coils with a Kuhn statistical segment length of 325 nm.     

Chemical structural and chain conformational characterization of some bioactive polysaccharides isolated from natural sources

Some polysaccharides isolated from natural sources show various important biological activities, such as antitumor, immunomodulatory, and anti-inflammatory effects, which are strongly affected by their chemical structures and chain conformations. This article attempts to review the current development on structural and conformational characterization of some importantly bioactive polysaccharides isolated from natural sources. The chemical structures were analyzed by FTIR, liquid-state NMR (one and two dimensions), solid-sate NMR, Raman spectroscopy, gas chromatography (GC), GC–Mass (GC–MS), and high-performance liquid chromatography (HPLC). The chain conformations of polysaccharides in solutions were investigated using static and dynamic light scattering, viscosity analysis based on the theory of dilute polymer solution, circular dichroism analysis, atomic force microscopy (AFM) including single molecular AFM and AFM-based single-molecule force spectroscopy, fluorescence correlation spectroscopy and NMR spectroscopy.     

Imaging polysaccharides by atomic force microscopy

Techniques have been developed for the routine reliable imaging of polysaccharides by atomic force microscopy (AFM). The polysaccharides are deposited from aqueous solution onto the surface of freshly cleaved mica, air dried, and then imaged under alcohols. The rationale behind the development of the methodology is described and data is presented for the bacterial polysaccharides xanthan, acetan, and the plant polysaccharides 1-carrageenan and pectin. Studies on uncoated polysaccharides have demonstrated the improved resolution achievable when compared to more traditional metal-coated samples or replicas. For acetan the present methodology has permitted imaging of the helical structure. Finally, in addition to data obtained on individual polysaccharides, AFM images have also been obtained of the network structures formed by κ-carrageenan and gellan gum. © 1996 John Wiley & Sons, Inc.     

Molecular weight of scleroglucan and other extracellular microbial polysaccharides by size-exclusion chromatography and low angle laser light scattering

High-performance aqueous size-exclusion chromatography coupled to a low angle laser light scattering detector has been applied to the analysis of scleroglucan and various other extracellular microbial polysaccharides. Emphasis has been focused on three main findings. (1) The molecular weight of these macromolecules is not very sensitive to changes in fermentation conditions. This is specially true in the case of scleroglucan and related (1 → 3)-β- -glucans including schizophyllan, which all exhibited a constant weight-average molecular weight of 5·7×10⁶±5%. (2) In contrast to plant polysaccharides, polydispersity is very low, usually near unity. (3) The molecular weight levels off quickly during biosynthesis since the molecular weight is constant from the middle of fermentation, if not before.     

Characterisation of heterogeneous arabinoxylans by direct imaging of individual molecules by atomic force microscopy

Atomic force microscopy has been used to characterise populations of extracted water-soluble wheat endosperm arabinoxylans. The adsorbed molecules are extended structures with an estimated Kuhn statistical segment length of 128 nm, suggesting that they adopt an ordered helical structure. However, estimates of the molecular weight distribution, coupled with size exclusion data, suggest that, in solution, the polysaccharides behave as semi-flexible coils, with a Kuhn length of 16 nm. These data imply that adsorption of the arabinoxylan structures onto mica promotes formation of the helical structure. Adoption of this ordered structure is fortunate because it has permitted characterisation of branching observed in a small proportion (approximately 15%) of the population of otherwise linear molecules. The degree of branching has been found to increase with the contour length of the molecules. Degradation of the polysaccharides with xylanase has been used to confirm that both the backbone and branches are based on beta-(1-->4) linked D-xylopyranosyl residues.     

Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus

The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production.     

Water-soluble polysaccharides with pharmaceutical importance from Durian rinds (Durio zibethinus Murr.): isolation, fractionation, characterisation and bioactivity

Crude, pharmaceutically useful water-soluble polysaccharides have been isolated from durian rinds (Durio zibethinus) by hot water extraction followed by ethanol precipitation. The polysaccharides were fractionated by anion exchange chromatography and size exclusion chromatography. Characterisation of the sub-fractions by methanolysis, methylation analysis and NMR spectroscopy revealed that the principal components were pectic polysaccharides with starch as a contaminant. Physical features namely molecular weight (M_w) and intrinsic viscosity of the main fractions were investigated by size exclusion chromatography coupled to multi angle laser light scattering (SEC/MALLS) and capillary viscometer, respectively. The main fractions were subjected to the complement-fixation assay and the relationship of the chemical features of the polysaccharide fractions with their activity was also considered.     

Effect of particle size on the enzymatic hydrolysis of polysaccharides from ultrafine lignocellulose particles

The efficiency of the enzymatic hydrolysis of wood polysaccharides ground into ultrafine particles (UFPs) has been investigated. The content of reducing sugars (RS’s) in powdered raw materials and the yield of sugars during enzymatic hydrolysis have been shown to depend on the particle size. Laser interference microscopy and dynamic light scattering studies have shown that increasing the grinding time from 20 to 40 min resulted in the formation of particles ranging from 2 to 200 nm in size. Enzymatic hydrolyzates of UFPs mostly contained glucose and galactose. The grinding intensity (mill rotation rate) and time had a significant effect on the extent of the enzymatic hydrolysis of wood.     

Effect of particle size on the enzymatic hydrolysis of polysaccharides from ultrafine lignocellulose particles

The efficiency of the enzymatic hydrolysis of wood polysaccharides ground into ultrafine particles (UFPs) has been investigated. The content of reducing sugars (RS's) in powdered raw materials and the yield of sugars during enzymatic hydrolysis have been shown to depend on the particle size. Laser interference microscopy and dynamic light scattering studies have shown that increasing the grinding time from 20 to 40 min resulted in the formation of particles ranging from 2 to 200 nm in size. Enzymatic hydrolyzates of UFPs mostly contained glucose and galactose. The grinding intensity (mill rotation rate) and time had a significant effect on the extent of the enzymatic hydrolysis of wood.     

Sugar Beet Pectin–Protein Complexes

The physical structure of pectin extracted from fresh sugar beet has been examined by atomic force microscopy (AFM). The images obtained reveal that these extracts contain a mixture of pectin polysaccharides and protein–pectin complexes. The AFM data demonstrate, for the first time, that these protein–pectin complexes consist of pectin molecules with protein attached to one end of the pectin chain.     

Effects of substrate branching characteristics on kinetics of enzymatic depolymerization of mixed linear and branched polysaccharides: II. Amylose/glycogen alpha-amylolysis

Enzymatic depolymerization of polysaccharides with alpha-amylase has been studied in mixed aqueous dimethylsulfoxide (DMSO)/water solvents. Polysaccharide substrate chemical compositions, configurational structures, and bonding pattersn are known to affect observed enzymatic reaction kinetics. The branching structures of polysaccharides and their effects on the kinetic mechanisms of depolymerization reactions via endo-acting hydrolyzing enzyme was studied via size exclusion chromatography coupled to low angle laser light scattering (SEC/LALLS). The glycogen branching structure is a heterogeneously distributed "cluster" structure rather than a homogeneously distributed "treelike" structure. The action pattern of alpha-amylase on glycogen, which is composed of highly branched clusters, as end-products, has a "pseudo-exo-attack" in contrast to an expected "endoattack" as seen in the hydrolysis of amylose or amylopectin substrates. These effects of branched substrates for mixed amylose/glycogen alpha-amylolysis have been predicted and demonstrated by both experimental and theoretical analysis using the kinetic model presented in this report. The "lumped" kinetic model employed, assumes that the enzyme simultaneously attacks both linear and branched substrates. In general, excellent agreement between the model predictions and the experimental observations, both qualitatively and quantitatively, was obtained. (c) 1995 John Wiley & Sons, Inc.     

Structural properties of colloidal complexes between condensed tannins and polysaccharide hyaluronan

Interactions of plant tannins with polysaccharide hyaluronan are studied by means of light scattering and small-angle X-ray scattering (SAXS). In this paper, we show that (1) the tannin-polysaccharide complexes remain stable in colloidal suspension; (2) the masses and structures of colloidal tannin-polysaccharide objects depend on the tannin degree of polymerization; and (3) the densities of tannin-polysaccharide aggregates are about 7 times lower than the density of a single solvated polysaccharide molecule. Short tannins and polysaccharides are aggregated in loose oligomeric structures whose sizes are comparable to a single polysaccharide molecule. Tannins longer than 10 nm and polysaccharides are aggregated in larger microgel-like particles whose sizes exceed 200 nm.     

Molecular dynamics simulation of dextran extension by constant force in single molecule AFM

The extension of 1-6 polysaccharides has been studied in a series of recent single molecule AFM experiments. For dextran, a key finding was the existence of a plateau in the force-extension curve at forces between 700 and 1000 pN. We studied the extension of the dextran 10-mer under constant force using atomistic simulation with various force fields. All the force fields reproduce the experimental plateau on the force-extension curve. With AMBER94 and AMBER-GLYCAM04 force fields the plateau can be explained by a transition of the glucopyranose rings in the dextran monomers from the chair ((4)C(1)) to the inverted chair ((1)C(4)) conformation while other processes occur at smaller (rotation around C5-C6 bond) or higher (chairs to boat transitions) forces. The CHARMM force field provides a different picture which associates the occurrence of the plateau to chair-boat transitions of the glucopyranose rings.     

Toward mechanical manipulations of cell membranes and membrane proteins using an atomic force microscope

Recent advances in the use of the atomic force microscope (AFM) for manipulating cell membranes and membrane proteins are reviewed. Early pioneering work on measurements of the magnitude of the force required to create indentations with defined depth on their surfaces and to separate interacting pairs of avidin-biotin, antigen-antibody, and complementary DNA pairs formed the basis of this field. The method has subsequently been applied to map the presence of cell surface receptors and polysaccharides on live cell membranes by force measurement, with promising results. Attempts to extract phospholipids and proteins from lipid bilayers and live cell surfaces have been reported, providing a new tool for the manipulation of cellular activities and biochemical analysis at the single-cell level. An increasing awareness of the effect of the pulling speed (nm/s or microm/s), or more accurately, the force loading rate (pN/s or nN/s) on the magnitude of the rupture force, has led researchers to construct energy diagrams of rupture events based on the parameters available from such studies. Information on such nature of the interplay of force and loading rate is vital for nanomanipulation of living cells and cell membranes. Some relevant work for membrane manipulation using other methods is also reviewed in relation to AFM-based methodology.     

Average molecular weight and molecular weight distribution of agarose and agarose-type polysaccharides

A procedure to determine the absolute weight-average molecular weight and molecular weight distribution of agarose and agarose-type polysaccharides by aqueous size-exclusion chromatography coupled with low-angle laser light scattering is described. The molecular weights of the majority of the commercial samples investigated were between 80 000 and 140 000 with a polydispersity lower than 1·7. In contrast, most of the laboratory-extracted agarose-type polysaccharides had lower molecular weights.     

Plant cell wall characterization using scanning probe microscopy techniques

Lignocellulosic biomass is today considered a promising renewable resource for bioenergy production. A combined chemical and biological process is currently under consideration for the conversion of polysaccharides from plant cell wall materials, mainly cellulose and hemicelluloses, to simple sugars that can be fermented to biofuels. Native plant cellulose forms nanometer-scale microfibrils that are embedded in a polymeric network of hemicelluloses, pectins, and lignins; this explains, in part, the recalcitrance of biomass to deconstruction. The chemical and structural characteristics of these plant cell wall constituents remain largely unknown today. Scanning probe microscopy techniques, particularly atomic force microscopy and its application in characterizing plant cell wall structure, are reviewed here. We also further discuss future developments based on scanning probe microscopy techniques that combine linear and nonlinear optical techniques to characterize plant cell wall nanometer-scale structures, specifically apertureless near-field scanning optical microscopy and coherent anti-Stokes Raman scattering microscopy.     

Localized dynamic light scattering: a new approach to dynamic measurements in optical microscopy.

We present a new approach to probing single-particle dynamics that uses dynamic light scattering from a localized region. By scattering a focused laser beam from a micron-size particle, we measure its spatial fluctuations via the temporal autocorrelation of the scattered intensity. We demonstrate the applicability of this approach by measuring the three-dimensional force constants of a single bead and a pair of beads trapped by laser tweezers. The scattering equations that relate the scattered intensity autocorrelation to the particle position correlation function are derived. This technique has potential applications for measurement of biomolecular force constants and probing viscoelastic properties of complex media.     

Hydration of pectic polysaccharides

The hydration and swelling of pectic polysaccharides was examined at different pHs and ionic strengths as a function of osmotic stress. For weakly charged pectic polysaccharides at low concentrations of a monovalent salt (20 mM), the main driving force for swelling originates from a polyelectrolyte effect due to the translational entropy of ions within the film. Swelling is reduced at higher salt concentrations and lower pHs. Polyelectrolyte collapse and minimal swelling is observed for more highly charged pectic polysaccharides. Replacement of the Na(+) counterion with Ca(2+) results in minimal swelling and the formation of network structures even for the weakly charged pectic polysaccharides.     

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