Monte Carlo study of cycloamylose: chain conformation, radius of gyration, and diffusion coefficient

Cyclic (1 --> 4)-alpha-D-glucan chains with or without excluded volume have been collected from a huge number (about 10(7)) of linear amylosic chains generated by the Monte Carlo method with a conformational energy map for maltose, and their mean-square radii of gyration and translational diffusion coefficients D (based on the Kirkwood formula) have been computed as functions of x (the number of glucose residues in a range from 7 to 300) and the excluded-volume strength represented by the effective hard-core radius. Both /x and D in the unperturbed state weakly oscillate for x < 30 and the helical nature of amylose appears more pronouncedly in cyclic chains than in linear chains. As x increases, these properties approach the values expected for Gaussian rings. Though excluded-volume effects on them are always larger in cycloamylose than in the corresponding linear amylose, the ratios of and the hydrodynamic radius of the former to the respective properties of the latter in good solvents can be slightly lower than or comparable to the (asymptotic) Gaussian-chain values when x is not sufficiently large. An interpolation expression is constructed for the relation between the gyration-radius expansion factors for linear and cyclic chains from the present Monte Carlo data and the early proposed asymptotic relation with the aid of the first-order perturbation theories.     

Solution properties of (1-->3)-alpha-D-glucan and its sulfated derivative from Poria cocos mycelia via fermentation tank

From Poria cocos mycelia yielded via a pilot scale facility-fermentation tank, a water-insoluble (1-->3)-alpha-D-glucan coded as Pi-PCM3-I was isolated by extraction with 0.5 M NaOH/0.01 M NaBH(4) aqueous solution. Nine fractions from F1 to F9 with a weight-average molecular mass (M(w)) range from 7.75 x 10(4) to 57.3 x 10(4) were prepared from the Pi-PCM3-I sample by a nonsolvent addition method. The fractions were reacted with chlorosulfonic acid-pyridine complex to product water-soluble sulfated derivatives coded as S1 to S8 with M(w) from 2.36 x 10(4) to 14.5 x 10(4) and degree of substitution (DS) of 0.86-1.38. M(w), z-average radius of gyration (s(2) (z) (1/2)), the second virial coefficient (A(2)), and the intrinsic viscosity ([eta]) of the native and sulfated Pi-PCM3-I were measured by laser light scattering (LLS), size-exclusion chromatography combined with LLS (SEC-LLS), and viscometry at 25 degrees C. The Mark-Houwink equations for Pi-PCM3-I in 0.25 M LiCl/dimethylsulfoxide (DMSO) (Me(2)SO) and for its sulfated derivative in 0.15 M NaCl aqueous solution at 25 degrees C were established to be [eta] = 1.33 x 10(-2) M(w) (0.75+/-0.01) (mL g(-1)) and [eta] = 1.46 x 10(-4) M(w) (1.13+/-0.01) (mL g(-1)), respectively. On the basis of theories for a wormlike cylinder model, the conformational parameters of the native and sulfated Pi-PCM3-I were calculated to be 760 +/- 50 and 1060 +/- 30 nm(-1) for the molar mass per unit contour length (M(L)), 6.3 +/- 0.5 and 13.1 +/- 1 nm for the persistence length (q), and 14.9 +/- 0.2 and 31.8 +/- 1 for the characteristic ratio (C( proportional, variant)), respectively. The results revealed that Pi-PCM3-I existed as an extended flexible chain in 0.25 M LiCl/Me(2)SO, and its sulfated derivative existed as a semistiff chain in 0.15 M NaCl aqueous solution. Furthermore, Pi-PCM3-I possessed similar structure and molecular parameters to wc-PCM3-I from a rotary shaker; this suggests promising industrialization of Poria cocos polysaccharides.     

Cyclization reaction catalyzed by glycogen debranching enzyme (EC 2.4.1.25/EC 3.2.1.33) and its potential for cycloamylose production

Glycogen debranching enzyme (GDE) has 4-alpha-glucanotransferase and amylo-1,6-glucosidase activities in the single polypeptide chain. We analyzed the detailed action profile of GDE from Saccharomyces cerevisiae on amylose and tested whether GDE catalyzes cyclization of amylose. GDE treatment resulted in a rapid reduction of absorbance of iodine-amylose complex and the accumulation of a product that was resistant to an exo-amylase (glucoamylase [GA]) but was degraded by an endo-type alpha-amylase to glucose and maltose. These results indicated that GDE catalyzed cyclization of amylose to produce cyclic alpha-1,4 glucan (cycloamylose). The formation of cycloamylose was confirmed by high-performance anion-exchange chromatography, and the size was shown to range from a degree of polymerization of 11 to a degree of polymerization around 50. The minimum size and the size distribution of cycloamylose were different from those of cycloamylose produced by other 4-alpha-glucanotransferases. GDE also efficiently produced cycloamylose even from the branched glucan substrate, starch, demonstrating its potential for industrial production of cycloamylose.     

Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: production of cycloamylose

The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75 degrees C in a 10-min reaction with maltotriose and was stable at temperatures up to 85 degrees C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic alpha-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.     

Cyclization Reaction Catalyzed by Glycogen Debranching Enzyme (EC 2.4.1.25/EC 3.2.1.33) and Its Potential for Cycloamylose Production

Glycogen debranching enzyme (GDE) has 4-α-glucanotransferase and amylo-1,6-glucosidase activities in the single polypeptide chain. We analyzed the detailed action profile of GDE from Saccharomyces cerevisiae on amylose and tested whether GDE catalyzes cyclization of amylose. GDE treatment resulted in a rapid reduction of absorbance of iodine-amylose complex and the accumulation of a product that was resistant to an exo-amylase (glucoamylase [GA]) but was degraded by an endo-type α-amylase to glucose and maltose. These results indicated that GDE catalyzed cyclization of amylose to produce cyclic α-1,4 glucan (cycloamylose). The formation of cycloamylose was confirmed by high-performance anion-exchange chromatography, and the size was shown to range from a degree of polymerization of 11 to a degree of polymerization around 50. The minimum size and the size distribution of cycloamylose were different from those of cycloamylose produced by other 4-α-glucanotransferases. GDE also efficiently produced cycloamylose even from the branched glucan substrate, starch, demonstrating its potential for industrial production of cycloamylose.     

Optical properties of sugars. VI. Circular dichroism of amylose and glucose oligomers

The CD spectra of amylose, the maltose oligomers, cellobiose, and the cyclodextrins are measured in aqueous solution to 1640 Å. Two bands are found in this region. An examination of chromophorically equivalent but conformationally different glucans demonstrates that the CD of glucans is sensitive to conformation. However, the conformation of the subunits in the maltose oligomer series appears independent of chainlength. The conformations of the interior maltosyl groups of this series are all approximately equivalent. A comparison of the amylose and cycloamylose spectra indicates that amylose is substantially biased in its chirality. Since there is little chainlength dependence, the oligomers should have a similar chirality bias. The butanol complex of amylose, which is believed to have a V-form helical conformation, has a CD similar to free aqueous amylose. This is consistent with conformational calculations on amylose which predict that the maltosyl subunits of amylose are restricted to a small region on its conformational map which includes the V-form helix. It is also consistent with the idea that amylose a loosely wound and extended helix in aqueous solution.     

TreX from Sulfolobus solfataricus ATCC 35092 displays isoamylase and 4-alpha-glucanotransferase activities

A treX in the trehalose biosynthesis gene cluster of Sulfolobus solfataricus ATCC 35092 has been reported to produce TreX, which hydrolyzes the alpha-1,6-branch portion of amylopectin and glycogen. TreX exhibited 4-alpha-D-glucan transferase activity, catalyzing the transfer of alpha-1,4-glucan oligosaccharides from one molecule to another in the case of linear maltooligosaccharides (G3-G7), and it produced cyclic glucans from amylopectin and amylose like 4-alpha-glucanotransferase. These results suggest that TreX is a novel isoamylase possessing the properties of 4-alpha-glucanotransferase.     

Effect of various acceptors on the rates of the cyclization and chain-shortening of amylose catalyzed by the cyclodextrin glycosyltransferase from Klebsiella pneumoniae M 5 al. Improvement of new photometric assay methods

The effect of various acceptors on the cyclization and chain-shortening reaction of amylose catalyzed by cyclodextrin glycosyltransferase [(1 leads to 4)-alpha-D-glucan: [(1 leads to 4)-alpha-D-glycopyranosyl]transferase (cycling) EC 2.4.1.19] from Klebsiella pneumoniae M 5 al was studied by use of photometric-assay methods. The requirements for the acceptor were the same for both cyclization and chain-shortening, indicating the close relationship between both reactions. Maltose proved to be the most effective (2.48- and 5-fold acceleration of the cyclization and chain-shortening, respectively, in the presence of 584 micrometers maltose). The dependence of the chain-shortening reaction on the conformational state of the amylose molecules is discussed.     

Exploring the (1 → 3)‐β‐D‐glucan conformational phase diagrams to optimize the linear to macrocycle conversion of the triple‐helical polysaccharide scleroglucan

The immunologically important (1 → 6) comb‐like branched (1 → 3)‐β‐D ‐glucans scleroglucan, schizophyllan, lentinan, and others, exist mainly as linear triple‐helical structures in aqueous solution. Partial interconversion from linear to circular topology has been reported to take place following conformational transition of the triple‐helical structure and subsequent regeneration of the triplex conformation. We here report on experimental data indicating that complete strand separation of the triple‐helical structure is required for this interconversion. NaOH or dimethylsulfoxide was used to induce dissociation of the triplex at combinations of concentrations and temperatures shown by calorimetry to yield a conformational transition of the triplex structures. For the alkaline treatment at 55°C, it is found that up to about 30% of the material readily can be converted to the cyclic topology. This fraction increased to about 60% when the subsequent annealing of the scleroglucan in aqueous solution at pH 7 was carried out at 100°C. Further increase of the annealing temperature yielded a smaller relative amount of cyclic species. The data indicate that the lower molecular weight fraction of the molecular weight distributions can be converted selectively to the macrocyclic topology by conditions that do not yield complete strand separation of the whole sample. These findings add to previous reports by providing more details about how the conditions required for the linear triplex to macrocycle interconversion relate to the conformational properties of the triple‐helical structure. © 1999 John Wiley & Sons, Inc. Biopoly 50: 496–512, 1999     

Structure and chain conformation of beta-glucan isolated from Auricularia auricula-judae

From Auricularia auricula-judae, a water soluble beta-D-glucan, named as AAG, was isolated by extraction with 70% ethanol/water solution. Its chemical structure was analyzed by gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), matrix-assisted laser desorption /ionization (MALDI)-time of flight (TOF), and 1D, 2D NMR. AAG was detected, for the first time, to be composed of a main chain of (1-->4)-linked D-glucopyranosyl with glucopyranosyl side groups at O6. With the help of MALDI-TOF-MS, the sequence and the distribution of glucuronic acid were determined and the content of glucuronic acid is about 19%. Five fractions were prepared from the AAG sample in water by ultrasonic degradation method. Their molecular weight, size, and shape (chain conformation) were studied by dynamics light scattering (DLS), static laser light scattering (LLS), size exclusion chromatography combined LLS (SEC-LLS) and viscometry in 0.1M NaCl aqueous solution at 25 degrees C. The dependence of intrinsic viscosity ([eta]) on Mw for this polysaccharide was established to be [eta] = 1.22 x10(-3)Mw (1.00) (cm3 g(-1)) in the range of Mw from 3.40 x 10(4) to 2.88 x 10(5). The conformational parameters of the AAG polysaccharide were found to be 820 nm(-1) for molar mass per unit contour length (ML), 12.3 nm for persistence length (q) and 2.1 for rho (s2(1/2)/Rh). The results suggested that the polysaccharide exists as extended chains in 0.1M NaCl aqueous solution. The chemical structure of AAG containing glucuronic acid and side groups led to steric hindrance, resulting in the increased stiffness of the chains.     

Determination of beta-amylase activity by a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate flow-injection analysis (2, 6-TNS-FIA) method, using amylose and amylopectin as substrates

2-p-Toluidinylnaphthalene-6-sulfonate (2,6-TNS) is a compound which is barely fluorescent in pure water but whose fluorescence can be strongly enhanced if the environment becomes hydrophobic, i.e. by the addition of suitable substrates such as proteins or 1, 4-alpha-D-glucans. The enhancement of fluorescence results from the formation of a 2,6-TNS/substrate complex. For linear and ramified 1, 4-alpha-D-glucans, the fluorescence intensities of the complexes depend linearly on their concentrations but nonlinearly on their average molecular weights (AMW). Thus, the fluorescence detector acts simultaneously as a linear detector concerning the concentration of 1,4-alpha-D-glucan and as a nonlinear mass-selective detector concerning its AMW. These properties have been used for the development of a fluorimetric 2,6-TNS-FIA methodology for the determination of beta-amylase activity, using amylose and amylopectin as substrates. The experimental data points, corresponding to the concentration of "detectable" substrate vs depolymerization time, were fitted using a two-parameter exponential decay curve, and the depolymerization rates at time zero were calculated. The depolymerization rates at time zero vs the corresponding initial substrate concentrations were fitted using the Michaelis-Menten hyperbola and the enzymic constants k(3) and K(m) for amylose (5.93 x 10(-3) g/microKat. min and 1.49 g/L, respectively) and for amylopectin (7.40 x 10(-3) g/microKat+. min and 1.65 g/L, respectively) were determined.     

Chain stiffness of heteropolysaccharide from Aeromonas gum in dilute solution by dynamic light scattering

Dynamic light scattering measurements have been made on 15 fractions of aeromonas (A) gum, an extracellular heteropolysaccharide produced by the strain Aeromonas nichidenii, with dimethylsulfoxide containing 0.2M lithium chloride as the solvent at 25 degrees C. Data for the translational diffusion coefficient D covering a molecular weight range from 4.5 x 10(5) to 2.1 x 10(6) and ratios of the z-average radius of gyration (z) (1/2) to the hydrodynamic radius R(H) (calculated with previous (z) data) suggest that the polymer behaves like a semiflexible chain in this solvent similar to the stiffness of cellulose derivatives. Thus the D data are analyzed on the basis of the Yamakawa-Fujii theory for the translational friction coefficient of a wormlike cylinder by coarse-graining the heteropolysaccharide molecule. Excluded-volume effects are taken into account in the quasi-two-parameter scheme, as was done previously for (z) and [eta] (the intrinsic viscosity) of A gum in the same solvent. The molecular weight dependence of R(H) is found to be explained by the perturbed wormlike chain with a persistence length of 10 nm, a linear mass density of 1350 nm(-1), an excluded-volume strength parameter of 1.3 nm, and a chain diameter of 2.8 nm. These parameters are in substantial agreement with those estimated previously from (z) and [eta] data, demonstrating that the solution properties (D, (z), and [eta]) of the heteropolysaccharide are almost quantitatively described by the current theories for wormlike chains in the molecular weight range studied.     

Molecular conformation of polysaccharides in solution. Changes in the optical rotation and in the elution pattern of gel filtration

Molecular conformation of some polysaccharides in aqueous solution in evidenced by changes in the optical rotation and in the elution pattern of gel filtration. The changes in the specific rotation against that in water are expressed as a molar conformational value [K]: ?495° for colominic acid in 1.0 N NaOH solution, and ?180° for hyaluronate (HA), +85° for corneal keratin sulfate, and +234° for amylose in 8 M urea solution. The gel filtration of amylose and HA dissolved in 8 M urea solution shows elution patterns distinctly different from those dissolved in water. The phenomena are attributable to a molecular conformational transition of polysaccharide molecules in aqueous solution.     

Quasielastic neutron scattering measurements of fast local translational diffusion of lipid molecules in phospholipid bilayers

Quasielastic incoherent neutron scattering has been used to investigate the rate of local translational diffusion of lipid molecules in phospholipid bilayers of dipalmitoyl-phosphatidylcholine. The measured translational diffusion constants (4 x 10(-10) m2 s-1 at 63 degrees C and 1.4 x 10(-11) m2 s-1 at 30 degrees C) are considerably faster than those deduced using other less direct methods, but are in agreement with those measured in soap-water lyotropic liquid crystals, and with calculated values. This disagreement is attributed to differences in the time and distance scales characterising the various measurements. Quasielastic neutron scattering experiments observe fast motions over molecular distances, whereas other methods tend to measure a rate of diffusion which is averaged over macroscopic distances, and may thus contain contributions from long distance slow diffusive motions such as diffusion between the bilayers.     

Chemical structure and chain conformation of the water-insoluble glucan isolated from Pleurotus tuber-regium

A water-insoluble polysaccharide (TM8) was isolated from sclerotium of Pleurotus tuber-regium by extraction with 0.5M NaOH aqueous solutions at 120 degrees C. Its chemical structure was confirmed by infrared, high performance liquid chromatography, gas chromatography, and (13)C NMR in dimethylsulfoxide (DMSO) to be composed of beta-(1 --> 3)-D-glucan backbone chain linked with a branched glucose, one out of every three glycosyl units being substituted at C6 position. The glucan TM8 in DMSO was fractionated by nonsolvent addition method into ten fractions, and the solution properties were studied by size exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS) and viscometry in DMSO at 30 degrees C. The dependencies of intrinsic viscosity [eta] and radius of gyration [(s(2)(1/2)(z-2)] on weight-average molecular mass M(w) for this glucan were found to be [eta] = (9.24 +/- 0.2) x 10(-2)M(w)(0.51 +/- 0.02) (cm(3)g(-1)) and [(s(2)(1/2)(z-2)] = (3.67 +/- 0.3) x 10(-2)M(w)(0.56 +/- 0.02) (nm) in the range of M(w) from 1.07 x 10(4) to 77.4 x 10(4). Based on current theories for a wormlike chain, the conformational parameters of the glucan TM8 were found to be 408 (nm(-1)) for M(L), 3.1 (nm) for q, and 16.8 for C(infinity), suggesting that the polysaccharide exists as a dense random-coil chain in DMSO, due to branched structure.     

Solution properties of antitumor sulfated derivative of alpha-(1-->3)-D-glucan from Ganoderma lucidum

Four fractions of a water-insoluble alpha-(1-->3)-D-glucan GL extracted from fruiting bodies of Ganoderma lucidum were dissolved in 0.25 M LiCl/DMSO, and then reacted with sulfur trioxide-pyridine complex at 80 degrees C to synthesize a series of water-soluble sulfated derivatives S-GL. The degree of substitution of DS was measured by using IR infrared spectra, elemental analysis, and 13C NMR to be 1.2-1.6 in the non-selective sulfation. Weight-average molecular weight Mw and intrinsic viscosity [eta] of the sulfated derivatives S-GL were measured by multi-angle laser light scattering and viscometry. The Mw value (2.4 x 10(4)) of sulfated glucan S-GL-1 was much lower than that (44.5 x 10(4)) of original alpha-(1-->3)-D-glucan GL-1. The Mark-Houwink equation and average value of characteristic ratio C(infinity) for the S-GL in 0.2 M NaCl aqueous solution at 25 degrees C were found to be: [eta] = 1.32 x 10(-3) Mw(1.06) (cm3 g(-1)) and 16, respectively, in the Mw range from 1.1 x 10(4) to 2.4 x 10(4). It indicated that the sulfated derivatives of the alpha-(1-->3)-D-glucan in the aqueous solution behave as an expanded chain, owing to intramolecular hydrogen bonding or interaction between charge groups. Interestingly, two sulfated derivatives synthesized from the alpha-(1-->3)-D-glucan and curdlan, a beta-(1-->3)-D-glucan, all had significant higher antitumor activity against Ehrlich ascites carcinoma (EAC) than the originals. The effect of expanded chains of the sulfated glucan in the aqueous solution on the improvement of the antitumor activity could not be negligible.     

Molecular mass and antitumor activities of sulfated derivatives of alpha-glucan from Poria cocos mycelia

Two kinds of water-insoluble (1-->3)-alpha-D-glucan samples, ab-PCM3-I and ac-PCM3-I, isolated from different Poria cocos mycelia were sulfated, to produce two series of water-soluble derivatives ab-PCM3-I-S1-S5 and ac-PCM3-I-S1-S5, respectively. The derivatives having different weight-average molecular mass (Mw) were produced by changing reaction temperature and time as well as molar ratios between chlorosulfonic acid and number of hydroxyl groups in the glucan. The degrees of substitution (DS) of the sulfated derivatives were analyzed by elemental analysis (EA) to be 0.39-0.67 for ab-PCM3-I-S and 0.73-0.96 for ac-PCM3-I-S, respectively. The Mw and the intrinsic viscosity ([eta]) of the samples ab-PCM3-I-S and the ac-PCM3-I-S were measured by size exclusion chromatography combined with laser light scattering (SEC-LLS) and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The results indicated that their Mw ranged from 2.0 to 11.3 x 10(4) for the samples ab-PCM3-I-S, and 4.7 to 40.0 x 10(4) for the samples ac-PCM3-I-S. Moreover, the antitumor activities of the sulfated derivatives ab-PCM3-I-S and ac-PCM3-I-S against Sarcoma 180 tumor cell tested both in vitro and in vivo are significantly higher than those of the native alpha-D-glucans. Therefore, a moderate range of molecular mass from 2.0 x 10(4) to 40.0 x 10(4), relatively high chain stiffness and good water solubility of the sulfated derivatives are beneficial to the enhancement of their antitumor activities.     

Translational and Rotational Diffusion Constants of Tobacco Mosaic Virus from Rayleigh Linewidths

The translational and rotational diffusion constants of tobacco mosaic virus (TMV) have been determined from homodyne and heterodyne measurements of the spectrum of laser light scattered from dilute aqueous solutions of TMV. Our results for the translational and rotational constants respectively, reduced to 20 degrees C, are: D(T) = 0.280 +/- 0.006 x 10(-7) cm(2)/sec, and D(R) = 320 +/- 18 sec(-1). We include a theoretical derivation of the spectrum of light scattered from rod-shaped molecules which reproduces results obtained previously by Pecora, but which is specialized at the outset to the problem of dilute solutions so that simple single-particle correlation functions may be utilized. An analysis of the photocurrent spectrum for both the homodyne and heterodyne detection schemes is given. Various data reduction schemes utilized in the analysis of our spectra are described in some detail, and our results are compared with values of the diffusion constants obtained from other experiments.     

Translational diffusion of fluorescent proteins by molecular fourier imaging correlation spectroscopy

The ability to noninvasively observe translational diffusion of proteins and protein complexes is important to many biophysical problems. We report high signal/noise (>or=250) measurements of the translational diffusion in viscous solution of the fluorescent protein, DsRed. This is carried out using a new technique: molecular Fourier imaging correlation spectroscopy (M-FICS). M-FICS is an interferometric method that detects a collective Fourier component of the fluctuating density of a small population of fluorescent molecules, and provides information about the distribution of molecular diffusivities. A theoretical analysis is presented that expresses the detected signal fluctuations in terms of the relevant time-correlation functions for molecular translational diffusion. Furthermore, the role played by optical orientational degrees of freedom is established. We report Fickian self-diffusion of the DsRed tetramer at short timescales. The long-time deviation of our data from Fickian behavior is used to determine the variance of the distribution of the protein self-diffusion coefficient. We compare our results to the expected outcomes for 1), a bi-disperse distribution of protein species, and 2), dynamic disorder of the host solvent.     

Linear beta-mannose-containing polysaccharide,beta-xylan,and amylose from the cultured photobiont Trebouxia sp. of the ascolichen Ramalina celastri

The cultured photobiont Trebouxia sp. of Ramalina celastri was successively extracted at 100 degrees C with hot water, 2% aqueous KOH, and 10% aqueous KOH to give polysaccharide-containing fractions A (2.9%), B (3.9%), and C (0.9% yield) respectively. The intact biont contained 3.8% amylose, which was present in each fraction, and was identified by a blue color formed with iodine solution. In fraction A, and following retrogradation from aqueous solution, it was characterized by (13)C-NMR spectroscopy. Fraction B was treated with alpha-amylase to give a water-soluble fraction consisting mainly of beta-mannose-containing polysaccharides (1.5% yield), whose main component had dn/dc 0.162 and M(r) 17 kDa. Fraction C was subjected to freeze-thawing and the precipitate was treated with alpha-amylase to give a resistant, linear, low molecular mass (1-->4)-linked beta-xylan. The beta-D-mannopyranan preparation contained mainly of 3-O- (28%), 4-O- (11%), and 6-O-substituted Manp units (35%), with 3-O-substituted Rhap units (11%). A controlled Smith degradation provided a beta-mannan with nonreducing end- (8%), 3-O- (85%) and 6-O-substituted units, showing (1-->3)- and (1-->6)-linked structures in the original polysaccharide. These could be present as block-type structures.