Determination of structural peculiarities of dexran,pullulan and gamma-irradiated pullulan by Fourier-transform IR spectroscopy

Deconvoluted IR-absorbance spectra of dextran, pullulan and gamma-irradiated pullulan were analyzed in order to find the most specific spectral peculiarities that allow one to obtain information about the structure and conformation of these macromolecules in solvents that exhibit different influences on the system of intra- and intermolecular interactions. The changes in intensity and width of the IR bands at about 1040, 1020 and, in the case of pullulan, also at 996 cm(-1), were related to changes in conformation and short-range interactions of the polysaccharides. Furthermore, certain bands within the 1200-900 cm(-1) region were considered as a characteristic for the type of glycosidic linkage. The results of the FTIR spectroscopy study allowed one to suggest a predominant cleavage of the alpha-(1-->4) linkages upon the radiation-chemical destruction of pullulan.     

Development of aqueous two‐phase systems comprising poly ethylene glycol and dextran for purification of pullulan: Phase diagrams and fiscal analysis

Pullulan is a commercially important Exopolysaccharide (EPS) with wide‐spread applications which is produced by Aureobasidium pullulans. The alternative α (1 4) & α (1 6) configuration in pullulan provides it the specific structural and conformational properties. Pullulan is currently being exploited in food, health care, pharmacy, lithography, cosmetics. The fermented broth is processed by organic solvent precipitation for isolation and purification of pullulan. In this study, we have tried to analyze the potential of aqueous two phase system as an alternate technique to extract pullulan from fermented broth. Including this viability of ATPS was also compared with conventional organic solvent precipitation system in terms of cost and time. It was found that ATPS process produced a higher yield of pullulan (80.56%) than organic solvent precipitation method (71.6%). ATPS was also found more economical and less time consuming method.     

Downstream processing and characterization of pullulan from a novel colour variant strain of Aureobasidium pullulans FB-1

Exopolysaccharide produced by a new novel colour variant strain of Aureobasidium pullulans FB-1 was purified by cell harvesting and precipitation of the polymer. Various organic solvents were screened for pullulan precipitation. Isolation and purification of pullulan from fermentation broth was carried out using single-step purification strategy by isopropyl alcohol precipitation. Ratio of culture supernatant to isopropyl alcohol and time of precipitation were optimized for pullulan precipitation. Maximum yield (4.47%, w/v) of polysaccharide was obtained when two volumes of ice-cold isopropyl alcohol were added to one volume of supernatant with precipitation time of 12 h. IR spectra as well as carbon-13 and proton NMR spectra in aqueous solution of intact polysaccharide obtained from A. pullulans FB-1 and commercially available pullulan (Sigma, USA) revealed solely α-(1 → 6) linked maltosyl units, in accord with the generally accepted structure of pullulan. Maximum hydrolysis (94.25%) of purified pullulan at 50 °C by pullulanase was achieved under agitation (150 rpm) after 360 min.     

Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the alpha-amylase family

Cyclomaltodextrinase (CDase, EC 3.2.1.54), maltogenic amylase (EC 3. 2.1.133), and neopullulanase (EC 3.2.1.135) are reported to be capable of hydrolyzing all or two of the following three types of substrates: cyclomaltodextrins (CDs); pullulan; and starch. These enzymes hydrolyze CDs and starch to maltose and pullulan to panose by cleavage of alpha-1,4 glycosidic bonds whereas alpha-amylases essentially lack activity on CDs and pullulan. They also catalyze transglycosylation of oligosaccharides to the C3-, C4- or C6-hydroxyl groups of various acceptor sugar molecules. The present review surveys the biochemical, enzymatic, and structural properties of three types of such enzymes as defined based on the substrate specificity toward the CDs: type I, cyclomaltodextrinase and maltogenic amylase that hydrolyze CDs much faster than pullulan and starch; type II, Thermoactinomyces vulgaris amylase II (TVA II) that hydrolyzes CDs much less efficiently than pullulan; and type III, neopullulanase that hydrolyzes pullulan efficiently, but remains to be reported to hydrolyze CDs. These three types of enzymes exhibit 40-60% amino acid sequence identity. They occur in the cytoplasm of bacteria and have molecular masses from 62 to 90 kDa which are slightly larger than those of most alpha-amylases. Multiple amino acid sequence alignment and crystal structures of maltogenic amylase and TVA II reveal the presence of an N-terminal extension of approximately 130 residues not found in alpha-amylases. This unique N-terminal domain as seen in the crystal structures apparently contributes to the active site structure leading to the distinct substrate specificity through a dimer formation. In aqueous solution, most of these enzymes show a monomer-dimer equilibrium. The present review discusses the multiple specificity in the light of the oligomerization and the molecular structures arriving at a clarified enzyme classification. Finally, a physiological role of the enzymes is proposed.     

Hydroxyl radical induced cross-linking of cytosine and tyrosine in nucleohistone

Hydroxyl radical induced formation of a DNA-protein cross-link involving cytosine and tyrosine in nucleohistone in buffered aqueous solution is reported. The technique of gas chromatography-mass spectrometry was used for this investigation. A gamma-irradiated aqueous mixture of cytosine and tyrosine was first investigated in order to obtain gas chromatographic-mass spectrometric properties of possible cytosine-tyrosine cross-links. One cross-link was observed, and its structure was identified as the product from the formation of a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. With the use of gas chromatography-mass spectrometry with selected-ion monitoring, this cytosine-tyrosine cross-link was identified in acidic hydrolysates of calf thymus nucleohistone gamma-irradiated in N2O-saturated aqueous solution. The yield of this DNA-protein cross-link in nucleohistone was found to be a linear function of the radiation dose in the range of 100-500 Gy (J.kg-1). This yield amounted to 0.05 nmol.J-1. Mechanisms underlying the formation of the cytosine-tyrosine cross-link in nucleohistone were proposed to involve radical-radical and/or radical addition reactions of hydroxyl adduct radicals of cytosine and tyrosine moieties, forming a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. When oxygen was present in irradiated solutions, no cytosine-tyrosine cross-links were observed.     

Synthesis, physicochemical and spectroscopic characterization of copper(II)-polysaccharide pullulan complexes by UV-vis, ATR-FTIR, and EPR

Bioactive copper(II) complexes with polysaccharides, like pullulan and dextran, are important in both veterinary and human medicine for the treatment of hypochromic microcitary anemia and hypocupremia. In aqueous alkaline solutions, Cu(II) ion forms complexes with the exopolysaccharide pullulan and its reduced low-molecular derivative. The metal content and the solution composition depend on pH, temperature, and time of the reaction. The complexing process begins in a weak alkali solution (pH >7) and involves OH groups of pullulan monomer (glucopyranose) units. Complexes of Cu(II) ion with reduced low-molecular pullulan (RLMP, M_w 6000 g mol⁻¹) were synthesized in water solutions, at the boiling temperature and at different pH values ranging from 7.5 to 12. The Cu(II) complex formation with RLMP was analyzed by UV–vis spectrophotometry and other physicochemical methods. Spectroscopic characterizations (ATR-FTIR, FT-IRIS, and EPR) and spectra–structure correlation of Cu(II)–RLMP complexes were also carried out.     

Preparation and solution properties of pullulan fractions as standard samples for water-soluble polymers

A series of pullulan fractions with molecular weights in the range 5 × 10³ to 8 × 10⁵ were prepared. The weight-average molecular weight (M_w) of all the samples was determined by sedimentation equilibrium. The hydrodynamic properties of pullulan in aqueous solution were investigated by viscometry and ultracentrifugation. The experimental results indicate that pullulan molecules in water are fairly stable and behave as expanded random coils when M_w is above 2 × 10⁴. The molecular weight distributions of the fractions were measured by gel filtration. The ratio M_w/M_n was close to 1·1, except for a sample with the highest M_w.It is concluded that the pullulan fractions prepared by the present work are well characterized and have a narrow molecular weight distribution. They may be useful as standard samples for studies of water-soluble polymers.     

Conjugation of heparin into carboxylated pullulan derivatives as an extracellular matrix for endothelial cell culture

A carboxylated pullulan, for use as a structural material for a number of tissue engineering applications, was synthesized and conjugated with heparin. By immobilization of heparin to pullulan, endothelial cells (ECs) attached on the heparin-conjugated pullulan were more aggregated than when attached to other pullulan derivatives. Attachments were 50, 45, 49, and 90% for a polystyrene dish, pullulan acetate, carboxylated pullulan, and heparin-conjugated pullulan, respectively. Heparin-conjugated pullulan inhibited the proliferation of smooth muscle cells (SMCs) in vitro. Heparin-conjugated pullulan material can thus be used for the proliferation of vascular ECs and to inhibit the proliferation of SMCs.     

Polyelectrolyte microstructure in chitosan aqueous and alcohol solutions

This work deals with chain ordering in aqueous and water-alcohol solutions of chitosan. The so-called polyelectrolyte peak is investigated by small-angle synchrotron X-ray scattering. The polyelectrolyte microstructure was characterized by the position of the maximum of the polyelectrolyte scattering peak qmax, which scales with the polymer concentration cp as qmax approximately cp alpha. An evolution of the power law exponent alpha is observed as a function of the degree of acetylation (DA) of chitosan, which is responsible for changes of both the charge density (f) and the hydrophobicity of the polymer chains. The results highlighted the two organization regimes of the theory of Dobrynin and Rubinstein, investigated here for the first time for a natural polymer. At low DAs, alpha approximately 1/2, in agreement with a pearl necklace organization where the structure is controlled by the string between pearls. For higher DA, alpha approximately 1/3, and the correlation revealed by the polyelectrolyte peak is controlled by the pearls. This analysis offers a way to study quantitatively the balance between solvophobic-solvophilic interactions that play an important role in the solution properties of natural polymers. In addition, the role of several parameters acting on the interaction balance were evidenced, such as the nature of the counterion, the composition of the solvent (amount of alcohol in the aqueous solution), and the screening of Coulombic forces by salt addition. Finally, the nanostructure transition from a polyelectrolyte solution to a physical gel is discussed. The gel state is reached when the solvophobic interactions are favored, but depending on the gelation route the polyelectrolyte ordering could be preserved or not.     

DWS microrheology of a linear polysaccharide

Diffusing wave spectroscopy has been used to measure the rheological behavior of pullulan (M(w) = 1 x 10(5)) aqueous solutions up to concentration of 40 g/dL. It was found that these solutions were mainly viscous, with the loss modulus G' higher than the elastic modulus G'. The plot of the specific viscosity eta(sp) as a function of pullulan concentration showed two critical concentrations c = 4 g/dL and c = 15 g/dL. For c < c, eta( sp) approximately c(1.25+/-0.05); for c < c < c, eta( sp) approximately c(2+/-0.05); and for c > c, eta( sp) approximately c(4.5+/-0.5). These results are in very good agreement with those reported in the literature.     

Flow birefringence of xanthan and other polysaccharide solutions

A xanthan sample with molecular weight M = 2.2 x 10(6) was investigated in three solvents: bidistilled water, 0.2 M aqueous NaCl and cadoxen by flow birefringence and viscometry methods in dilute solutions. It was shown that the optical shear rate coefficients of xanthan in aqueous and cadoxen media differ by two orders of magnitude. An estimation of xanthan optical anisotropy in different conformational states has been made and compared with values for other polysaccharides: dextran, pullulan, cellulose and chitosan. The process of denaturation and the flow birefringence of renaturated xanthan in aqueous solutions (after heat treatment at 121 degrees C) have also been studied.     

Coarse-grained simulations of the salt dependence of the radius of gyration of polyelectrolytes as models for biomolecules in aqueous solution

The salt dependent radius of gyration of a polyelectrolyte in aqueous solution is calculated in an environment where the polyelectrolyte is surrounded by a permeable membrane that exchanges only solvent particles with the bulk. We obtain additionally the scaling exponent of the gyration radius as a function of the polymerization degree, and find that the polyelectrolyte retains a stretched conformation during the condensation and re-expansion process, indicating that these effects are of an electrostatic nature. The solvent quality is also shown to affect the polyelectrolyte conformation, especially for the poor solvent case. These results are obtained using a hybridized Monte Carlo technique with the coarse-grained, dissipative particle dynamics method with fluctuating number of solvent particles. The full range of the electrostatic interactions is included in the simulations, using the Ewald sum method, and the counterions and solvent molecules are included explicitly. In the complex systems mentioned above, the electrostatic interactions and the solvent quality play a key role in understanding phenomena that do not occur in uncharged systems. Our results are compared and validated with the behavior of some biomolecules under similar environments.     

Estimation of pullulan by hydrolysis with pullulanase

A novel method for the estimation of pullulan was developed in which pullulan was hydrolysed by pullulanase. The hydrolysed product was mainly maltotriose and was determined colorimetrically using 3,5-dimethylsalicylic acid. This gave good linearity with respect to the concentration of pullulan in the fermentation broth. The content of pullulan determined in this way was less than that determined by a coupled enzyme assay and was comparable to that determined by an HPLC method. The new method was specific for estimation of pullulan, demonstrated high accuracy, and could assay pullulan from up to 3.2 mg/ml.     

Comparative study of antifungal activity of two preparations of green silver nanoparticles from Portulaca oleracea extract

The green silver nanoparticles (green AgNPs) exhibit an exceptional antimicrobial property against different microbes, including bacteria and fungi. The current study aimed to compare the antifungal activities of both the crude aqueous extract of Portulaca oleracea or different preparations of green AgNPs biosynthesized by mixing that aqueous extract with silver nitrate (AgNO₃). Two preparations of the green AgNPs were synthesized either by mixing the aqueous extract of P. oleracea with silver nitrate (AgNO₃) (normal AgNPs) or either irradiation of the AgNPs, previously prepared, under ⁶⁰Co γ-ray using chitosan (gamma-irradiated AgNPs). Characterization of different AgNPs were tested by Zeta potential analyzer, Ultraviolet (UV) Visible Spectroscopy, and Fourier-Transform Infrared (FTIR) spectrometry. Three different plant pathogenic fungi were tested, Curvularia spicifera, Macrophomina phaseolina, and Bipolaris sp. The antifungal activities were evaluated by Transmission Electron Microscope (TEM) for either the crude aqueous extract of P. oleracea at three doses (25%, 50%, and 100%) or the newly biosynthesized AgNPs, normal or gamma-irradiated. With a few exceptions, the comparative analysis revealed that the irradiated green AgNPs at all three concentrations showed a relatively stronger antifungal effect than the normal AgNPs against all the three selected fungal strains. UV–visible spectroscopy of both preparations showed surface plasmon resonance at 421 nm. TEM results showed that both AgNPs were aggregated and characterized by a unique spherical shape, however, the gamma-irradiated AgNPs were smaller than the non-irradiated AgNPs (0.007–0.026 µM vs. 0.009–0.086 µM). TEM photographs of the fungal strains treated with the two AgNPs preparations showed flaccid structures, condensed hyphae, and shrunken surface compared with control cells. The data suggested that the biosynthesized P. oleracea AgNPs have antifungal properties against C. spicifera, M. phaseolina, and Bipolaris sp. These AgNPs may be considered a fungicide to protect different plants against phytopathogenic fungi.     

二价阳离子对短梗霉多糖发酵的影响

就二价阳离子对短梗霉多糖和黑色素的影响进行了分析和研究。结果表明 ,二价阳离子对短梗霉多糖的合成和黑色素的形成均有较大的影响。通过对培养基中二价阳离子含量和种类的控制不仅可以抑制细胞黑色素的形成 ,而且还保持了很高的多糖发酵水平 ,在 30L生物反应器中短梗霉多糖的产量和转化率分别达到了 59 8g/L和 61 5%。     

Aqueous two-phase extraction and polyelectrolyte precipitation combination: A simple and economically technologies for pepsin isolation from bovine abomasum homogenate

The combination of two bioseparation techniques, partition in aqueous two-phase systems and polyelectrolyte precipitation of the target enzyme from the phase where it is present, was assayed to purify pepsin from bovine abomasum homogenate. Pepsin was partitioned in favor of the polyethyleneglycol-rich phase in an aqueous two-phase system of polyethyleneglycol 600 and 1450-sodium phosphate; however, a great amount of impure proteins were present. Chitosan (a cationic natural polyelectrolyte) was added to precipitate this acid enzyme as a form of insoluble complex. The addition of this second step increased the purity of the enzyme significantly while the yield was not significantly decreased. The combination of both partition in polyethyleneglycol 1450-phosphate system and chitosan precipitation produced a pepsin recovery of 48.5% with a purification factor of 9.0. The biological activity of the recovered enzyme remained unaltered.     

出芽短梗霉色素变异菌株R45的普鲁蓝糖发酵研究

用正交实验确定了出芽短梗霉（Aureobasidiumpullulans）色素变异菌株R45的发酵优化条件。在此条件下,摇瓶培养的普鲁蓝糖产量最高可达82．4g／L,转化率为54．9％。实验表明,CaCO3是多糖发酵的重要影响因素,多糖的合成与发酵pH值及细胞形态密切相关。     

Diffusion-ordered NMR spectroscopy: a versatile tool for the molecular weight determination of uncharged polysaccharides

Diffusion-ordered NMR spectroscopy (DOSY) experiments have been carried out on dilute aqueous solutions of uncharged saccharide systems and, in particular, on six well characterized pullulan fractions of different molecular weights. The values of diffusion coefficients and hydrodynamic radii determined for the pullulan fractions are in good agreement with the results obtained with other methodologies such as light scattering. Fitting the diffusion coefficients data as a function of the molecular weight allows for the determination of a calibration curve that can be applied to a wide range of mono-, oligo-, and polysaccharides. Therefore, DOSY is proposed as a versatile tool for achieving a simple estimation of the molecular weight of uncharged polysaccharides. Mixtures of homopolymers of different molecular weight can be nicely separated. An advantage of the method is that the same sample used for the NMR characterization can be used for the molecular weight determination without any further manipulation. Other water soluble polymers, such as poly(ethylene oxide) and poly(vinylpyrrolidone), can be roughly characterized using the same calibration curve.     

Optimization of physico‐chemical and nutritional parameters for a novel pullulan‐producing fungus,Eurotium chevalieri

Aims: To isolate the novel nonmelanin pullulan‐producing fungi from soil and to optimize the physico‐chemical and nutritional parameters for pullulan production. Methods and Results: A selective enrichment method was followed for the isolation, along with development of a suitable medium for pullulan production, using shake flask experiments. Pullulan content was confirmed using pure pullulan and pullulanase hydrolysate. Eurotium chevalieri was able to produce maximum pullulan (38 ± 1·0 g l^?1) at 35°C, pH 5·5, 2·5% sucrose, 0·3% ammonium sulfate and 0·2% yeast extract in a shake flash culture medium with an agitation rate of 30 rev min^?1 for 65 h. Conclusions: The novel pullulan‐producing fungus was identified as E. chevalieri (MTCC no. 9614), which was able to produce nonmelanin pullulan at from poorer carbon and nitrogen sources than Aureobasidium pullulans and may therefore be useful for the commercial production of pullulan. Significance and Impact of the Study: Eurotium chevalieri could produce pullulan in similar amounts to A. pullulans. Therefore, in future, this fungus could also be used for commercial pullulan production, because it is neither polymorphic nor melanin producing, hence its handling during pullulan fermentation will be easier and more economical.     

Partition of polysaccharide-coated liposomes in aqueous two-phase systems

Hydrophobized polysaccharides such as cholesterol-bearing pullulan (CHP), dextran (CHD) and mannan (CHM) effectively coat the liposomal surface. Partition of the hydrophobized polysaccharide-coated liposomes in an aqueous two-phase system (PEO (top)/pullulan (bottom) or PEO (top)/dextran (bottom)) was investigated (PEO = poly(ethylene oxide)). Conventional liposomes without a polysaccharide coat mostly locate at the interface between the two polymer phases. The polysaccharide-coated liposomes, on the other hand, were partly partitioned to the bottom polysaccharide phase depending on the structure of the hydrophobized polysaccharide on the liposomal surface. The affinity between the polysaccharide on the liposomal surface and that in the bulk bottom phase controls the efficiency of partition. The sequence of interaction strength between the two carbohydrates as the following: for the PEO/dextran two-phase system, dextran_(liposome)-dextran_(bulk) > mannan_(liposome)-dextran_(bulk) > pullulan_(liposome)-dextran_(bulk); while for the PEO/pullulan system, the sequence of interaction strength was pullulan_(liposome)-pullulan_(bulk) > dextran_(liposome)-pullulan_(bulk) ≈ mannan_(liposome)-pullulan_(bulk).