Interaction of N-acylated and N-alkylated chitosans included in liposomes with lipopolysaccharide of gram-negative bacteria

The interactions of lipopolysaccharide (LPS) with the polycation chitosan and its derivatives — high molecular weight chitosans (300 kDa) with different degree of N-alkylation, its quaternized derivatives, N-monoacylated low molecular weight chitosans (5.5 kDa) — entrapped in anionic liposomes were studied. It was found that the addition of chitosans changes the surface potential and size of negatively charged liposomes, the magnitudes of which depend on the chitosan concentration. Acylated low molecular weight chitosan interacts with liposomes most effectively. The binding of alkylated high molecular weight chitosan with liposomes increases with the degree of its alkylation. The analysis of interaction of LPS with chitoliposomes has shown that LPS-binding activity decreased in the following order: liposomes coated with a hydrophobic chitosan derivatives > coated with chitosan > free liposomes. Liposomes with N-acylated low molecular weight chitosan bind LPS more effectively than liposomes coated with N-alkylated high molecular weight chitosans. The increase in positive charge on the molecules of N-alkylated high molecular weight chitosans at the cost of quaternization does not lead to useful increase in efficiency of binding chitosan with LPS. It was found that increase in LPS concentration leads to a change in surface ζ-potential of liposomes, an increase in average hydrodynamic diameter, and polydispersity of liposomes coated with N-acylated low molecular weight chitosan. The affinity of the interaction of LPS with a liposomal form of N-acylated chitosan increases in comparison with free liposomes. Computer simulation showed that the modification of the lipid bilayer of liposomes with N-acylated low molecular weight chitosan increases the binding of lipopolysaccharide without an O-specific polysaccharide with liposomes due to the formation of additional hydrogen and ionic bonds between the molecules of chitosan and LPS.     

The molecular mechanism of adsorption immobilization of inulinase on polymer matrices

The conditions and mechanisms of the immobilization of inulinase on polymeric carriers were studied using the VION KN-1 and KU-2 cation-exchangers, VION AN-1 and AV-17-2P anion-exchangers, and the ampholyte KOPAN-90. The calculated data showed a significant role of van der Waals interactions and hydrogen bonding in the formation of virtually all inulinase complexes with the immobilization matrices. The AV-17-2P anion-exchanger was the only one of the studied polymer matrices that was unable to form hydrogen bonds with inulinase. The mechanisms of the interaction between inulinase and various ampholytes and cation and anion exchange resins differ from each other. The strongest differences are observed in mechanisms of the sorption of inulinase on VION KN-1 and chitosan matrices. Approximately 87% of the identical amino-acid residues are involved in the interaction of the enzyme with the KU-2 and AV-17-2P resins and the VION AN-1 and KOPAN-90 fibers.     

Relevance of molecular weight of chitosan and its derivatives and their antioxidant activities in vitro

The antioxidant potency of different molecular weight (DMW) chitosan and sulfated chitosan derivatives was investigated employing various established in vitro systems, such as superoxide (O(2)(.-))/hydroxyl ((-.)OH) radicals scavenging, reducing power, iron ion chelating. As expected, we obtained several satisfying results, as follows: firstly, low molecular weight chitosan had stronger scavenging effect on O(2)(.-) and (-.)OH than high molecular weight chitosan. For example the O(2)(.-) scavenging activity of low molecular weight chitosan (9 kDa) and high molecular weight chitosan (760 kDa) were 85.86% and 35.50% at 1.6 mg/mL, respectively. Secondly, comparing with DMW chitosan, DMW sulfated chitosans had the stronger inhibition effect on O(2)(.-). At 0.05 mg/mL, the scavenging activity on O(2)(.-) reached 86.26% for low molecular weight chitosan sulfate (9 kDa), but that of low molecular weight chitosan (9 kDa) was 85.86% at 1.6 mg/mL. As concerning chitosan and sulfated chitosan of the same molecular weight, scavenging activities of sulfated chitosan on superoxide and hydroxyl radicals were more pronounced than that of chitosan. Thirdly, low molecular weight chitosan sulfate had more effective scavenging activity on O(2)(.-) and (-.)OH than that of high molecular weight chitosan sulfate. Fourthly, DMW chitosans and sulfated chitosans were efficient in the reducing power, especially LCTS. Their orders were found to be LCTS>CTS4>HCTS>CTS3>CTS2>CTS1>CTS. Fifthly, CTS4 showed more considerable ferrous ion-chelating potency than others. Finally, the scavenging rate and reducing power of DMW chitosan and sulfated derivatives increased with their increasing concentration. Moreover, change of DMW sulfated chitosans was the most pronounced within the experimental concentration. However, chelating effect of DMW chitosans were not concentration dependent except for CTS4 and CTS1.     

Interaction of chitosans and their N-acylated derivatives with lipopolysaccharide of gram-negative bacteria

The interactions of lipopolysaccharide (LPS) with the natural polycation chitosan and its derivatives--high molecular weight chitosans (80 kD) with different degree of acetylation, low molecular weight chitosan (15 kD), acylated oligochitosan (5.5 kD) and chitooligosaccharides (biose, triose, and tetraose)--were studied using ligand-enzyme solid-phase assay. The LPS-binding activity of chitosans (80 kD) decreased with increase in acetylation degree. Affinity of LPS interaction with chitosans increased after introduction of a fatty acid residue at the reducing end of chitosan. Activity of N-monoacylated chitooligosaccharides decreased in the order: oligochitosan --> tetra- > tri- --> disaccharides. The three-dimensional structures of complexes of R-LPS and chitosans with different degree of acetylation, chitooligosaccharides, and their N-monoacylated derivatives were generated by molecular modeling. The number of bonds stabilizing the complexes and the energy of LPS binding with chitosans decreased with increase in acetate group content in chitosans and resulted in changing of binding sites. It was shown that binding sites of chitooligosaccharides on R-LPS overlapped and chitooligosaccharide binding energies increased with increase in number of monosaccharide residues in chitosan molecules. The input of the hydrophobic fragment in complex formation energy is most prominent for complexes in water phase and is due to the hydrophobic interaction of chitooligosaccharide acyl fragment with fatty acid residues of LPS.     

Study of the oligomeric structure and some physicochemical properties of inulinase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> Y-303

It has been found using a combination of atomic force microscopy with infrared spectroscopy, gel chromatography, and electrophoresis that inulinase from Kluyveromyces marxianus Y-303 has oligomeric structure, which includes two subunits differing in size, molecular mass, and catalytic activity. It has been shown that the division of the inulinase dimer into monomers leads to an increase in the number of irregular regions by 6% for subunit 1 (54.8 kDa) and by 10% for subunit 2 (8.4 kDa) compared with the native enzyme.     

Effect of Molecular Weight and Degree of Acetylation on Adjuvantive Properties of Chitosan Derivatives

The hemostatic and immunostimulating activity and cytotoxicity were determined for a number of chitosans differing in molecular weight (from 3 to 510 kDa) and degree of acetylation (from 1 to 25 mol%) that were used as adjuvants in inactivated poliomyelitic, influenza, and live influenza vaccines. It has been shown that the hemostatic activity of chitosan increased sharply with an increase in its molecular weight. In oligochitosan with a molecular weight of <16 kDa, it was smaller by a factor of 15–100 than in chitosan with a molecular weight of 20–510 kDa. The level of increase in the immunogenicity of vaccines containing oligochitosan as adjuvants was not lower than that for the vaccine including high-molecular chitosan. However, the immunostimulatory activity of oligochitosan depended on the degree of acetylation, reaching a maximum value at 6 mol%. It was shown that all oligochitosans and chitosans with a molecular mass below ~50 kDa showed almost no cytotoxicity at a concentration of ≤2.5 mg/mL, which enable their use as adjuvants for inactivated and live vaccines at the optimal ratio of molecular weight to the degree of acetylation.     

Optimization for high-level expression of the Pichia guilliermondii recombinant inulinase in Pichia pastoris and characterization of the recombinant inulinase

The inulinase gene cloned from the marine-derived yeast Pichia guilliermondii strain 1 was expressed in Pichia pastoris X-33 and the conditions for overexpression of the inulinase were optimized. After the optimization of the conditions for production of the recombinant inulinase, 286.8 ± 5.4 U/ml and 8873 ± 55.3 U/mg of the recombinanat inulinase in the supernatant of the culture of 2-l fermentor were attained at 120 h of the fermentation and fermentation efficiency was 13.04 μg ± 0.4 of protein/ml/d. The recombinant inulinase was purified and characterized. The molecular weight of the purified recombinant inulinase was 57.6 kDa, which was higher than that of the native iunlinase. The optimal pH and temperature of the purified recombinant inulinase were 6.0 and 60 °C, respectively. Other biochemical characteristics of the purified recombinant inulinase were the same as those of the native inulinase produced by the marine-derived P. guilliermondii strain 1. The purified recombinant inulinase also had high exoinulinase activity. Therefore, the recombinant inulinase may have highly potential applications in food and pharmaceutical industies.     

Chitosan-cholesterol and chitosan-stearic acid interactions at the air-water interface

We report in this work the isotherms of cholesterol and stearic acid at the air-water interface modified by different chitosans (chitosan chloride, hydrophobic modified chitosan, and medium and high molecular weight chitosans) in the aqueous subphase. The Langmuir-Blodgett films of the complexes cholesterol-chitosan and stearic acid-chitosan are analyzed by atomic force microscopy (AFM), and a molecular simulation was performed to visualize the chitosan-lipid interactions. Strong modifications are obtained in the isotherms as a result of the chitosan interactions with cholesterol and stearic acid at the air-water interface. These modifications were dependent on the type and concentration of chitosan. Severe modifications of all phases were noticed with larger molecular areas, and the observed changes in the compressional modulus were dependent on the type of chitosan used. The complexes of chitosan-stearic acid were more flexible than the ones of chitosan-cholesterol. The AFM images demonstrated that chitosan was disaggregated by the cholesterol and stearic acid interactions producing more homogeneous surfaces in some cases. The hydrophobic chitosan showed more affinity with stearic acid, while both medium and high molecular weight chitosans produced homogeneous surfaces with cholesterol. The simulated chitosan chains interacting with cholesterol and stearic acid demonstrated the possibility of specific sites of electrostatic bonds between these molecules. Adsorption of cholesterol on the different powdered chitosans, performed by HPLC, showed that the medium and high molecular weight chitosans could retain higher proportions of cholesterol compared with the other analyzed samples.     

Antibacterial effects of water-soluble low-molecular-weight chitosans on different microorganisms

Low-molecular-weight chitosans with a viscosity-average molecular weight (Mv) of 5 to 27 kDa and equal degree of deacetylation (DD, 85%) were highly active against Pseudomonas aureofaciens, Enterobacter agglomerans, Bacillus subtilis, and Bifidobacterium bifidum 791, causing death of 80 to 100% of cells. An exception to this tendency was Escherichia coli, for which the rate of cell death, induced by the 5-kDa chitosan, was 38%. The antibacterial effect was manifested as early as 10 min after incubation of 12-kDa chitosan with B. subtilis or E. coli cells. Candida krusei was almost insensitive to the above crab chitosans. However, Candida krusei was highly sensitive to chitosans with Mv 5, 6, 12, 15.7, and 27 kDa: the minimum inhibitory concentration (MIC) varied from 0.06 to 0.005%. Chitosans with M, 5, 12, and 15.7 kDa exerted an antibacterial effect on Staphylococcus aureus. Chitosans with Mv 5, 15.7, and 27 kDa had no effect on Bifidobacterium bifidum ATCC 14893. The antibacterial effect of the 4-kDa chitosan on E. coli and B. bifidum 791 increased with DD in the range 55-85%.     

Microwave-assisted degradation of chitosan for a possible use in inhibiting crop pathogenic fungi

Degradation of chitosan by H(2)O(2) under microwave irradiation was investigated. The oxidative degradation of chitosan was highly accelerated by microwave irradiation under the condition of low temperature and low concentration of H(2)O(2). The degraded chitosans with low molecular weight (M(w)) were characterized by gel permeation chromatography, Fourier-transform infrared spectroscopy, ultraviolet-visible spectroscopy, X-ray diffraction and elemental analysis. The decrease of M(w) led to transformation of crystal structure and increase of water solubility, whereas no significant chemical structure change in the backbone of chitosan was observed. Antifungal activities of chitosans with different M(w) against crop pathogenic fungi Phomopsis asparagi, Fusarium oxysoporum f. sp. Vasinfectum and Stemphylium solani were investigated at the concentrations of 100, 200 and 400mg/L. All degraded chitosans with low M(w) exhibited enhanced antifungal activity compared with original chitosan and the chitosan of 41.2kDa showed the highest activity. At 400mg/L, the chitosan of 41.2kDa inhibited growth of P. asparagi at 89.3%, stronger than polyoxin and triadimefon, the inhibitory effects of which were found to be 55.5% and 68.5%. All the results indicated that oxidative degradation under microwave irradiation was a promising technique for large-scale production of low M(w) chitosan for use in crop protection.     

Polyelectrolyte Complexes: Interactions between Lignosulfonate and Chitosan

The interactions between high molecular weight chitosans (fraction of acetylated units (F(A)) = 0.10 or 0.50) and lignosulfonates of varying molecular weights (5000-400000 g/mol) and degrees of sulfonation (0.39-0.64) were studied. Lignosulfonates and chitosans form primarily insoluble polyelectrolyte complexes when mixed at pH 4.5, where the polymers are oppositely charged. In contrast, no complex formation occurred at pH 8, as shown by using a chitosan with F(A) = 0.50, which is soluble at this pH. Thus, a positively charged chitosan is a prerequisite for interactions leading to insoluble complexes with lignosulfonates. It is therefore unlikely that complex formation involves the formation of covalent sulfonylamide linkages as proposed in the literature. The composition of the complexes varied to some degree with the mixing ratio and molecular weight of lignosulfonate, but in most cases compact complexes with a sulfonate/amino ratio close to 1.0 were formed, suggesting that all sulfonate groups are accessible for interactions with chitosan. The influence of the ionic strength and temperature on the complex formation and the behavior of the precipitated complexes were in agreement with that expected for classical polyelectrolyte complexes where the associative phase separation is primarily governed by the increase in entropy due to the release of counterions.     

Catalytic activity and the stability of horseradish peroxidase increase as a result of its incorporation into a polyelectrolyte complex with chitosan

The incorporation of horseradish peroxidase into polyelectrolyte complexes with chitosans of different molecular weights (MW 5–150 kDa) yielded highly active and stable enzyme preparations. As a result of the selection of optimal conditions for the formation of peroxidase-chitosan complexes, it was found that 0.1% chitosan with a MW of 10 kDa had the strongest activatory effect on peroxidase (activation degree, >70%) in the reaction of o-dianisidine oxidation by hydrogen peroxide. The complex formed by 0.001% chitosan with a molecular weight of 150 kDa was most stable: when immobilized on foamed polyurethane, it retained at least 50% of the initial activity for 550 days. The highest catalytic activity was exhibited in a 0.05 M phthalate buffer (pH 5.9–6.2) by the complex containing 0.006–0.009% chitosan in the indicator reaction. The activatory effect of the polysaccharide on the enzyme was determined by its influence on the binding and conversion of the reducting substrate peroxidase.     

Production and properties of inulinase from Penicillium sp. NFCC 2768 grown on inulin-rich vegetal infusions

Inulinase production by Penicillium sp. NFCC 2768 isolated from the rhizosphere soil of dahlia was studied on media containing inulin-rich plant extracts. The maximum inulinase activity (64.54 nkat/ml) was observed with the tuber extract of dahlia (Dahlia pinnata). The fungus produced substantial inulinase activity on asparagus root powder (45.23 nkat/ml) and garlic extracts (41.32 nkat/ml). The apparent molecular weight of the purified inulinase was 68 kDa. The optimum pH and temperature for enzyme activity were 5.0 and 50°C, respectively. Mn²⁺ and Ca²⁺ were found to enhance the inulinase activity, while Hg²⁺ was found to be a strong inhibitor. Inulinase liberated fructose, glucose, sucrose, kestose (GF₂), nystose (GF₃), and inulooligosaccharides (IOS). This study suggested the use of dahlia tuber extract and asparagus root powder as suitable substrates for inulinase production by the newly isolated Penicillium sp. NFCC 2768, and its application in the generation of fructose and IOS.     

The preparation of low-molecular-weight chitosan using chitinolytic complex from Streptomyces kurssanovii

Streptomyces kurssanovii are Gram-positive mycelial bacteria ubiquitous in soil. They have a saprophytic way of life and produce many extracellular enzymes with polymer-degrading properties, for example, chitinase (EC 3.2.1.14) and N-acetyl-β- -glucosaminidase (EC3.2.1.30). Biochemical aspects of chitosan degradation were presented. Low-molecular-weight (LMW) chitosans with molecular weight 4–8 kDa were prepared from commercial crab chitosan by means of chitinolytic a complex from S. kurssanovii. The optimum conditions of process in solution (temperature, pH, enzyme-substrate ratio) have been determined. Yields of LMW chitosan were 70–80%.     

Cloning and characterization of the inulinase gene from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after the hydrolysis of inulin with the crude recombinant inulinase.     

Characterization of molecular-sieve-bound inulinase

The enzyme inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7), prepared from Kluyveromyces marxianus has been immobilized using an inorganic solid support, molecular sieve 4A via the metal link method. The immobilized enzyme had around 22 units of inulinase activity per g of the support with retention of 72% of the original activity. The optimum protein to molecular sieve ratio for the maximum retention of inulinase activity was 9 mg/g molecular sieve. The properties of soluble and immobilized enzyme differed in many respects. The optimum pH of the enzyme shifted from 6 to 5 and the optimum temperature of enzyme activity changed from 50 to 55°C. K_m values were 6.7 mM for soluble enzyme and 10 mM for immobilized enzyme. The heat stability of the enzyme was improved by immobilization. Immobilized enzyme retained about 76% of the original activity after 40 days of storage at room temperature (30±2°C).     

Effect of chitosan chain architecture on gene delivery: comparison of self-branched and linear chitosans

Chitosan possesses many characteristics of an ideal gene delivery system. However, the transfection efficiency of conventional chitosans is generally found to be low. In this study, we investigated the self-branching of chitosans as a strategy to improve its gene transfer properties without compromising its safety profile. Self-branched (SB) and self-branched trisaccharide-substituted (SBTCO) chitosans with molecular weights of 11-71 kDa were synthesized, characterized, and compared with their linear counterparts with respect to transfection efficiency, cellular uptake, formulation stability, and cytotoxicity. Our studies show that in contrast with unmodified linear chitosans that were unable to transfect HeLa cells, self-branched chitosans mediated high transfection efficiencies. The most efficient chitosan, SBTCO30, yielded gene expression levels two and five times higher than those of Lipofectamine and Exgen, respectively, and was nontoxic to cells. Nanoparticles formed with SBTCO chitosans exhibited a higher colloidal stability of formulation, efficient internalization without excessive cell surface binding, and low cytotoxicity.     

壳聚糖抑菌机制的初步研究

壳聚糖在医学、食品、环保、日化用品等领域有着广泛而重要的应用.近年来,壳聚糖由于对不同的菌类都具有良好的抑菌效果而被研究者们密切关注.然而,有关壳聚糖抑菌机制的研究却并不多,其抑菌机制也没有被完全阐明.在本研究中,我们发现很多金属离子可以对壳聚糖的抑菌效果产生影响,高浓度金属离子(0.5%)可以使壳聚糖完全丧失抑菌活性.还发现金黄色葡萄球菌和白色念珠菌在壳聚糖的作用下会发生钾离子和ATP的渗漏,而且五万分子量的壳聚糖引起钾离子和ATP的渗漏大约比五千分子量壳聚糖多2到4倍.不同分子量的壳聚糖对金黄色葡萄球菌和白色念珠菌都具有较好的抑菌效果,但是引起钾离子和ATP的渗漏量却存在很大差异,这说明小分子量壳聚糖很可能存在与大分子量壳聚糖不同的抑菌机制.     

Low-molecular-weight chitosans derived from β-chitin: preparation, molecular characteristics and aggregation activity

Preparation, molecular characteristics, and aggregation activity of low-molecular-weight chitosans derived from β-chitin have been studied in comparison with those of chitosans from -chitin. Chitosan derived from β-chitin was partially degraded with alkali and acid to prepare chitosans with reduced molecular weights. The reaction was also conducted with chitosan from -chitin, but it was less susceptible to the degradation than chitosan from β-chitin. The resulting two series of chitosans had molecular weights ranging from 11 to 436 kDa. GPC analysis showed similar changes in the molecular weight distribution in the progress of main chain cleavage of the two kinds of chitosans. The polydispersity values were 2.01–4.16, indicating relatively narrow molecular weight distributions. These chitosans aggregated bovine serum albumin efficiently, and the aggregation behavior was dependent on the molecular weight and concentration of chitosan in addition to the pH of the media and concentration of sodium chloride. The aggregation activity of chitosans from β-chitin was found to be somewhat higher than that of chitosans from -chitin.     

Investigation of mechanisms of interaction between inulinase from Kluyveromyces marxianus and the matrices of ion-exchange resins and fiber

It is established that ion exchange resins AV-17-2P, KU-2, AV-16-GS, AM 21A, IMAC-HP, PUROLITE and fiber VION KN-1 can be applied as carriers for inulinase immobilization. The analysis of IR spectra for an enzyme, carriers and heterogeneous enzyme preparations showed that inulinase binding to matrices of various carriers occurs in general through electrostatic interactions. It is assumed that the mechanisms of interaction between inulinase from Kluyveromyces marxianus and the matrices of cation and anion exchange polymers differ essentially from each other: different sites of protein molecule take part in adsorption that causes various conformational reorganizations in an enzyme molecule.