The degrees of polymerization and N-acetylation of chitosan determine its ability to elicit callose formation in suspension cells and protoplasts of Catharanthus roseus

Partially and fully deacetylated chitosan fragments and oligomers were compared for their potency to elicit formation of the 1.3--glucan callose in suspension-cultured cells and protoplasts of Catharanthus roseus (line 385). Chitosan oligomers induced little callose formation, while callose synthesis increased with the degree of polymerization of chitosan up to several thousand corresponding to a molecular mass near 10⁶ Da. At a comparable degree of polymerization, partially N-acetylated chitosan fragments were less effective. Colloidal chitin and chitin oligomers induced only trace callose synthesis in protoplasts. These results indicate that the primary interaction involved the amino groups of chitosan and numerous negative charges at the surface of the plasma membrane with spacing in the nanometer range and occurring regularly over micrometer stretches. Charged phospholipid head-groups may fulfill these requirements. The resulting alteration of membrane fluidity may lead to the changes in ion transport known to be associated with the induction of callose formation.Abbreviations DP degree of polymerization - FDA fluorescein diacetate - PE pachyman equivalents     

Studies on chitosan: 3. Evidence for the presence of random and block copolymer structures in partially N-acetylated chitosans

The chemical structures of moderately N-deacetylated chitosans (MDC) derived from chitin under heterogeneous reaction conditions and partially N-acetylated chitosans (PAC) derived from highly N-deacetylated chitosans (HDC) under homogeneous reaction conditions were deduced from the data of the stability of their solutions in alkaline media, the swelling behaviour and X-ray diffraction patterns of their films in connection with the degree of N-acetylation of them. The solutions of PAC with more than 51% acetyl content, which were prepared from HDC by N-acetylation, were stable and remained clear and homogeneous by adding 1.2 equivalents of NaOH. On the contrary the solutions of PAC with more than 52% acetyl content, which were prepared from MDC, became turbid by neutralization with less than 1.15 equivalents of NaOH. The films of PAC prepared from HDC were highly swollen in water. The degree of swelling of the chitosan film with 51% acetyl content, prepared from the 6% acetyl content chitosan, was 121% while that of the 53% acetyl content chitosan, prepared from the 30% acetyl content chitosan, was 28%. From these data it was possible to set up a hypothesis that PAC prepared from HDC were considered as random-type copolymers of N-acetyl-glucosamine and glucosamine units whereas MDC were considered as block-type copolymers.     

Structural analysis of chitosan mediated DNA condensation by AFM: influence of chitosan molecular parameters

Chitosan is a nontoxic and biodegradable polysaccharide that has recently emerged as a promising candidate for gene delivery. Here the ability of various chitosans, differing in the fractional content of acetylated units (F(A)) and the degree of polymerization (DP), to compact DNA was studied. Polyplexes made from mixing plasmid DNA with chitosan yielded a blend of toroids and rods, as observed by AFM. The ratios between the fractions of toroids and rods were observed to decrease with increasing F(A) of the chitosan, indicating that the charge density of chitosan, proportional to (1 - F(A)), is important in determining the shape of the compacted DNA. The amount of chitosan required to fully compact DNA into well-defined toroidal and rodlike structures were found to be strongly dependent on the chitosan molecular weight, and thus its total charge. A higher charge ratio (+/-) was needed for the shorter chitosans, showing that an increased concentration of the low DP chitosan could compensate for the reduced interaction strength of the individual ligands with DNA. Employing chitosans with different molecular parameters offers the possibility of designing DNA-chitosan polyplexes with various geometries, reflecting various chitosan-DNA interaction strengths, which is necessary for the evaluation of efficient gene delivery vehicles.     

Characterization of Some Fungal Chitosans

Chitosan-like materials were extracted from five different fungal cells with NaOH and acetic acid, with the yields varying from 1.2 to 10.4% of the dry fungal cell weight. The degree of N-acetylation of the extracts measured by the colloidal titration method varied considerably depending on the individual species. By IR measurements and the Elson-Morgan method, four kinds of the extracts were characterized as chitosan while another one was not.

The degree of N-acetylation and the Cu²⁺ adsorption capacity of the fungal chitosans were measured and compared with those of authentic samples with various degrees of N-acetylation, which were prepared by chemical treatment of authentic chitin and chitosan derived from Crustacea. The Cu²⁺ adsorption capacity of the fungal chitosans was higher than that of the authentic chitosan samples with similar degrees of N-acetylation and independent of the molecular weight of the chitosans from the various sources.     

PEGylated chitosan complexes DNA while improving polyplex colloidal stability and gene transfection efficiency

Chitosan is widely explored as a gene delivery vehicle due to its ability to condense DNA, facilitate transport, and subsequent release allowing gene expression, as well as protecting the DNA. Here, we investigate the enhancement of chitosan–DNA dispersion stability while maintaining transfection efficacy by PEGylation of chitosan. Molecular properties of fully deacetylated chitosans and degree of PEGylation were investigated with respect to compaction of DNA, stability and transfection efficacy. Each of the three chitosan samples with varying chain lengths was PEGylated at three different degrees. The chitosans with degree of PEGylation from 0.6 to 1.9% made polyplexes with DNA. PBS induced colloidal aggregation of polyplexes with initial radius of about 100 nm observed for nonPEGylated chitosans was suppressed for 1.9% PEGylated chitosans. The observed increase in transfection efficacy coinciding with increased polyplex colloidal stability suggests that aggregation of gene-delivery packages may reduce the transfection efficacy.     

N-(carboxymethylidene)chitosans and N-(carboxymethyl)chitosans: Novel chelating polyampholytes obtained from chitosan glyoxylate

Glyoxylic acid, added to aqueous suspensions of chitosan, causes immediate dissolution of chitosan and gel formation within 3–4 h if the pH is 4.5–5.5. Solutions at lower pH values gel after 2 min of warming at 60–80°. Chitosan glyoxylate solutions brought to alkaline pH with sodium hydroxide do not precipitate chitosan. Evidence is given that a Schiff base, namely N-(carboxymethylidene)chitosan, is formed. N-(Carboxymethylidene)chitosans are reduced by sodium cyanoborohydride at room temperature to give N-(carboxymethyl)chitosans, obtained as white, free-flowing powders, soluble in water at all pH values. A series of N-(carboxymethyl)chitosans having various degrees of acetylation and N-carboxymethylation was obtained, and characterized by viscometry, elemental analysis, and i.r. spectrometry. For the fully substituted N-(carboxymethyl)chitosans, the pK′ is 2.3, the pK″ is 6.6, and the isoelectric point is 4.1. The addition of N-(carboxymethyl)chitosan to solutions (0.2–0.5mm) of transition-metal ions produces immediate insolubilization of N-(carboxymethyl)chitosan-metal ion chelates.     

Preparation and characterisation of oligosaccharides produced by nitrous acid depolymerisation of chitosans.

Two chitosans with widely different chemical composition (fraction of N-acetylated units (F(A))<0.001 and F(A)=0.59), were degraded by nitrous acid, to obtain the reactive 2,5-anhydro-D-mannose- (M-) unit at the new reducing end. The fully N-acetylated and fully N-deacetylated oligomers were separated by size-exclusion chromatography. Both the chemical structure and purity were studied by one- and two-dimensional 1H and 13C NMR methods. The fully N-acetylated oligomers were found to be stable, whereas the N-deacetylated oligomers reacted intermolecularly by a Schiff base reaction between the 2-amino group on the N-deacetylated units and the M-units, facilitating the cleavage of the glycosidic bond next to the M-unit and the formation of 5-hydroxymethylfurfural (HMF).     

Antitumor activity of chitosan from mayfly with comparison to commercially available low,medium and high molecular weight chitosans

Insects’ cuticles have a potential to be evaluated as a chitin source. Especially adults of aquatic insects like mayflies (order Ephemeroptera) swarm in enormous numbers in artificially lit areas while mating in spring and then die by leaving huge amounts of dead insects’ bodies. Here in this study, mayfly corpses were harvested and used for production of low MW chitosan. Dried mayfly bodies had 10.21% chitin content; mayfly chitin was converted into chitosan with efficiency rate of 78.43% (deacetylation degree, 84.3%; MW, 3.69 kDa). Cytotoxicity and anti-proliferative activity of mayfly and commercially available shrimp chitosans (low, medium, and high MW) were determined on L929 fibroblast and three different cancer types including HeLa, A549, and WiDr. Apoptosis and necrosis stimulating potential of mayfly and commercial chitosans were also evaluated on A549 and WiDr cells using acridine orange and propidium iodide dual staining to observe morphological changes in nuclei and thus to reveal the predominant cell death mechanism. The effects of chitosans have varied depending on cell types, concentration, and chitosan derivatives. Mayfly and low MW chitosans had a cytotoxic effect at a concentration of 500 μg mL^?1 on non-cancer cells. At concentrations below this value (250 μg mL^?1), mayfly and commercial chitosans except high MW one exhibited strong inhibitory activity on cancer cells especially A549 and WiDr cells. Mayfly chitosan induced early and late apoptosis in A549 cells, but late apoptosis and necrosis in WiDr cells. This study suggests that dead bodies of mayflies can be used for production of low MW chitosan with anti-proliferative activity.     

Synthesis, characterization, and antibacterial activity of N,O-quaternary ammonium chitosan

N,N,N-Trimethyl O-(2-hydroxy-3-trimethylammonium propyl) chitosans (TMHTMAPC) with different degrees of O-substitution were synthesized by reacting O-methyl-free N,N,N-trimethyl chitosan (TMC) with 3-chloro-2-hydroxy-propyl trimethyl ammonium chloride (CHPTMAC). The products were characterized by ¹H NMR, FTIR and TGA, and investigated for antibacterial activity against Staphylococcus aureus and Escherichia coli under weakly acidic (pH 5.5) and weakly basic (pH 7.2) conditions. TMHTMAPC exhibited enhanced antibacterial activity compared with TMC, and the activity of TMHTMAPC increased with an increase in the degree of substitution. Divalent cations (Ba²⁺ and Ca²⁺) strongly reduced the antibacterial activity of chitosan, O-carboxymethyl chitosan and N,N,N-trimethyl-O-carboxymethyl chitosan, but the repression on the antibacterial activity of TMC and TMHTMAPC was weaker. This indicates that the free amino group on chitosan backbone is the main functional group interacting with divalent cations. The existence of 100 mM Na⁺ slightly reduced the antibacterial activity of both chitosan and its derivatives.     

A chitinase with high activity toward partially<Emphasis Type="Italic"> N</Emphasis>-acetylated chitosan from a new,moderately thermophilic,chitin-degrading bacterium,<Emphasis Type="Italic"> Ralstonia</Emphasis> sp. A-471

A moderately thermophilic bacterium, strain A-471, capable of degrading chitin was isolated from a composting system of chitin-containing waste. Analysis of the 16S rDNA sequence revealed that the bacterium belongs to the genus Ralstonia. A thermostable chitinase A (Ra-ChiA) was purified from culture fluid of the bacterium grown in colloidal chitin medium. Purification of the enzyme was achieved mainly by exploiting its binding to the colloidal chitin. The molecular mass of the enzyme was estimated to be 70 kDa and the isoelectric point approximately 4.7. N-terminal amino acid sequencing revealed a sequence of ADPYLKVAYYP, which had high homology (66% identity) with that of chitinase A1 from Bacillus circulans WL-12. The pH and temperature optima were determined to be 5.0 and 70°C, respectively. The enzyme was classified as a retaining glycosyl hydrolase and was most active against partially N-acetylated chitosans. Its activities towards the partially N-acetylated chitosans, i.e. chitosan 7B, chitosan 8B, and chitosan 9B, were about 11-fold, 9-fold, and 5-fold higher than towards colloidal chitin, respectively. Ra-ChiA cleaved (GlcNAc)₆ almost exclusively into (GlcNAc)_2. Activation of Ra-ChiA was observed by the addition of 1 mM Cu²⁺, Mn²⁺, Ca²⁺_, or Mg²⁺. Degradation of the partially N-acetylated chitosan produced oligosaccharides with a degree of polymerization ranging from 1–8; these are products that offer potential application for functional oligosaccharide production.     

Physicochemical characterization of chitin and chitosan from crab shells

Crab chitosan was prepared by alkaline N-deacetylation of crab chitin for 60, 90 and 120 min and the yields were 30.0-32.2% with that of chitosan C120 being the highest. The degree of N-deacetylation of chitosans (83.3–93.3%) increased but the average molecular weight (483–526 kDa) decreased with the prolonged reaction time. Crab chitosans showed lower lightness and WI values than purified chitin, chitosans CC and CS but higher than crude chitin. With the prolonged reaction time, the nitrogen (8.9–9.5%), carbon (42.2–45.2%) and hydrogen contents (7.9–8.6%) in chitosans prepared consistently increased whereas N/C ratios remained the same (0.21). Crab chitosans prepared showed a melting endothermic peak at 152.3–159.2 °C. Three chitosans showed similar microfibrillar crystalline structure and two crystalline reflections at 2θ = 8.8–9.0° and 18.9–19.1°. Overall, the characteristics of three crab chitosans were unique and differed from those of chitosan CC and CS as evidenced by the element analysis, differential scanning calorimetry, scanning electron microscopy and X-ray diffraction patterns.     

壳聚糖对植物病原细菌的抑制作用研究

本文通过测定最小抑制浓度和相对抑制率,观察了分子量和脱乙酰度对壳聚糖抑制植物病原细菌(胡萝卜软腐欧文氏菌Erwinia cartovara Var carotovara、油菜黄单孢菌绒毛草致病菌Xanthamonas campestris Pv holcicola、丁香假单孢菌黍致病变种Pseudomonas spyings Pv panici)作用的影响。结果表明：在一定范围内,随分子量和脱乙酰度的增大,壳聚糖的抑菌效果相应降低,而且各种病原细菌对不同,壳聚糖的敏感性也有很大差异。     

Antimicrobial Characteristics of Chitosans against Food Spoilage Microorganisms in Liquid Media and Mayonnaise

Four different kinds of chitosans were prepared by treating crude chitin with various NaOH concentrations. The antimicrobial activities of the chitosans were tested against four species of food spoilage microorganisms (Lactobacillus plantarum, Lactobacillus fructivorans, Serratia liquefaciens, and Zygosaccharomyces bailii). The initial effect of the chitosans was biocidal, and counts of viable cells were significantly reduced. After an extended lag phase, some strains recovered and resumed growth. The activities of chitosan against these microorganisms increased with the concentration. Chitosan-50 was most effective against L. fructivorans, but inhibition of L. plantarum was greatest with chitosan-55. There was no significant difference among the chitosans in their antimicrobial activity against S. liquefaciens and Z. bailii. The addition of chitosan to mayonnaise significantly decreased the viable cell counts of L. fructivorans and Z. bailii during storage at 25°C. These results suggest that chitosan can be used as a food preservative to inhibit the growth of spoilage microorganisms in mayonnaise.     

The effect of size and acetylation degree of chitosan derivatives on tobacco plant protection against Phytophthora parasitica nicotianae

Enzymatic degradation of chitosan polymer with Pectinex Ultra SPL was used to obtain derivatives with biological potential as protective agents against Phytophthora parasitica nicotianae (Ppn) in tobacco plants. The 24 h hydrolysate showed the highest Ppn antipathogenic activity and the chitosan native polymer the lowest. The in vitro growth inhibition of several Phytophthora parasitica strains by two chitosans of different DA was compared. While less acetylated chitosan (DA 1%) fully inhibited three P. parasitica strains at the doses 500 and 1000 mg/l the second polymer (DA 36.5%) never completely inhibited such strains. When comparing two polymers of similar molecular weight and different DA, again the highest antipathogenic activity was for the less acetylated polymer. However, degraded chitosan always showed the highest pathogen growth inhibition. Additionally, a bioassay in tobacco seedlings to test plant protection against Ppn by foliar application demonstrated that partially acetylated chitosan and its hydrolysate induced systemic resistance and higher levels of glucanase activity than less acetylated chitosan. Similarly, when treatments were applied as seeds coating before planting, about 46% of plant protection was obtained using chitosan hydrolysate. It was concluded that, while less acetylated and degraded chitosan are better for direct inhibition of pathogen growth, partially acetylated and degraded chitosan are suitable to protect tobacco against P. parasitica by systemic induction of plant resistance.     

Proteinase inhibitor synthesis in tomato leaves : induction by chitosan oligomers and chemically modified chitosan and chitin

Soluble chemical derivatives of chitin and chitosan including ethylene glycol chitin, nitrous acid-modified chitosan, glycol chitosan, and chitosan oligomers, produced from chitosan by limited hydrolysis with HCl, were found to possess proteinase inhibitor inducing activities when supplied to young excised tomato (Lycopersicon esculentum var Bonnie Best) plants. Nitrous acid-modified chitosans and ethylene glycol chitin exhibited about 2 to 3 times the activity of acid hydrolyzed chitosan and 15 times more activity than glycol chitosan. The parent chitin and chitosans are insoluble in water or neutral buffers and cannot be assayed. Glucosamine and its oligomers from degree of polymerization = 2 through degree of polymerization = 6 were purified from acid-fragmented chitosan and assayed. The monomer was inactive and dimer and trimer exhibited weak activities. Tetramer possessed higher activity and the larger pentamer and hexamer oligomers were nearly as active as the total hydrolyzed mixture. None of the fragments exhibited more than 2% acetylation (the limits of detection). The contents of the acid-fragmented mixture of oligomers was chemically N-acetylated to levels of 13% and 20% and assayed. The N-acetylation neither inhibited nor enhanced the proteinase inhibitor inducing activity of the mixture. These results, along with recent findings by others that chitinases and chitosanases are present in plants, provide further evidence for a possible role of soluble chitosan fragments as signals to activate plant defense responses.     

Utilization of partially N-succinylated derivatives of chitosan and glycolchitosan as supports for the immobilization of enzymes

Polymer ampholites, partially N-succinylated chitosans (PSC), and partially N-succinylated glycolchitosans (PSGC) were prepared from chitosan (an N-deacetylated chitin) and glycolchitosan (a partially O-2-hydroxyethylated chitosan), and they were utilized as novel supports for the immobilization of enzymes. The immobilization was conducted simultaneously with gelation of PSC and PSGC by reaction with water-soluble carbodiimide in the presence of enzymes. Enzymes were covalently bonded on PSC and PSGC chains. Maximum activity yields of glucoamylase, beta-fructosidase, and D-glucose isomerase were 58.8, 64.3, and 65.2%, respectively. Favorable activity yields of glucoamylase and beta-fructosidase were attained with PSC and PSGC having high degree of N-succinylation, but those of D-glucose isomerase were not affected by the degree of N-succinylation (DS). The activity of immobilized glucoamylase was retained up to 85.5% over 30 batch reactions.     

Antimicrobial characteristics of chitosans against food spoilage microorganisms in liquid media and mayonnaise.

Four different kinds of chitosans were prepared by treating crude chitin with various NaOH concentrations. The antimicrobial activities of the chitosans were tested against four species of food spoilage microorganisms (Lactobacillus plantarum, Lactobacillus fructivorans, Serratia liquefaciens, and Zygosaccharomyces bailii). The initial effect of the chitosans was biocidal, and counts of viable cells were significantly reduced. After an extended lag phase, some strains recovered and resumed growth. The activities of chitosan against these microorganisms increased with the concentration. Chitosan-50 was most effective against L. fructivorans, but inhibition of L. plantarum was greatest with chitosan-55. There was no significant difference among the chitosans in their antimicrobial activity against S. liquefaciens and Z. bailii. The addition of chitosan to mayonnaise significantly decreased the viable cell counts of L. fructivorans and Z. bailii during storage at 25 degrees C. These results suggest that chitosan can be used as a food preservative to inhibit the growth of spoilage microorganisms in mayonnaise.     

Influence of chitosan structure on the formation and stability of DNA-chitosan polyelectrolyte complexes

The interactions between DNA and chitosans varying in fractional content of acetylated units (FA), degree of polymerization (DP), and degree of ionization were investigated by several techniques, including an ethidium bromide (EtBr) fluorescence assay, gel retardation, atomic force microscopy, and dynamic and electrophoretic light scattering. The charge density of the chitosan and the number of charges per chain were found to be the dominating factors for the structure and stability of DNA-chitosan complexes. All high molecular weight chitosans condensed DNA into physically stable polyplexes; however, the properties of the complexes were strongly dependent on FA, and thereby the charge density of chitosan. By employing fully charged oligomers of constant charge density, it was shown that the complexation of DNA and stability of the polyplexes is governed by the number of cationic residues per chain. A minimum of 6-9 positive charges appeared necessary to provide interaction strength comparable to that of polycations. In contrast, further increase in the number of charges above 9 did not increase the apparent binding affinity as judged from the EtBr displacement assay. The chitosan oligomers exhibited a pH-dependent interaction with DNA, reflecting the number of ionized amino groups. The complexation of DNA and the stability of oligomer-based polyplexes became reduced above pH 7.4. Such pH-dependent dissociation of polyplexes around the physiological pH is highly relevant in gene delivery applications and might be one of the reasons for the high transfection activity of oligomer-based polyplexes observed.     

Studies on chitosan: 4. Lysozymic hydrolysis of partially N-acetylated chitosans.

The lysozymic digestibility of partially N-acetylated chitosans was studied by measuring the reducing sugars produced and the molecular weights of their hydrolysates. Moderately N-deacetylated chitosans (MDC), obtained by N-deacetylation of chitin under heterogeneous conditions, were about four times more digestible at an early stage than partially N-acetylated chitosans (PAC-H) with similar acetyl content, prepared by N-acetylation of highly N-deacetylated chitosans under homogeneous conditions. The molecular weights of the hydrolysates of MDC decreased rapidly but gradually reached a constant value in contrast to the behaviour of PAC-H. The Km was 0.14 mM for 30% N-acetylated MDC and 0.12 mM for 65% N-acetylated PAC-H although the degree of N-acetylation of the latter was twice as much as the former. These differences were due to the different distribution patterns of N-acetyl groups in two types of the chitosans. MDC with 20-30% acetyl content have the sequences of more than three N-acetyl-D-glucosamine residues but PAC-H with about 30% acetyl content are random-type copolymers of N-acetyl-D-glucosamine and D-glucosamine units. PAC-H with more than 50% acetyl content have the sequences of more than three N-acetyl-D-glucosamine residues.     

Determination of the degree of N-acetylation and the distribution of N-acetyl groups in partially N-deacetylated chitins (chitosans) by high-field n.m.r. spectroscopy.

The composition and sequence of 2-acetamido-2-deoxy-beta-D-glucose (GlcNAc) and 2-amino-2-deoxy-beta-D-glucose (GlcN) residues in partially N-deacetylated chitosans, prepared under homogeneous and heterogeneous conditions, have been determined by 1H-n.m.r. spectroscopy. It was necessary to depolymerise the chitosan slightly by treatment with nitrous acid before spectroscopy. A sequence-dependent deshielding of H-1 of the GlcNAc residues made it possible to determine the proportions of the four possible diads. Chitosan prepared by N-deacetylation under homogeneous conditions gave values for the diad frequencies that were roughly consistent with a random distribution of the N-acetyl groups. Samples prepared under heterogeneous conditions have a frequency of the GlcNAc-GlcNAc diad slightly higher than for a random (Bernoullian) distribution. The chitosans, prepared under both homogeneous and heterogeneous conditions, with a degree of acetylation of 50% were soluble at neutral pH.