V. P. Varlamov

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

Low-molecular-weight chitosans with a viscosity-average molecular weight (Mν) of 5 to 27 kDa and an equal degree of deacetylation (DD, 85%) were highly active against Pseudomonas aureofaciens, Enterobacter agglomerans, Bacillus subtilis, and Bifidobacterium bifidum791, causing death in 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 the 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 Mν 5, 6, 12, 15.7, and 27 kDa: the minimum inhibitory concentration (MIC) varied from 0.06 to 0.005%. Chitosans with Mν 5, 12, and 15.7 kDa exerted an antibacterial effect on Staphylococcus aureus. Chitosans with Mν 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%.

Enzymic preparation of acid-free-water-soluble chitosan

Abstract A chitinolytic enzyme complex produced by the Gram-positive bacterium Streptomyces kurssanovii was immobilized on macroporous cross-linked chitin by physical adsorption, and the biocatalyst obtained used for the preparation of acid-free-water soluble chitosan with a molecular weight of 2–9 kDa starting from commercial crab chitosan by two-step hydrolysis. The first hydrolysis at pH 4.6 lead to a 22–24 kDa chitosan of low solubility in water whereas the second hydrolysis at pH 6.2 allowed a 2–9 kDa acid-free-water soluble chitosan. Optimal conditions for the complex immobilization and chitinolytic hydrolysis are described. The method allows not less than 80-cyclic procedures of immobilized complex and chitin matrix utilization, and is suitable for a large-scale production of the chitosan free of protein admixtures.

[Chitosan-based polyelectrolyte complexes: a review].

This review focuses on the formation of polyelectrolyte chitosan complexes with biologically active compounds and the prospects of use thereof. The possibility of obtaining low-molecular-weight, water-soluble batches of chitosan, which differ in their degree of acetylation, is discussed, with emphasis on their use for binding nucleic acids into complexes.

[Modulation of plant resistance to diseases by water-soluble chitosan].

Low-molecular-weight water-soluble chitosan (5 kDa) obtained after enzymatic hydrolysis of native crab chitosan was shown to display an elicitor activity by inducing the local and systemic resistance of Solanum tuberosum potato and Lycopesicon esculentum tomato to Phytophthora infestans and nematodes, respectively. Chitosan induced the accumulation of phytoalexins in tissues of host plants; decreased the total content; changed the composition of free sterols producing adverse effects on infesters; activated chitinases, β-glucanases, and lipoxygenases; and stimulated the generation of reactive oxygen species. The activation of protective mechanisms in plant tissues inhibited the growth of taxonomically different pathogens (parasitic fungus Phytophthora infestans and root knot nematode Meloidogyne incognita).

Effect of the molecular weight of chitosan on its antiviral activity in plants

The effect of the molecular weight of chitosan on its ability to suppress systemic infection of bean mild mosaic virus in bean (Phaseolus vulgaris L.) plants was studied. The enzymatic hydrolysate of low-molecular-weight chitosan was successively fractionated by ultrafiltration through membranes with decreasing pore size. In total, four chitosan fractions with a weight-average molecular weight varying from 1.2 to 40.4 kDa were obtained. It was shown that the treatments of bean plants with these fractions (chitosan concentration, 10 or 100 μg/ml) inhibited virus accumulation and systemic propagation. The degree of chitosan-induced antiviral resistance increased as the molecular weight of chitosan decreased. The monomers comprising the chitosan molecule—glucosamine and N-acetylglucosamine—exhibited no antiviral activity.

Hydrolysis of Chitosan in Lactic Acid

The effects of molecular weight and the degree of acetylation on the hydrolysis of chitosan in dilute lactic acid were studied. It was demonstrated that the higher the values of both parameters, the more rapid the decreases in viscosity and the viscosity-average molecular weight of chitosan.

Isolation of Chitin and Chitosan from Honeybees

A procedure of isolation of chitin, chitosan, and water-soluble low-molecular-weight chitosan from the corpses of bees has been developed. This procedure includes deproteinization of bee corpses, discoloration of the chitin–melanin complex, deacetylation, and enzymatic hydrolysis of chitosan.

Ultrastructural study of chitosan effects on Klebsiella and staphylococci.

Antibacterial effect of chitosan on the morphofunctional organization of clinical strains of Klebsiella pneumoniae and Staphylococcus aureus was studied by transmission electron microscopy. Chitosan promoted aggregation of bacterial cells and disorganization of bacterial cell wall and cytoplasmic membrane, which leads to the release of bacterial contents into the environment. These structural changes result in bacterial death.

THE PREPARATION OF LOW-MOLECULAR-WEIGHT CHITOSAN USING CHITINOLYTIC COMPLEX FROM STREPTOMYCES KURSSANOVII

Abstract 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- d -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%.

Characterization of Protein and Peptide Binding to Nanogels Formed by Differently Charged Chitosan Derivatives

Chitosan (Chi) is a natural biodegradable cationic polymer with remarkable potency as a vehicle for drug or vaccine delivery. Chi possesses multiple groups, which can be used both for Chi derivatization and for particle formation. The aim of this work was to produce stable nanosized range Chi gels (nanogels, NGs) with different charge and to study the driving forces of complex formation between Chi NGs and proteins or peptides. Positively charged NGs of 150 nm in diameter were prepared from hexanoyl chitosan (HC) by the ionotropic gelation method while negatively charged NGs of 190 nm were obtained from succinoyl Chi (SC) by a Ca2+ coacervation approach. NGs were loaded with a panel of proteins or peptides with different weights and charges. We show that NGs preferentially formed complexes with oppositely charged molecules, especially peptides, as was demonstrated by gel-electrophoresis, confocal microscopy and HPLC. Complex formation was accompanied by a change in zeta-potential and decrease in size. We concluded that complex formation between Chi NGs and peptide/proteins is mediated mostly by electrostatic interactions.