Vladimir E. Tikhonov

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.

Metal-chelating chitin derivatives via reaction of chitosan with nitrilotriacetic acid

Abstract Chitosan was cross-linked to various extents with nitrilotriacetic acid in the presence of a water-soluble carbodiimide in homogeneous solution, and was then fully N-acetylated. The structure of the nitrilotriacetic acid residues attached to the chitin backbone was determined by acid-base titration. It was shown that the chitin derivatives contained both iminodiacetate and iminomonoacetate residues which form 1:1-type complexes with copper ions. The chitin gels obtained swelled reversibly with water and behaved like ampholytic cross-linked resins in aqueous solutions of acids and bases.

Antifungal activity of oligochitosans (short chain chitosans) against some Candida species and clinical isolates of Candida albicans: molecular weight-activity relationship.

A series of oligochitosans (short chain chitosans) prepared by acidic hydrolysis of chitosan and characterized by their molecular weight, polydispersity and degree of deacetylation were used to determine their anticandidal activities. This study has demonstrated that oligochitosans show a high fungistatic activity (MIC 8-512 μg/ml) against Candida species and clinical isolates of Candida albicans, which are resistant to a series of classic antibiotics. Flow cytometry analysis showed that oligochitosan possessed a high fungicidal activity as well. For the first time it was shown that even sub-MIC oligochitosan concentration suppressed the formation of C. albicans hyphal structures, cause severe cell wall alterations, and altered internal cell structure. These results indicate that oligochitosan should be considered as a possible alternative/additive to known anti-yeast agents in pharmaceutical compositions.

Hydrophobically modified low molecular weight chitosans as efficient and nontoxic gene delivery vectors.

Chitosan derivatives are potential candidates for gene delivery because they are biocompatible and low toxic. However, their use has been limited by their moderate transfection efficiency and the rather large sizes of DNA complexes with high molecular weight chitosans. To circumvent these limitations, we used low molecular weight (approximately 5 kDa) chitosans grafted at 3 and 18 mol% with N‐/2(3)‐(dodec‐2‐enyl)succinoyl groups (HM‐LMW‐ch) that exhibit surfactant‐like properties.

Preparation of affinity sorbents and isolation of individual chitinases from a crude supernatant produced by Streptomyces kurssanovii by a one-step affinity-chromatographic system.

The preparation of two affinity‐chromatography sorbents based on cross‐linked chitin are described. Both sorbents retained selectively one of the four extracellular chitinases in the culture supernatant produced by Streptomyces kurssanovii. Chitinases with molecular masses of 42 kDa and 26 kDa were isolated in homogeneous form using one‐step affinity‐chromatography procedures involving either a fully N‐acetylated or a partially N‐acetylated cross‐linked chitin‐type sorbent. Two other chitinases were not selectively bound by the sorbents and, therefore, were not isolated in a homogeneous form. The affinity sorbents were shown to be stable over the period of separation and could be used repeatedly.

Novel liposomal forms of antifungal antibiotics modified by amphiphilic polymers

A novel method was developed to incorporate macrocyclic polyene antibiotics, nystatin and amphotericin B, into liposomes prepared from the mixture of phosphatidylcholine and cholesterol (7: 3) or phosphatidylcholine, cholesterol, and cardiolipin (7: 3: 1). Membranes of the liposomes were modified using the amphiphilic polymer N-vinylpyrrolidone with the molecular mass (MM) of the polymer fragment of 4000 and a single terminal n-octadecyl group. The content of the antibiotic incorporated within such nanosize liposomal carriers can reach 17–22%. The obtained modified liposomes, 150–200 nm in size, were more stable during prolonged storage and more resistant to various destructive factors, such as destabilizing agents (Triton X-100, ethanol) and ultrasound. The liposomal preparations showed higher antifungal activity than non-immobilized antifungal antibiotics.

Anticoagulant activity of low-molecular-weight sulfated derivatives of galactomannan from Cyamopsis tetragonoloba (L.) seeds

Galactomannan from seeds of Cyamopsis tetragonoloba (L.) Taub. (guar) was depolymerized using immobilized enzymatic preparation celloviridin. A set of fragments whose molecular weights varied from 12.6 to 245.6 kDa was obtained. Sulfated derivatives of components of all fractions were synthesized, in which the content of HSO3−-groups was 48.05 ± 2.31%. All preparations exhibited anticoagulant activity, which was recorded in vitro in two tests—aIIa and aXa. The antithrombin activity (aIIa) was high (up to 65–87 U/mg) and did not depend on the molecular weight of a sulfated derivative; in the second test (aXa), the effect of molecular weight was observed. Biospecific electrophoresis allowed us to detect the ability of galactomannan sulfates to form complexes with protamine sulfate, a classic antidote to heparin.

Affinity purification of major chitinases produced by Streptomyces kurssanovii

A highly O-cross-linked chitin-based sorbent resistant toward digestion by the extracellular chitinolytic complex of Streptomyces kurssanovii was prepared by means of cross-linking of N-acetylchitosan with 2,3-epoxypropyl chloride. Not less than four stages of cross-linking were required for the matrix to be fully resistant toward the action of chitinolytic enzymes. The sorbent was shown to be specific for two major endochitinases which were isolated in homogeneous forms quantitatively.

One-step isolation of a chitinase by affinity chromatography of the chitinolytic enzyme complex produced by Streptomyces kurssanovii.

One of the four chitinases with a molecular mass of 42 kDa existing in the chitinolytic enzyme complex produced by Streptomyces kurssanovii was separated in homogeneous form using one‐step affinity chromatography on cross‐linked and phosphorylated chitin‐type sorbents. Three other chitinases were not selectively bound by the sorbents and were not obtained in homogeneous form. The affinity sorbents were shown to be stable over the time of separation and could be used repeatedly.

Influence of glucosamine on oligochitosan solubility and antibacterial activity

Light scattering studies indicate that oligochitosan (short-chain chitosan) solutions contain aggregates at pH values below the critical pH of phase separation, while at or above this point the gel phase coexists with the aggregate solution. This work demonstrates for the first time that the presence of D-glucosamine in an oligochitosan solution shifts the critical pH to a higher value and improves the oligochitosan antibacterial activity against Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermis in neutral and slightly alkaline aqueous media. By comparing the results of light scattering studies and antimicrobial assays one can conclude that the antimicrobial activity of oligochitosan is dependent on its unimolecular form, not its supramolecular structures. The widening of the homogeneity region of an oligochitosan solution could lead to promising biomedical applications.