Biomedical subjects
Publications and source records attributed to G E Aktuganov.
Low-molecular-weight (3-6 kDa) water-soluble chitosan was obtained by enzymatic depolymerization. Hydrolysis of crab chitosan was induced by O-glycoside hydrolase (EC 3.2.1), an extracellular chitinolytic complex from Bacillus sp. 739. The optimum conditions for hydrolysis were found (sodium-acetate buffer, pH 5.2; 55 degrees C; an enzyme/substrate ratio 4 U/g chitosan; 1 h).
Investigation of the crude extracellular chitinase of Bacillus sp. 739, an antagonist of phytopathogenic fungi, discerned a relationship between the chitinase and antifungal activities of this bacterium. Purified chitinase lost its ability to inhibit the growth of micromycetes. The antagonistic (antifungal) activity of crude chitinase was found to be located in a low-molecular-weight fraction of the enzyme, which does not possess chitinase activity. Both crude and purified chitinase were able to lyse the cell walls of intact mycelium. Accordingly, it may be inferred that the antagonistic activity of Bacillus sp. 739 against micromycetes is largely determined by low-molecular-weight nonenzymatic substances whereas the role of chitinase is to utilize chitin, which is ubiquitously present in soil.
Thermolabile peptides inhibiting the growth of Helminthosporium sativum, a facultative phytopathogen, have been isolated from the low-molecular-weight fraction of extracellular metabolites of the strain Bacillus sp. 739. Paper chromatography of the fraction, followed by bioautography, revealed the presence of three components exhibiting antifungal activity. These components were separated by gel chromatography on Toyopearl HW-40. SDS-PAGE (the Laemmli procedure) demonstrated that only one component was a protein (MW, approximately 14 kDa). The other two substances were polypeptides with molecular weights less than 6 kDa each. The protein factor inhibited the growth of H. sativum with a minimum effective concentration of 0.1 to 0.2 mg/ml.
Among the 70 tested Bacillus spp. strains antagonistic to phytopathogenic fungi, 19 were found to possess chitinolytic activity when grown on solid media with 0.5% colloidal chitin. The chitinolytic activity of almost all of these 19 strains grown in liquid cultures ranged from 0.1 to 0.3 U/ml. One of the 19 strains exhibited exochitinase activity. In addition to chitinase, two strains also produced chitosanase and one strain, beta-1,3-glucanase. No correlation was found between the antifungal activity of the bacillar strains studied and their ability to synthesize extracellular chitinase. Among the 19 chitinolytic strains, the correlation between these parameters was also low (r x,y = 0.45), although the enzymatic preparations of most of these strains inhibited the growth of the phytopathogenic fungus Helminthosporium sativum.
The specific nature of the chitosanase activity of the strain Bacillus sp. 739 has been determined. Maximum enzyme activity was observed in a medium containing the biomass of the fruiting bodies of the fungus Macrolepiota procera. The chitosanase was purified to homogeneity using chromatography on DEAE-Sephadex A-50 and Toyopearl HW-50. The molecular weight of the enzyme, assessed by electrophoresis (the Laemmli procedure) approximated 46 kDa. Temperature and pH optima of the purified chitosanase were in the ranges 45-55 degrees C and 6.0-6.5, respectively. Time to half-maximum inactivation of the enzyme at 50 degrees C was equal to 1 h. With colloidal chitosan as the substrate, the value of K(M) of the purified chitosanase was equal to 25 mg/ml. The enzyme also exhibited a weak ability to hydrolyze colloidal chitin.
Species composition of micromycete complexes colonizing aspen, birch, and pine wood was studied. Calculation of the Sorens-Tchekanovsky similarity coefficients showed that these complexes shared a high degree of similarity. They were dominated by the representatives of the genera Penicillium, Paecilomyces, Trichoderma, and Rhizopus. Some antagonistic bacilli inhibited growth and development of wood-decay fungi in vitro and led to the formation of spheroplasts on growing hyphae. A study of possible involvement of bacillary mycolytic enzymes in the inhibition of wood-decay fungi demonstrated selectivity of their lytic effect, which was determined by the genus and species of micromycetes and did not correlate with their relative resistance to antagonists.