Constitutive chitosanase from Bacillus thuringiensis B-387 and its potential for preparation of antimicrobial chitooligomers.

The article proves the ability of the entomopathogenic strain B. thuringiensis var. dendrolimus B-387 to high the constitutive production (3-12.5 U/mL) of extracellular chitosanase, that was found for the first time. The enzyme was purified in 94-fold by ultrafiltration, affinity sorption and cation-exchange chromatography and characterized biochemically. The molecular mass of the chitosanase determined using SDS-PAGE is 40 kDa. Temperature and pH-optima of the enzyme are 55 °C and pH 6.5, respectively; the chitosanase was stable under 50-60 °C and pH 4-10.5. Purified chitosanase most rapidly (Vmax ~ 43 µM/mL × min, KM ~ 0.22 mg/mL, kcat ~ 4.79 × 104 s-1) hydrolyzed soluble chitosan of the deacetylation degree (DD) 85% by endo-mode, and did not degrade colloidal chitin, CM-cellulose and some other glucans. The main reaction products of the chitosan enzymolysis included chitobiose, chitotriose and chitotetraose. In addition to small chitooligosaccharides (CHOs), the studied chitosanase also generated low-molecular weight chitosan (LMWC) with average Mw in range 14-46 kDa and recovery 14-35%, depending on the enzyme/substrate ratio and incubation temperature. In some cases, the chitosan (DD 85 and 50%) oligomers prepared using crude chitosanase from B. thuringiensis B-387 indicated higher antifungal and antibacterial activities in vitro in comparison with the initial polysaccharides. The data obtained indicate the good prospect of chitosanase B-387 for the production of bioactive CHOs.

The Novel Strain Acidomyces acidophilum Isolated from Acidophilic Biofilms (Snottites) Located in the Sheki-Heh Cave (North Caucasus).

A novel acidophilic fungal strain isolated from snottites in the active sulfuric acid speleogenesis (SAS) Sheki-Heh Cave (North Caucasus, Chechen Republic) was identified and characterized. The Sheki-Heh Cave is one of three cavities of the joint SAS speleosystem; to date, it remains the only of such cave explored in Russia. Highly acidic biofilms termed snottites are found sporadically on the cave roof in sulfurous water degassing zones. Only dark-colored micromycete colonies were isolated from these microbial biofilms using direct inoculation onto Czapek agar. The dominant fungal isolate was selected for further characterization. This work aimed to identify the micromycete strain isolated from cave snottites and explore its growth characteristics. Based on the phylogenetic analysis of the rDNA ITS region (540 bp), the novel fungal strain was identified as Acidomyces acidophilum with a similarity level of 99.26%. The physiological properties of the strain were examined; the optimal pH and temperature for its growth were pH 3 and 20-28 °C, respectively. Strain IB-G85 is able to grow under NaCl concentrations up to 3%. Although IB-G85 was isolated from an oligotrophic environment and was growing under nutrient deficiency, it could utilize some sugars and proteins as well as recalcitrant substrates, such as chitin and tannin. Compared to base Czapek-Dox Agar, lactic acid and colloidal chitin as the sole carbon sources enhanced fungal growth by 100 and 59%, respectively. The occurrence of A. acidophilum and closely related fungal species within acidophilic microbial communities inhabiting sulfur-containing ecosystems is discussed in view of their contribution to snottite structure formation in SAS caves.

Purification and characterization of exo-ß-1,4-glucosaminidase produced by chitosan-degrading fungus, Penicillium sp. IB-37-2A.

Chitosan-degrading fungal strain, Penicillium sp. IB-37-2A, produced mainly extracellular chitosanolytic enzymes under submerged agitating cultivation in presence of soluble chitosan or colloidal chitin as main carbon source. Significant N-acetyl-ß-D-glucosaminidase activity (8-18 × 103 U·ml-1) was also detected in culture filtrate of the fungal strain. Alone major exo-chitosanase from culture filtrate of Penicillium sp. IB-37-2A was purified in 46-fold using ultrafiltration, affinity sorption on colloidal chitosan and hydrophobic chromatography on Phenyl-Sepharose CL 4B and characterized. Molecular weight of the exo-ß-1.4-glucosaminidase is 41 kDa according to SDS-PAGE. The purified enzyme has optima pH and temperature 4.0 and 50-55 °C, respectively, pI 4.9; it is stable under pH 3.0-8.0 and 55 °C. Activity of the enzyme is strongly inhibited by 1 mM Hg2+ and Ag+, in less degree-10 mM Cu2+, Zn2+, Ni+ and Fe2+, slightly activated-with 1 mM Mg2+, 10 mM Ca2+, tween-80 (10 mM) and Triton X-100 (1 mM). Viscosimetric assay confirmed reported earlier exo-splitting manner of the enzyme activity. Soluble chitosan (deacetylation degree (DD) 80-85%) is most rapidly hydrolyzed by the enzyme (Vmax = 7.635 µM × min-1 × mg-1, KM ~ 0.83 mg/ml). Purified exo-chitosanase also degraded laminarin, ß-glucan, colloidal chitin and showed significant chitobiohydrolase activity (V ~ 50 µM × ml-1 × min-1 for pNP-GlcNAc2) but no hydrolyzed CMC, cellulose, xylan and galactomannan. It is found that crude and partially purified exo-ß-1.4-glucosaminidase inhibits in vitro the growth of some phytopathogenic fungi that is first report for antifungal activity of exo-chitosanase.

Cytokinin producing bacteria stimulate amino acid deposition by wheat roots.

Phytohormone production is one mechanism by which rhizobacteria can stimulate plant growth, but it is not clear whether the bacteria gain from this mechanism. The hypothesis that microbial-derived cytokinin phytohormones stimulate root exudation of amino acids was tested. The rhizosphere of wheat plants was drenched with the synthetic cytokinin trans-zeatin or inoculated with Bacillus subtilis IB-22 (which produces zeatin type cytokinins) or B. subtilis IB-21 (which failed to accumulate cytokinins). Growing plants in a split root system allowed spatial separation of zeatin application or rhizobacterial inoculation to one compartment and analyses of amino acid release from roots (rhizodeposition) into the other compartment (without either microbial inoculation or treatment with exogenous hormone). Supplying B. subtilis IB-22 or zeatin to either the whole root system or half of the roots increased concentrations of amino acids in the soil solution although the magnitude of the increase was greater when whole roots were treated. There was some similarity in amino acid concentrations induced by either bacterial or zeatin treatment. Thus B. subtilis IB-22 increased amino acid rhizodeposition, likely due to its ability to produce cytokinins. Furthermore, B. subtilis strain IB-21, which failed to accumulate cytokinins in culture media, did not significantly affect amino acid concentrations in the wheat rhizosphere. The ability of rhizobacteria to produce cytokinins and thereby stimulate rhizodeposition may be important in enhancing rhizobacterial colonization of the rhizoplane.