Abstract
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.
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This work was funded by Russian Foundation of Basic Research (RFBR), project number 19-34-90119 and was performed under government contract 075-00326-19-00 of the Ministry of Science and Higher Education of the Russian Federation on the subject AAAA-A18-118022190098-9.
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The main conceptualization and supervision by GA and AM; GA and VS carried out researches, analyzed the data and prepared the paper. NG, EG, LK, AB and SL contributed to experiments, methodologies, and resources. GA, VS and NG were responsible for discussion and editing.
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Aktuganov, G.E., Safina, V.R., Galimzianova, N.F. et al. Constitutive chitosanase from Bacillus thuringiensis B-387 and its potential for preparation of antimicrobial chitooligomers. World J Microbiol Biotechnol 38, 167 (2022). https://doi.org/10.1007/s11274-022-03359-5
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DOI: https://doi.org/10.1007/s11274-022-03359-5