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.