Constitutive Chitosanase from Bacillus Thuringiensis B-387 and its Potential for Preparation of Antimicrobial Chitooligomers

Abstract Ability of the insecticide strain B. thuringiensis var. dendrolimus B-387 to high constitutive production (3-12.5 U/mL) of extracellular chitosanase was found for first time. The enzyme was purified in 94-fold by ultrafiltration, affinity sorption and cation-exchanged chromatography and characterized biochemically. 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 deacetylation degree (DD) 85% by endo-mode, and no degraded colloidal chitin, CM-cellulose and some other glucans. Main reaction products of the chitosan enzymolysis included chitobiose, chitotriose and chitotetraose. In addition to small chitooligosaccharides (CHOs), studied chitosanase generated also low-molecular weight chitosan (LMWC) with average Mw in range 14–46 kDa and recovery 14–35%, depending of enzyme / substrate ratio and incubation temperature. 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 initial polysaccharides. The findings evidence the chitosanase B-387 promising for bioactive CHOs preparation.