A novel halophilic and solvent-stable chitinase from Enterobacter sp. SFM-22 with biofungicidal potential

With the development of eco-friendly technology, bacterial chitinolytic enzymes are effectively used to degrade chitin-based wastes into various valuable products. This study aims to isolate, identify, and characterize a new chitin-degrading bacterium, Enterobacter sp. SFM-22, for producing extracellular chitinase. Various submerged fermentation parameters (physical and nutritional) were evaluated to boost extracellular chitinase production, resulting in a 3.51-fold increase when 1.5% (v/v) primary culture was used to inoculate a modified 2NB (pH 6.5) medium [1.5% (w/v) colloidal chitin, 1% (w/v) urea, and sucrose], followed by incubation for 72 h under shaking (150 rpm) at 40 °C. Purified chitinase has a molecular weight of 40 kDa and showed optimal activity at 55 °C and pH 6.5 (McIlvaine buffer), with a specific activity of 281.62 U mg^-1 using colloidal chitin as a substrate. Chitinase exhibited notable pH (5.0-8.5) and thermal stability (20-60 °C) for 2 h, along with tolerance toward various organic solvents (10-40% v/v), metallic ions (10-20 mM), surfactants, chemical inhibitors, and NaCl. SFM-22 chitinase activity was significantly stimulated by 4% (w/v) NaCl, as well as 10 mM Ca²⁺, Mg²⁺, Mn²⁺, urea, Na⁺, EDTA, Fe²⁺, and EGTA. The kinetic parameters, including V_max, K_m, k_cat, and k_catK_m^-1, were 356.41 μmol mg^-1 min^-1, 0.76 mg mL^-1, 971 s^-1, and 1277.63 mL mg^-1 s^-1, respectively, using colloidal-chitin. The chitinase demonstrated biocatalytic activity against various fungal pathogens; therefore, it is a promising candidate for use as a biofungicide in agriculture. Additionally, this solvent-, thermo-, and halo-tolerant chitinase could also be applied in other industries (such as food, pharmaceuticals, and chitinous waste management) to support ecological sustainability.