Separation, Purification and Characterization of Chitinase of the Endophytic Bacterium Bacillus thuringiensis Bt028 Isolated from Sour Orange

In order to reveal the basic enzymatic properties of the chitinase produced by an endophytic bacterium Bacillus thuringiensis Bt028 isolated from sour orange fruit, the chitinase in the strain fermentation broth were purified by centrifugation, ammonium sulfate precipitation, dextran gel G-100 chromatography and SDS-polyacrylamide gel electrophoresis and the optimum temperature, optimum pH and catalytic kinetic parameters of the chitinase were also investigated. The results showed that electrophoretic pure chitinase was obtained by centrifugation, ammonium sulfate precipitation and dextran G-100 gel chromatography from fermentation broth of the strain Bt028, with the specific activity of 681.78 U/mg, the purification factor of 3.21 and enzymatic activity recovery of 15.52%, and the molecular mass of the chitinase was determined to be 65 kDa by SDS-polyacrylamide gel electrophoresis. The enzymatic characteristics study results showed that the optimum reaction temperature of the chitinase was 60 ℃ with good stability when temperature lower than 60 ℃; and the optimum reaction pH was 6.5 with good stability at pH5.5~7.5. Mg²⁺, Ca²⁺, Hg²⁺ and Co²⁺ had inhibitory effect on the enzyme activity, while Cu²⁺ and Fe³⁺ had a certain promotion effect. Low concentrations of methanol, ethanol, n-propanol and dimethyl sulfoxide increased the enzymatic activity, however, the chitinase would be inhibited when the concentration of these organic solvents were increased to a certain level. The chitinase could be activated by acetone but inhibited by formaldehyde. Under the optimal catalytic conditions, the value of Km, Vmax and Kcat of the chitinase-catalyzed reaction were 29.533 mg/mL, 108.696 μmoL/(L·min) and 0.527/min, respectively. Research results provide technical parameters for the practical application of the chitinase.