Purification and characterization of chitinase from Paenibacillus sp. 

The chitinase-producing bacteria Paenibacillus sp. was isolated from soil samples. The chitinase was purified successively by ammonia sulfate fractional precipitation followed by chromatography on DEAE 52-cellulose column and then on Sephadex G-75 column. The chitinase has a molecular weight of ca. 30 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. Its optimum pH is 4.5, and its optimum temperature is 50 °C with colloidal chitin as a substrate. The enzyme is stable below 45 °C and in pH ranges between 4.5 and 5.5. It is activated by glucosamine, glucose, N-acetylglucosamine, and metal ions including Ca²⁺ , Fe²⁺ , Fe³⁺ , and Ni²⁺ . It is inhibited by SDS, H₂ O₂ , ascorbic acid, Cu²⁺ , Mg²⁺ , Ba²⁺ , Sn²⁺ , Cr³⁺ , and K⁺ . With colloidal chitin as substrate, the K_m and the V_max of the chitinase are 4.28 mg/mL and 14.29 μg/(Min·mL), respectively, whereas the end products of the enzymatic hydrolysis are 14.33% monomer and 85.67% dimer of N-acetylglucosamine. The viscosity of carboxymethyl chitin decreased rapidly at the initial stages when subjected to chitinase hydrolysis, which indicates that the chitinase acts in an endosplitting pattern.