Covalent immobilization of Dothideomycetes sp. NRC-SSW chitosanase and its application in chitosan hydrolysis

Chitosanases are chitosan specific glycosyl hydrolases that can exploit in several biotechnological applications. In the current study, a modified bead of carrageenan and carboxymethyl cellulose was used for the covalent immobilization of Dothideomycetes sp. NRC-SSW chitosanase. The immobilization process was optimized and the immobilization recovery was increased up to 69%. The stepwise different bead formulations were examined under Scanning Electron Microscope, Fourier Transform Infrared Spectroscopy and Thermal Gravimetric analysis, confirming the success of the immobilization process. Additionally different properties including enzyme storage, thermal and pH stabilities were examined and an improvement in all of these properties was estimated. Although the same Vmax value (23.8U/min/mg protein) was recorded for the free and the immobilized enzymes, the Km was 10mg chitosan/ml and 16.67mg chitosan/ml for the immobilized and free form respectively. The activation energy for chitosan hydrolysis using the immobilized enzyme (12.497kJmol−1) was less than the third of that recorded for the free one (42.196kJmol−1). Furthermore, the immobilized enzyme was stable until the 5th cycle at which it retained more than 50% of its original activity. All the tested parameters confirmed that the immobilized Dothideomycetes sp. NRC-SSW chitosanase was more applicable for chitosan hydrolysis than the native enzyme.