Achieving regio-specific hydroxylation of aromatic compounds remains a major challenge in synthetic chemistry.

A MONG the methods used for the hydrolysis of chitosan, enzymatic hydrolysis using chitosanase was selected in order to produce high yield of specific chitooligosaccharides with less environmental pollutions. The production of Dothideomycetes sp. NRC-SSW extracellular chitosanase was statistically optimized in which a two -phase experimental design was applied. Plackett -Burman design was used to evaluate the relative importance of culture conditions and medium components for chitosanase production. Chitosan concentration, agitation speed and incubation period were found to be the most significant variables that affected the chitosanase production and their optimal values were obtained by applying Box-Behnken design. The optimized medium composed of (g/L) chitosan, 30; K 2 HPO 4 , 1.5; MgSO 4 , 0.4; KCl, 4.0; yeast extract, 18.5 and FeSO 4 ,0.01; at pH 5.5, 30°C and 180rpm for 96h gave 13.9U/mL with 36.3% increase in the activity. The R 2 value was 0.954 and this indicated the aptness of the model. The optimization of the hydrolytic conditions required for chitooligosaccharides production was also performed by Box-Behnken design. The highest yield of chitooligosaccharides was obtained with enzyme/ substrate ratio 0.05U/mg in 0.2M Tris HCl buffer incubation at 60 ο C for 5h. The cytotoxic activity of the chitooligosaccharides was tested in vitro against Hep-G2 and MCF7.

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 5 th 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.