Single step hydrolysis of chitin using thermophilic immobilized exochitinase on carrageenan-guar gum gel beads

Abstract

Abstract Chitinases are chitin glycosyl hydrolases that can be employed in various biotechnological applications. In the present study, exochitinase produced by Alternaria sp. strain Sha was immobilized on carrageenan-guar gum gel beads with immobilization yield of 43% improved to reach 100% by statistical optimization using central composite design. The coefficient of determination value for the applied model was 0.903 and the absolute average deviation was 8.9% confirming that the applied model was successfully used in the optimization process. Fourier Transform Infrared Spectroscopy, Thermal Gravimetric analysis and Scanning Electron Microscopy was used to examine the stepwise different formulations of the beads as well as the immobilized one, confirming the success of the immobilization process. The immobilized enzyme activity was optimized in respect to the effect of temperature, pH and substrate concentration indicating that the immobilized enzyme was optimally active at 60οC and pH 5 with suitable activation energy (20.287 ± 0.159kJmol−1), Km and Vmax values of 2 mg/mL and 5.9U/mg protein/min respectively. Moreover, the immobilized enzyme showed high thermal and operational stabilities, it retained more than 90% of its activity after 2 h at 65οC and it retained 100% of its activity for more than 15 cycles as well as it kept its complete activity at 4οC for more than one month. Finally, the immobilized enzyme was successfully employed for the direct hydrolysis of crab chitin and production of chitooligosaccharides as it produced reducing sugars of 193.8 μg/mg chitin after 12 h, confirmed by thin layer chromatography analysis and Fourier Transform Infrared Spectroscopy.