Alternaria alternata, a local isolate from fishery polluted soil, was used successfully for the biodegradation of shrimp shellfish waste in favor of the production of highly active chitinase enzyme. Chitinase production was noticeably influenced by the culture medium and the highest enzyme production was attained through the log growth phase (96 h). Pronounced increase in chitinase production was concomitant with the finest waste size. Stagnant culture conditions were more adequate than shake cultures. Statistically based experimental designs were applied to optimize the production of chitinase by A. alternata. Plackett-Burman factorial design revealed that concentrations of glucose, MnSO4.H2O and CoCl2 were the most significant factors affecting the process of enzyme production. Maximum enzyme activity (8.12 U/min), which is approximately 1.8 folds the activity expressed in the basal medium, has been assayed at concentrations (g/l): glucose, 9; MnSO4.2H2O, 3.2 and CoCl2, 2, after only 90 h of fermentation, when the second optimization step of Box-Behnken design was applied. The crude chitinase was characterized and maximum activity (19.53 U/min) was attained in reaction mixture of 50°C incubation temperature, 1.5 ml crude enzyme, 0.5 ml of 1% colloidal chitin, pH5 and reaction time of 10 min. The enzyme is thermostable and lost 20% of its activity when heated at 60°C for 60 min. The effect of metal ions in enzyme activity revealed that the enzyme have specific requirement of Ca and K ions for its activity. The results indicated that A. alternata is highly efficient fungus to produce highly active chitinase when grown in statistically optimized medium containing shrimp shellfish waste.
Key words: Alternaria alternate, chitinase, shellfish, statistical optimization.
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