Improvement of Extracellular Chitinase Enzyme Produced by Citrobacter freundii Through Nano Preparation Technology

A chitinolytic enzyme has been isolated from the marine bacterium Citrobacter freundii after a bioremediation process on fish wastes. The microbe was grown on nutrient broth medium supplemented with 1, 5, and 10% wastes. Enzyme production reached 70 u/ml after 9 days of incubation. At 40oC, the activity was raised to almost three times making the whole process a thermophilic bioremediation. The extracellular enzyme showed a maximum activity when the pH value was slightly alkaline (pH 8). Salinity greatly affects the enzymatic activity which was at least doubled at different pH. Maximum extracellular chitinase activity was obtained when 0.5% fish waste was used as the microbiological medium for the bioremediation process. The extracellular enzyme was purified by salting out using ammonium sulfate at 65% concentration followed by fractionation on Sephadex G-100 column. The gel filtration step resulted in more than 85 purification fold. The enzyme retained more than 70% of its activity at 60oC and pH 8.0 making it a very good candidate for almost all industrial processes. The activity of purified chitinase was greatly affected after enzyme nanoparticle formation on ferric oxide. Enzyme immobilization kept more than 90% of its activity for more than 6 weeks at 35oC. Moreover, 55oC was the optimum temperature for both purified and immobilized enzyme samples. The activity of enzyme-nanoparticles preparation reached more than double the activity of the purified enzyme alone at the same temperature.