Thesis (Ph.D.) - Cairo University - Faculty of Pharmacy - Department of Microbiology and Immunology

Over 13 microorganisms screened for chitosanase production, an isolate identified as Dothideomycetes sp. css035 secreted a considerable value of inducible chitosanase (1.27 U/ml). The optimization of the medium components and the culture conditions was performed by using one factor at a time technique followed by statistical methods including plackett-burman design and Box-Behnken design. The highest chitosanase production was induced by the medium composition g/L: soluble chitosan, 30; K₂HPO₄, 1.15; MgSO₄, 0.4; KCl, 4.0; yeast extract, 18.5; FeSO₄, 0.01; at pH 5.5, 30 {u00B0}C and 180 rpm for four days. The optimization study was markedly enhancing the production of the extracellular and the cell bound chitosanase from 1.27 U/ml and 11.9 U/ dry weight to 13.9 U/ml and 215.83 U/ dry weight. The crude extracellular chitosanase was optimally active (21.5 U/ml) at pH 4.5 and temperature 60{u00B0}C with 1% soluble chitosan and it exhibited a good stability over pH range from 4 to 5.5 and thermostability below 45{u00B0}C in the absence of the chitosan since the enzyme had half life time higher than 2 h. The immobilization of 30-70% ethanol precipitated Dothideomycetes sp. css035 chitosanase by using covalent binding to carrageenan: carboxymethyl cellulose carrier that showed improved pH and thermal stability in comparison to the partial pure enzyme. Thin-layer chromatography plate (TLC) clearly visualized the efficiency of the produced chitosanase to hydrolyze chitosan to chitooligosaccharides and the highest concentration of chitooligosaccharides was produced after 5 h reaction time at 50 {u00B0}C using 2% chitosan and E/S ratio 0.05 U/mg. The biological activities evaluation of the produced chitooligosaccharides mixture (eluted from the TLC) indicated that the chitooligosaccharides mixture showed fibrinolytic and anticancer activity

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