5273
Shaymaa Abdalla Ismail
Physiological and Biochemical Studies of Microbiological Production of Chitosanase
chitosanase, production, optimization, immobilization, chitooligosaccharides
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 °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°C with 1% soluble chitosan and it exhibited a good stability over pH range from 4 to 5.5 and thermostability below 45 °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 οC 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.
2016
Ph.d
Cairo
Pharmacy