The aim of this research was to find a new microbial source for chitosanase production. Optimization of the fermentation conditions and medium compositions to maximize the enzyme production using one variable at a time technique, characterization of the crude enzyme as well as studying the ability of this enzyme to produce chitooligosaccharides from chitosan at different times that evaluated by thin-layer chromatography plate. 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 highest chitosanase production was induced by the medium composition g/L: soluble chitosan, 2; K2HPO4, 1.0; MgSO4, 0.3; KCl, 4.0; yeast extract, 15.0; FeSO4, 0.01; at pH 5, 30°C and 180 rpm for three days. The optimization study was markedly enhanced the production of the extracellular and the cell bound chitosanase from 1.27 U/ml and 11.9 U/dry weight to 10.2 U/ml and 298.56 U/dry weight. The crude extracellular chitosanase was optimally active at pH 4.5 and temperature 60°C with 1% soluble chitosan. The produced Dothideomycetes sp. css035 chitosanase exhibited a good stability over pH range from 4 to 5.5 and thermostable below 45°C in the absence of the chitosan. Thin-layer chromatography plate clearly visualized the efficiency of the produced chitosanase to hydrolyze chitosan to chitooligosaccharides. The highest concentration of chitooligosaccharides was produced after 4 h at pH 5 and 60°C with 0.15U/mg substrate.
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