Characterization And Fermentation Of Chitosanase From Serratia Ficaria CH0203

The model for the screening of chitosanase-producing bacteria was established as follows: by using chitosan as sole carbon source in PH6.0 culture medium, holos around colonys growing on plates may be observed. By this method, we screened 27 soil samples for chitosanase on plates, by comparing the diameters of holos around the colonies and by further shaking culture screening. A strain of bacteria which may secrete higher activity chitosanase was selected and was identified as Serratia ficaria afterwards. It was named CH-0203. It was the first time that Serratia ficaria was found by the research to produce chitosanase. The chitosanase is secreted extracellularly, and can be effectively induced by chitosan and glucosamine, whereas chitosan is better than glucosamine, other kinds of carbon sources act poorly on inducing the bacteria to yield chitosanase.The optimal condition for yielding chitosanase is as follows: using 0.5% highly deacetylated chitosan as sole carbon source and nitrogen source, adding 0.10% KH₂PO₄, 0.05% Na₂HPO₄·12H₂O, 0.05% MgSO₄·7H₂O, 0.001% CaCl₂, 0.15% KC1, 0.15% Na₂SO₄, 0.05% tween 80, PH value of culture medium is 6.5, cultivating temperature is 30℃, filling in 40ml culture medium in a 250 ml conical flask, inoculating age and volume are 24 hours and 2.0ml separately in a 250 ml conical flask. Under such conditions, the chitosanase activity of fermenting liquor may reach 5.4U/ml.The chitosanase is found mainly to exist in the 60-90% saturation part, after the fermenting liquor was precipitated fractionally by ammonia sulphate. By further purification by superdex75 gel column chromatography and carboxymethyl cellulose ion exchange column chromatography, the chitosanase is purified 6.22 times, and is proved homogenous by SDS-PAGE electrophoresis. Its molecular weight is 29 kdal measured by SDS-PAGE electrophoresis. Its specific activity is 69.7U/mg, and the overall recovery is 23.5%. It is is an endo-type chitosanase, with its molecular weight 29 kdal and its isoelectric point 9.4. The chitosanase is stable below 45℃ and within the PH range 4.0-7.5. Its optimal temperature is 45℃ and optimal PH is 3.6. Mn²⁺ ,Co²⁺ activate and Pb²⁺ Cu²⁺ Ni²⁺ Cr³⁺ inhibit the chitosanase. The chitosanase may act most effectively on 85% deacetylated chitosan, and can't act on chitosan deacetylated lower than 45% or carboxymethyl chitin or carboxymethyl...