Protoplast Mutagenesis Of Serratia Marcescens And Chitinase Research

In this paper, some questions such as further enhancing the chitinase activity produced by RZ21, a strain separated and stored from Serratia marcescens, optimizing the culture medium and culure conditions were studied to produce some basis for its application in agriculture as a biocontrol bacterium.By means of the radiation of protoplast ultraviolet, NaNO2 respectively and the synergy, at the same time, by through screening of clear zones and rescreening of flasks, one strain of Serratia marcescens mutant NU2 with a high chitinase yield 2.75 times more than the parent screened and identified in our laboratory was isolated. Then, the culture medium and culture conditions were optimized, the properties of the chitinase secreted by the mutant were also researched in the last.In the experiment of radiation, single factor testes such as protoplast ultraviolet and NaNO2 was first studied. It was tested that the effect of protoplast ultraviolet was superior to effect of NaNO2. However, in the experiment of complex radiation, the method of radiation by initial protoplast ultraviolet and then NaNO2 markedly improved the chitinase activity than any other means in this study. Compared with the control, its HC value was 60.8% higher more than the parent and the mutant obtained a a better genetic stability.In the experiment of medium and condition culturing, the optimal constituent gradient of medium, corn flour 7.5g/L, silkworm pupa powder 5.0g/L, NaCl 7.5g/L, was determined by single-factor, multi-factor testes as well as orthogonal test. With the prior knowledge of the inducible ability of chitinase, the effect of micro-supplementary chitin was also studied besides the conventional carbon and nutrition optimization. It was found that the inducing effect of colloidal chitin was much better than that of granular chitin due to its tiny diameter and large accessible surface area. On the other hand, the inducing effect of 150-mesh chitin was slightly better than that of 50-mesh. In the culture condition optimizing test, the optimum culturing condition,30℃, initial pH8.0,4 mLmedium/250 mL flask,150rpm,30h, was decided. On this condition, the ability of chitinase produced by Serratia marcescens was 410U,4.23 times more than the initial medium.The chitinase produced by mutant NU2 of Serratia marcescens cultured in 25L fermenter, was purified by high pressure cell cracking, ammonium sulfate precipitation and dialysis respectively. It was found that the chitinase produced by mutat NU2 obtained the advantages of heat and pH stability studied by the enzymatic research with the colloidal chitin. The optimal reaction temperature was 37℃and the enzyme keeped its stable catalytic activity in the range of 4℃to 60℃and pH 5.0 to 9.0. The optimum reaction system was phosphate buffer pH8.0. Magnesium ion greatly improved the chitinase ability, nevertheless, metal ion chelating agent EDTA inhibited the chitinase ability completely, all of these showed that the chitinase was a kind of metal ion activated enzyme and needed magnesium ion to be used as an auxiliary group. The SDS-PAGE showed that five Chitinases were produced by the mutant.