Study On Separation And Purification Of A Group Of Nucleases From Lumbricus Bimastus And Their Preparation And Purification Technics

Our recent years'studies on Lumbricus Bimastus showed that there were a group of components within it that can strongly kill microbes and viruses, further research indicated that these components could degrade many kinds of DNA and RNA that were characters possessed by nucleases. In order to determine the kinds, number, Enzymatic kinetics characters, structures, biological functions and action mechanisms of these nucleases, this study set up a series of accurate and sensitive activity measuring methods that suitable for nucleases of different characters, stabilities of rude Lumbricus Bimastus extracts under different conditions were measured and Preparation and Purification Technics of Nucleases from Lumbricus Bimastus were studied, several nucleases were purified.1. Set up of activity measuring methods system of nucleases from Lumbricus BimastusSingle radial enzyme diffusion (SRED), agarose gel electrophoresis, ultraviolet spectrophotometric method, imidazole-Zinc Chloride reverse staining ,PAGE-DNA activity measuring method and dried agarose film overlay method were utilized to set up enzyme-catalyzed reaction system with DNA and RNA as substrates. Through analysis of the extents and manners of degradation of the substrates or the increase of the products (hyperchromic effect), the activities of nucleases in the preparations were measured and the enzymatic mechanisms were speculated.2. Studies on stabilities and physical and chemical characters of rude Lumbricus Bimastus nucleases extractsSingle radial enzyme diffusion (SRED) and agarose gel electrophoresis were utilized to study the Influencing factors of nucleases activities during the preparation of rude extraction liquids of Lumbricus Bimastus, the stabilities under acid and alkaline conditions, the extents of heat resistance and influence of physical and chemical factors on nucleases activities during PAGE and SDS-PAGE. Mechanical extraction method was at last designated as a quick and effective method, 0.1mol/L NaAc/HAc (pH4.6) was proved to be the optimum extraction buffer. Results showed the optimum pH was between 4.0 and 5.4, absolute ethanol and acetone did not inhibit the activities of nucleases. The active components were observed mainly exist in the sediments when the contents of ethanol and acetone reached 50% and mainly exist in supernatant when encountering 55% ammonium sulfate. Methanol and acetate in the working concentrations...