Study On Separation And Purification Of A Kind Of Nuclease With High Isoionic Point From Lumbricus Bimastus And Its Activity

Objectives1.To optimize the preparation technics of rude extracts of Lumbricus Bimastus nucleases2.To establish a technique system to purify the nucleases3.To characterize the DNase.Methods1.Optimization of preparation technics of rude extracts of nucleasesHeat the rude extracts in different conditions.At least two groups of nucleases with different characters lies in the extracts.PAGE-DNA activity measuring method was established as the monitor system of the activity and variety change in the process of separation and purification.Different preparation technics of rude extracts was made according to their characters.2.Purification of nucleases through column chromatographyCommon-pressure liquid chromatography,anion exchange;High performance liquid chromatography including anion exchange,hydrophobic interaction,reverse phase were utilized to purify the nucleases.3 Purification of nucleases through electrophoresis and the study of the characters and activity of EWD.Based on the results of chromatography purification,PACE,2D-PACE,DNA-casting PACE were utilized to purify one kind of DNase.The molecular weight of DNase was determined by SDS-PACE and gel filtration,respectively.The purified DNase mixed with circular or liner DNA for 30minutes to examine the mixture. To examine its activity when the enzyme was mixed with the calf thymus DNA or plasmid at different temperature/pH.The effects of various cations were examined using plasmid as a substrate.Results1.Optimization on preparation technics of rude extracts of nucleasesPAGE-DNA activity measuring method was established as the monitor system of the activity and variety change in the process of separation and purification.The group of low migration nucleases were prepared by extracting with ammonium carbonate,60℃for 15 minutes,low pH buffer and rinsing with acetone.The group of high migration nucleases were prepared by extracting with ammonium carbonate,50℃for 10 minutes. 2.Purification of nucleases through column chromatographyThrough four steps of column chromatography including anion exchange(CM FF and POROS 20CM),hydrophobic interaction(POROS 20 HP2),reverse phase(POROS R1)etc,a kind of mixture contained a small quantity of other protein was obtained.3.Purification of nucleases through electrophoresis and the study on the characters and activity of EWD.Based on the results of chromatography purification,PAGE,2D-PAGE,DNA-casting PAGE were utilized to purify one kind of Dnase,EWD(Earthworm DNase)with electrophoresis-grade purities;EWD could decompose the circular DNA completely and degrade liner DNA.Its optimum temperature is 41.5℃,optimum pH is 5.2 when the substrate was the calf thymus DNA.The molecular weigh of EWD was 28.8KD which was examineed by SDS-PAGE.The PI value was higher than 7.Mg²⁺,Ca²⁺,Mn²⁺could activate EWD and the activity would decline if Zn²⁺,EDTA,Cu²⁺,K⁺,iminazole was contained in the buffer.Conclusion1.Preparation technics of rude extractions of Lumbricus Bimastus is optimized.One kind of nuclease was obtained by chang the preparation technics and the purification tactics.2.EWD could degrade the circular DNA completely and degrade liner DNA.Its optimum temperature is 41.5℃,optimum pH is 5.2 when the substrate was the calf thymus DNA and plasmid.The molecular weigh of EWD was 32KD.The PI value was higher than 7 Mg²⁺,Ca²⁺,Mn²⁺could activate EWD and Zn²⁺,EDTA,Cu²⁺,K⁺,iminazole were inhibiter.