Preparation And Characterization Of A New Type DNAase From Bimastos

1. To establish a biochemical method to find DNAase in earthworm;2. To establish a technique system to purify the DNAase;3. To study the physical and chemical character of the DNAase.Methods1. To find DNAase in the earthwormThe tissue extract of earthworm with different concentration reacted with RNA, circular DNA, linear DNA for an hour at 37℃, then the producetion was detected by 1% agrose gel. The tissue extract of earthworm, the tissue protein extract of earthworm and the tissue extract of earthworm without protein reacted with pBV220-r-INF for an hour at 37℃, then the producetion was detected by 1% agrose gel.2. To isolate and purify DNAase in the earthwormFirst, the tissue extract of earthworm was prepared by dissolving the earthworm with sucrose and denaturing the protein with low pH buffer. Then DNAase was purified by denaturing the protein with higher temperature. The followingsteps were ammonium sulfate precipitation, DEAE-cellulose(DE52) chromatography and filtration by ultra- filter membrane.3. To study physical and chemical property of DNAaseThe purified DNAase reacted with circular pBV220- y -INF foran hour at 37℃, then the producetion was detected by 1%agrose gel.The enzyme reacted with the calf thymus DNA at differenttemperature, then its activity unit was detected.The targeting enzyme reacted with the calf thymus DNA atdifferent pH, then its activity unit was detected.After the purified DNAase was exposed to different temperaturefor 3 hours, it reacted with the calf thymus DNA, then its activityunit was detected.After the interesting DNAase was exposed to different pH for 24hours, it reacted with the calf thymus DNA, then its activity unitwas detected.The different bivalent cations with the same final concentrationwere put into the DNAase reacting system for 15 minute atpH5.4,at 41 ℃, then DNAase's activity unit was detected.The purified DNAase reacted with the calf thymus DNA atdifferent NaCI concentration at pH5.4, at 41 ℃, then its activityunit was detected.SDS-PAGE and Sephcryl S-200 gel filtration detected themolecular weight of DNAase. Results1. The finding of DNAase in the earthwormThe tissue extract of earthworm which had been diluted 28 times could digest 4 ul pBV220- r -INF (6.66 u g/ ul) completely at 37@ in a hour but it had no effect on RNA. The crude sample which had been diluted 54 times could decompose 1 ul DNA (0.596 ug/ul) completely at 37@ in a hour. Under the same condition, the tissue extract of earthworm and the tissue protein extract of earthworm could decompose the pBV220- r -INF completely but the tissue extract of earthworm without protein couldn't.2. The isolation and purification of DNAase in theearthwormThe earthworm DNAase was purified from the tissue extract of earthworm by denaturing the protein with low pH buffer,high temperature, ammonium sulfate precipitation, DEAE-cellulose (DE52) chromatography and ultra- filter membrane. The enzyme appeared homogenous and the specific activity is 4640U/mg, the yield is 5%.3. The study of physical and chemical character of DNAaseThe earthworm DNAase could decompose the circular DNAcompletely. Its optimum temperature is 41℃, optimum pH is 5.4 when the substrate was the calf thymus DNA. Its activity became declining when the temperature was above 70℃ and when the temperature is 90℃, its residual activity is 38.4%. Its activity became declining when the pH was above 7.0, when the pH is 9.0, its residual activity is only 1%. But when the pH was 2.2, its activity had no obvious decline. Mg2+,Ba2+,Mn2+ could activate DNAase. When the concentration of NaCI was below 0.1mol/L, the activity of DNAase was increasing along with the increasing of the concentration of NaCI. Its activity was most when the concentration of NaCI was 0.1 mol/L. When the concentration of NaCI was above 0.1 mol/L, the activity of DNAase was declining along with the increasing of the co...