Isolation,Purification And Exploitation As A Tool Enzyme Of Nuclease P1

Nuclease P1 is a zinc-containing metalic enzyme produced by Penicillium citrinum,which is mainly used for industrial production of nucleotides.Generally,the yield and purity of nuclease P1 produced in China were not enough,relying heavily on imports.In this thesis,the nuclease P1 produced by Penicillium citrinum was purified and the enzymatic properties were investigated,and the application of nuclease P1 as a tool enzyme in the field of genetic engineering was further explored.The purification processes of nuclease P1 were as follows:the crude enzyme was subjected to ammonium sulfate fractionation and dialysis after heat-treatment,followed by Sephadex gel G75 filtration,DEAE-cellulose 52 ion exchange chromatography and Sephadex gel filtration to obtain pure enzyme solution.After the whole purification process,the total enzyme activity recovery was about 20-30%,nuclease P1 was purified by 3.05 times,and it was detected as a single band with a molecular weight of about 36kDa by SDS-PAGE.The study on the enzymatic properties of nuclease P1 showed that the optimal reaction temperature of nuclease P1 was 70?,and the recovery rate of enzyme activity was 87%after being treated at 70?for 1 h.The most suitable reaction buffer system was an acetate buffer solution at pH 5.4.Low concentration of Zn²⁺,Mg²⁺and Ca²⁺could slightly promote the activity of nuclease P1,and high concentration of metal ions had an inhibitory effect on nuclease P1,of which Cu²⁺and Fe³⁺showed the most obvious inhibitory effect.The Michaelis-Menten constant Km and Vmax of nuclease P1 were19.40 mmol/L and 3.23 mmol/?mL�min?,respectively,and the correlation coefficient R²was 0.9925.To study the effect of additives on the preservation stability of nuclease P1,it was found that when 15%?w/v?sucrose or 0.1%?w/v?potassium sorbate was added to the nuclease P1 solution at 4?for a month,the enzyme was almost completely deactivated,while the enzyme activities with other additives at 4?were maintained above 90%.The purified nuclease P1 was used to degrade contaminated RNA in DNA,detected by agarose gel electrophoresis,indicating that the nuclease P1 could digest the RNA contamination in genomic DNA extracted from E.coli.This had promoted our country to obtain nuclease P1 pure enzyme solution,reduce import dependence,improve nucleotide production efficiency and further expand the application of nuclease P1.