Research On Purification Of A Kind Of Nuclease From Lumbricus Bimastus And Its Substrate Specificity

Objectives1. Preparation of rude extracts of Lumbricus Bimastus nucleases and its purification.2. To establish the agarose gel electrophoresis system.3. Study on enzymology qualities of the nuclease.4. A preliminary study on substrate specificity of Lumbricus Bimastus nuclease.Methods1. Preparation of rude extracts of Lumbricus Bimastus nucleases and its purification Using DNA-casting SDS-PAGE and agarose gel to monitor the activity of the nuclease in the process of extraction.After ammonium carbonate processing, thermal denaturation, acid denaturation and acetone precipitation,the rude extracts of nucleases were purified by Column chromatography and electrophoresis.2. To establish the agarose gel electrophoresis systemUsing agarose gel electrophoresis system to monitor the activity of the nuclease, to study its optimum reaction conditions.3. Study on enzymology qualities of the nucleaseUsing SDS-PAGE gel electrophoresis to measure molecular weight, CE-IEF to measure pI, assay of enzyme activity to determine optimum pH, optimum temperature, temperature stability, activator, inhibitor, and to calculate its Km values.4. A preliminary study on substrate specificity of Lumbricus Bimastus nucleaseTo measure degrade rate of DNase E to several different substrate in optimal reaction condition to demonstrate the substrate selectivity of DNase E. Agarose gel electrophoresis was used to investigate the products of DNase E processing double strands cyclic DNA to investigate the action of DNase E.Results1. Preparation of rude extracts of Lumbricus Bimastus nucleases and its purification Two groups of nucleases with different characters lied in the DNA-casting SDS-PAGE. The group of low migration nucleases were prepared by ammonium carbonate processing +acid denaturation + thermal denaturation + acetone extraction. Through four steps of column chromatography and electrophoresis purification, one kind of nuclease(named DNase E) was obtained. 2. To establish the agarose gel electrophoresis systemTo identify the agarose gel electrophoresis system that using plasmid 2μl +10μl as samples(sample dissolved in 0.1 M pH 5.2NaAc buffer (containing 20 mM Mg²⁺)), then incubated for 30 min in 37℃.3. Study on enzymology qualities of the nucleaseThe enzymatic study results of DNase E showed that the molecular weight of DNase E was approximately 32 kDa, its isoelectric point was about 6.05. When the substrate was the calf thymus DNA, its optimum temperature was 42℃, optimum pH was 5.2, and its Km values was 0.152mg/ml. 20mM Mg²⁺,Ca²⁺,Mn²⁺ could activate its activity respectively. Zn²⁺, iminazole, EDTA, K+, Cu²⁺ could inhibit its activity respectively. The SDS-PAGE showed that DNase E was a oligomeric protein, it was stable to acid, alkali, and organic solvents.4. A preliminary study on substrate specificity of Lumbricus Bimastus nuclease DNase E degradation studies showed that there were differences in degradation rate of the DNase E to different substrates. The results indicated that DNase E belonging to endonuclease.Conclusion1. Established a preparation technics of rude extracts of Lumbricus Bimastus nucleases. One kind of nuclease was obtained by a series of purification methods, named DNase E (electrophoresis pure).2. The molecular weight of DNase E was approximately 32kDa, its isoelectric point was about 6.05. When the substrate was the calf thymus DNA, its optimum temperature was 42℃, optimum pH was 5.2. 20mM Mg²⁺,Ca²⁺,Mn²⁺ could activate its activity respectively. Zn²⁺, iminazole, EDTA, K+, Cu²⁺could inhibit its activity respectively. The experimental results showed that DNase E had no sequence specificity, it was a non-specific nuclease. There was difference in substrate hydrolysis. DNase E belonged to endonucleases.