Study On The Enzyme Activity And Content Determination Method Of Recombinant Nonspecific Nuclease

The Y.e.p non-specific nuclease(Nucyep)derived from Yersinia enterocolitica subsp.palearctica(Y.e.p)is a thermostable non-specific nuclease(NSN),which has no substrate specificity,and can degrade all kinds of nucleic acids including single-stranded(ss),double-stranded(ds),linear and cyclic ribonucleic acid(RNA)or deoxyribonucleic acid(DNA).At present,there are few reported assays for Nucyep.Therefore,the study on assays of recombinant Nucyep has important application value.In the first chapter,a rapid and simple method was established for the NSN enzyme activity assay.In this experiment,DNA was used as the substrate,and the gel-imaging system was used to determine the change in optical density(OD)caused by the reduction of the DNA,and the activity of NSN was calculated.By investigating the effects of several key factors in the experiment such as substrate concentration,enzyme activity assay range,reaction time and termination method,and the activity assay for NSN was established and performed methodology validation.The results showed that the optimal reaction time was 3 min,and the linear range of the enzyme activity was 4.17-9.01 U/?L.The concentration of DNA was linearly correlated with OD in the range of 0.02-0.40?g/?L(R~2=0.9942).The average recovery of three concentrations was 99.12%.The relative standard deviation(RSD)of enzyme activity was 1.90%for six samples.Above results revealed that the linearity,precision,accuracy and sensitivity of the NSN enzyme activity assay met the requirements of methodological development and evaluation.In the second chapter,the recombinant Nucyep with high enzyme activity was isolated and purified from the cell pellets of Escherichia coli(E.coli),and the enzyme was identified.The inclusion bodies were obtained from E.coli by homogenization,then washed and denatured with urea buffer.Finally,refolding by dilution,the recombinant Nucyep crude enzyme solution was obtained.The recombinant Nucyep(30.5 k Da)was purified by using DEAE FF anion exchange chromatography and Sephadex G-75 gel chromatography.The results revealed that the specific activity of the enzyme was 1.13×10~3 U/mg,the purification fold was 8.25 and the recovery rate was 13.75%.The physical and chemical identification of the enzyme was carried out.The SDS-PAGE electrophoresis showed a single band,and the protein content measured by Kjeldahl nitrogen method was 92.7%.The results of physico-chemical analyses experiments revealed that the recombinant Nucyep could be used as working references for content determination.In the third chapter,a HPLC method for the content determination of the recombinant Nucyep was developed and validated.The results showed that the resolution is 4.77,the concentration of recombinant Nucyep was linearly correlated with the area of the elution peak in the range of 0.12-1.58 mg/mL(R~2=0.9992),the average recovery of the three concentrations was 99.0%,the RSD value of six repeated determinations was 0.71%,and the RSD value of repeatability was 0.93%,and the RSD value of intermediate precision was 1.02%,and the LOD and LOQ were 10?g/mL and 20?g/mL.The specificity,linearity,precision,accuracy and sensitivity of the method were all in line with the requirements,indicated that it could be used for the content determination of recombinant Nucyep.