Cloning And Prokaryotic Expression Of Nitrite Reductase Gene From Alcaligenes Xylosoxidans

Nitrite has the function of color and anti-corrosion,while excessive nitrite accumulating in human body will result in teratogenic carcinogenic hazards,and therefore seeking a feasible way for the degradation of nitrite has been the hot points of research in the field.At present,the degradation of food nitrite method mainly include chemical and biological methods,the use of nitrite reductase(nitrite reductase,referred NiR)degradation of nitrite in food is an effective way in which the enzyme widely exists in plants or microorganisms,and the use of gene cloning technology from NiR enzyme is rarely reported.This study,the use of cloning technology,will NiR cloned into the prokaryotic expression vector induced efficient expression.The technology laid the foundation of industrial production for producing enzymes and the enzyme preparation,which has extensive application prospect.Detailed research contents are as follows:1.Total DNA of Alcaligenes xylosoxidans subsp xylosoxidans as templates,primers were designed according to Genbank gene sequence in NiR,amplify the expected band sizes with the use of PCR technology,the size of 1083bp,the amplified products were linked with T vector,to construct the recombinant plasmid of pMD18-T-nir and sequencing,the results of sequencing and Genbank sequence alignment,the homology was 98.43%.pMD18-T-nir plasmid expression vector pET-28a(+)was digested,the digestion product was transformed into competent cells successfully constructed pET-28a-nir.2.The recombinant plasmid pET-28a-nir was transformed into E.coliBL21(DE3)competent cells,namely,BL21/pET-28a-nir,with the final concentration of 5h expression induced by 0.6mmol/L IPTG 30?,expressed protein bands of about 37KDa certified induction,analysis of formation of inclusion bodies,cell breakage extraction of crude enzyme solution,a preliminary analysis of the enzymatic properties of the crude enzyme was conducted,the results show that the optimum of the enzyme pH value is 6.5,the optimum temperature is 35 ?,this is measured under the condition of crude enzyme activity was 51.74 U/mL.3.The cell fermentation liquid ultrasonic crushing frozen after centrifugation and inclusion,by dialysis method of inclusion body denaturation and renaturation,renaturation solution by freeze drying and filtration treatment,with the volume for the purification of the protein Ni-NTA column 1 mL,the final measured protein concentration is 0.06mg/mL,the pure enzyme activity was 13.04U/mL.