Cloning And Function Of Key Enzymes Of Aerobic Denitrification Pathway

Denitrification is an important process of N metabolism and N cycle in the natural world. This process can lead to the loss of N in farmland soil, and it is the main approach to wastewater purification and biological remediation of non-point source pollution.The intermediate metabolite of denitrification NO2- is an important factor in the death of aquatic organisms and human tumors;Nitrogen oxides are important factors in the formation of haze.Aerobic denitrification can resolve the limitation of traditional denitrification in anaerobic condition, which has become the mainstream of wastewater purification and polluted water restoration, and it also increases the loss of soil N.So cloning aerobic denitrification nitrification pathway genes and study their expression rules of wastewater purification,remediation of water pollution, reducing the loss of farmland N, reducing human food carcinogenic factors and atmospheric haze formation factors is of great significance.In this research, firstly, Sinorhizobium sp.NP1 which was isolated and purified by our laboratory had been used as the original strain,the full length sequence of napA, norB and nosZ genes were obtained for the first time by homologous cloning, the structures and composition characteristics of these three genes and gene products were also analyzed.(1) The periplasmic nitrate reductase gene(napA)(accession number is KR902902), whose length of the gene complete reading frame is 2505 bp, encoding 834 amino acids. The protein has a signal peptide sequence and the position of mature protein is between 31-32 of the AAQ-QP. It has a transmembrane signal in the first 100 amino acids.(2) The nitric oxide reductase(norB) gene(accession number is KR902903), whose complete reading frame sequence is 1347 bp, encoding 448 amino acids.According to analysis of bioinformatics, the protein has a signal peptide sequence and the position of mature protein is between 31-32 of the LAG-GT. The protein is a 12 transmembrane protein.(3) The nitrous oxide reductase gene(nosZ)(accession number is KR080373), whose whole length of the gene sequence is 1920 bp, encoding 639 amino acids, It has a complete reading frame. Bioinformatics analysis showed that the protein has a signal peptide sequence and the position of mature protein is between 30-31 of the VGA-GG. It dose not belong to the transmembrane protein.This research constructed the nitrite reductase, NIR gene(GenBank accession number: FJ598613)and nitrous oxide reductase NOSZ gene prokaryotic expression vector to obtain the expression products,and the physicochemical properties and enzymatic characteristics of the products were analyzed.(1)SDS-PAGE detection results of prokaryotic expression products of NIR gene showed that the NIR was about 40 KD, which was consistent with the molecular weight of the enzyme;Western blotting confirmed that the protein had a strong positive reaction with the polyclonal antibody which was synthesized by NIR;Enzymatic properties analysis showed that the activity of the enzyme was 247.15U/mL, the suitable reaction pH was 6.5, the suitable reaction temperature was 37 ℃, the tolerance to NaCl was 0.3 mol/L.(2)SDS-PAGE detection results of prokaryotic expression product of NOSZ gene showed that theNOSZ was approximately 70.7 KD, which was consistent with the molecular weight of the enzyme;in addition, the concentration of the soluble fusion expression of NOSZ is about 1.5 mg/mL by the method of coomassie brilliant blue can bind with protein.qRT-PCR analysis results showed that, the expression of NIR decreased compared with the control group under conditions pH6 and pH10 with the KNO3or(NH4)2SO4as the sole nitrogen source at12 h,but the expression of NIR was increased under condition pH6 at 24 h with the growth of NP1strain;Quantitative results were also found that under condition C/N5 the expression level of NIR increased compared to the control group at 12 h and 24 h,and the expression of NIR reduced under the condition of C/N20, which may be related with the growth and metabolism of NP1 strain, the high C/N value may inhibit the expression of the gene.