Construction Of The Engineering Escherichia Coli With Ammonia Oxidizing Function

Water pollution is a significant environmental problem, nitrogen is one of the main pollution comtaminants. At present, nitrification-denitrification process is used to remove nitrogen from sewage. Ammonia oxidizing bacteria (AOB) is major microbe in remove nitrogen which can convert ammonia nitrogen into nitrite. However, the growth rate of ammonia oxidizing bacteria is slow and it is difficult to cultivate and isolate, it is still a limiting factor in the process of wastewater treatment. Therefore, it is that building a strain that grows faster and better will promote the development of biological denitrification technology.In this paper, the two functional genes (amoA and hao) of AOB were cloned from the enrichment soil solution, then two genes were made to express simultaneously in the same Escherichia coli BL21(DE3), the engineering E.coli with ammonia oxidizing function was constructed. The main results were as follows:(1) With the total DNA extracted from enrichment soil solution, amoA and hao sequences were amplified by polymerase chain reaction (PCR) with the specific primers of AOB. Amplified products respectively connected with pMD18-T vector, after transformation of E.coli DH5a, the positive transformants were selected by colony PCR, sequenced and analyzed by Blast, two kinds of gene sequence were obtained. The result showed that the sequences of amoA and hao had 99% indentities with Nitrosomonas sp. GH22 and Nitrosomonas sp. ENI-11, respectively.(2) Bio informatics was used to analyze the structure and function of by AmoA and Hao, the analysis showed AmoA encoded 276 amino acids, its molecular weight was 31.94 kDa. It was a kind of unstable hydrophobic protein with potential transmembrane helix regions, and the tertiary structure prediction showed that spatial conformation was dissymmetric. Hao encoded 570 amino acids, its molecular weight was 64.27 kDa, it was a kind of stable hydrophilic protein with a potential transmembrane helix region, and its spatial conformation was dissymmetric.(3) The amoA and hao were respectively linked to the pQE30 vector and pET28a vector to construct recombinant prokaryotic expression vector (pQE30-amoA and pET28a-hao), transformed into E.coli BL21(DE3), the expression of two genes were confirmed by RT-PCR, and then the crude enzyme solution activity of AmoA and Hao were measured. With (NH4)2SO4 as substrate, the degradation rate of ammonia nitrogen of AmoA crude enzyme solution was 90.6%; With hydroxylamine hydrochloride as substrate, the degradation rate of hydroxylamine of Hao crude enzyme solution was 86.3%, it indicated that the crude protein enzyme solution of AmoA and Hao had certain activity of denitrification.(4) Plasmid pQE30-amoA and pET28a-hao were extracted and simultaneously transformed into E.coli BL21(DE3), the positive transformants were selected by double anti-flat and colony PCR, an engineering strain BL21(DE3)-pQE30-awoA-pET28a-hao were obtained, the co-expression of two genes were confirmed by RT-PCR. The ammonium degradation of the new strain was tested, its maximum ammonium degradation rate reached 92.0% after enlarged culture for 60 h, suggesting that BL21(DE3)-pQE30-amoA-pET28a-hao had certain activity of denitrification.To conclude, the construction of engineering E.coli with ammonia oxidizing function had certain application value in ammonia nitrogen sewage treatment.