Screening Of Nitrite-degrading Halophilic Archaea And Study On The Function Of Nitrite Reductase

Nitrite in high-salt preserved foods is potentially toxic,carcinogenic and teratogenic.And the most commonly used and effective way to degrade nitrite is biological method.In order to reduce the harm of nitrite in pickled food,the halophilic archaea distributed in natural hypersaline environments were selected,and the strains degrading nitrite were screened.Furthermore,the heterologous expression and function research of nitrite reductase from halophilic archaea were carried out to provide the basis for the application of nitrite reductase in salted food processing.High-throughput sequencing analysis was performed on 32 samples from hypersaline environments in different regions in order to clarify the distribution pattern of halophilic archaea in the main hypersaline environments of China.It was showed that the composition of archaea community was different between coastal and inland environments,northeast and northwest environments.Halophilic archaea were the dominant groups in the most coastal samples,with high abundance and different composition.Those samples from Xinjiang saline-alkali land?LT?,Yuncheng salt lake?YCN?,Yinggehai marine solar saltern?YGH?,Zhangpu marine solar saltern?ZP?and Tainan marine solar saltern?TNN?harboring the halophilic archaea as dominant groups were used to cultivate the main halophilic archaea.The strains of genus Halomicrobium and Haloarcula had the strongest degradation capacity of nitrite among the 88 strains of halophilic archaea from the selected samples.Strains YCN54 and ZP60 with 100%degradation rate were selected for whole genome sequencing,and the genes nirK_Hrs and nirK_Hmc encoding nitrite reductase were annotated respectively.The encoding region of nirK_Hrs was 1071 bp,encoding 356 amino acid residues and the encoding region of nirK_Hmc was 1095 bp,encoding 364 amino acid residues.Based on the prediction of conserved domain,signal peptide and 3D structure,it was deduced that NirK_Hrs and NirK_Hmc were both homologous trimeric Cu-Containing nitrite reductase containing Cu-nitrite-reductase domain and signal peptide.Enzyme proteins,NirK_Hrs and NirK_Hmc were respectively expressed by halophilic archaea heterologous expression system and the active recombinant nitrite reductase were obtained by Ni²⁺column affinity chromatography.The recombinant nitrite reductase NirK_Hrs with higher activity was selected for the enzymatic characterization.After Ni²⁺column affinity chromatography and Superdex 200 gel filtration chromatography,the SDS-PAGE result of NirK_Hrs showed that the apparent molecular weight of the protein was about 42 kDa and 48 kDa.Also,the result of mass spectrometry showed that the protein was the target protein.NirK_Hrs was found to be a blue copper-containing nitrite reductase by wave scanning.The optimum reaction temperature for the enzyme was 55?C,the optimum pH was 7.0 and the optimum concentration of NaCl was 4.5 M.And the enzyme possessed good thermal stability under 30?C,50?C and 70?C.NirK_Hrs had good tolerance to glycerol,DMSO,DMF,isopropanol,tween 20,tween 80 and 10%SDS.The food additive sodium disulfite?Na₂S₂O₄?could be used as an effective electron donor in the application of NirK_Hrs.The properties of recombinant nitrite reductase NirK_Hrs obtained in this study are different from those of reported nitrite reductase in halophilic archaea and its most significant salt tolerance provides a guarantee for the application in high-salt preserved foods.