Biodiversity And Nitrite Reductase Of Halophilic Archaea Isolated From Saline-alkali Lake In Tibet

As a food additive,nitrite is widely used in food,especially in various high-salt foods.Excessive intake of nitrite can lead to chronic carcinogenesis and acute poisoning.Controlling nitrite within safe limits has attracted increasing attention.In this study,the Kunzhongcuo Salt Lake and the Mandongcuo Salt Lake in Tibet were investigated to clarify the diversity of cultivable halophilic archaea and isolate new species.Novel nitrite reductase genes were screened from halophilic archaea through bioinformatics analysis.The novel nitrite reductases were heterologously expressed,purified and characterized.A total of 221 halophilic archaeal strains were isolated from Kunzhongcuo Salt Lake,belonging to 9 species of 7 genera.Halalkalicoccus paucihalophilus was the dominant species,accounting for 55.66%.In Mandongcuo Salt Lake,a total of 123 halophilic archaeal strains were isolated,belonging to 12 species of 8 genera.Halostagnicola larsenii was the dominant species,accounting for 52.04%.Novel halophilic archaea were isolated from the two salt lakes.The polyphasic taxonomic study of novel strains from Kunzhongcuo Salt Lake showed that KZCA68 is a new species of the genus Haloterrigena,named Haloterrigena tibetensis;KZCA101 and KZCA124 are two new genera in the family Natrialbaceae,named Natronorussus tibetensis and Natronorubellus tibetensis,respectively.Bioinformatics analysis of the whole genome of KZCA68,KZCA101 and KZCA124 revealed three potential nitrite reductase genes of KZCA68₀593,KZCA101₂193 and KZCA124₀481.They were subject to heterologous expression,purification and enzyme activity determination.KZCA68₀593 had the highest enzyme activity,named NirKHtg.NirKHtg is a Cu-containing dissimilatory nitrite reductase with Tat signal peptide and lipobox.Deletion of the Tat signal peptide and site-directed mutation of C26,the key amino acid residue of lipobox,were performed to investigate their effects on the enzyme activity.The results showed that the enzyme activity of the NirKHtgC26S mutant was not significantly affected,while the NirKHtg?Tat expressing strain lost the extracellular enzyme activity.The enzymatic properties of NirKHtg and NirKHtg?Tat were thus characterized.The optimal temperature and pH for NirKHtg and NirKHtg?Tat were 35? and pH 6.0,while the optimal NaCl concentration was 1.5 M and 2.5 M,respectively.They showed good thermal and salinity stability.They were relatively stable under pH 6.0 and pH 7.5.They displayed good tolerance to isoacetone,ethanol and Tween 80.Compared with NirKHtg?Tat,NirKHtg had better salinity tolerance,pH stability,organic solvent and metal ion tolerance.Therefore,NirKHtg is a novel Cu-containing dissimilatory nitrite reductase with excellent enzymatic properties,indicating potential applications in high-salt food industries.