Biodiversity And Aminopeptidase Of Culturable Halophilic Archaea Isolated From Chaganuoer Soda Lake In Inner Mongolia

China is rich in natural salt lake resources,which contain abundant halophilic archaea resources.The unique climate of the Inner Mongolia plateau gives birth to the largest number of salt lakes,including many soda lakes.In this study,the culturable halophilic archaeal diversity of Chagannuor Soda Lake in Inner Mongolia was mainly determined,new halophilic archaea resources were explored,and the taxonomic status of novel strains were determined by using taxonomic methods.The haloarchaea aminopeptidase genes were screened from the genome of halophilic archaea,and further studies on the heterologous expression and enzymatic properties of halophilic archaea aminopeptidase were conducted,hoping to provide a scientific basis for the development and utilization of enzyme preparations for food processing.Eighty-nine halophilic archaea were isolated from the soli of Chaganuoer soda lake in Inner Mongolia.Phylogenetic analysis based on 16S rRNA gene showed that these strains belonged to 15 species of 8 genera,including Natronococcus,Natronorubrum,Halovivax,Halovarius,Halkalicoccus,CGA53,CGA73 and CG73.Among these species,3 potential new genera and 8 potential new species were found,and the dominant group was Halkalicoccus tibetensis.Based on phylogenetic analysis of the 16S rRNA,rpoB'and orthologous genes,phenotypic characteristics and chemical composition,strain CGA30 represented a new species of the genus Halovivax?Halovivax salinus sp.nov.?,and strain CGA73 represented a new genus?Natronorubellus halophilus gen.nov.sp.nov.?,both of which were alkaliphilic haloarchaea.Among the new taxa obtained,strain CGA30 and CGA73 can produce aminopeptidases with highly activity.One M42 family aminopeptidase gene?CGA30₂942?was discovered from the genome of strain CGA30,and three aminopeptidase genes?CGA73₁020,CGA73₂402,CGA73₂818?were discovered from the strain CGA73 genome.It is predicted that these four aminopeptidases were all dodecameric metal aminopeptidase complex with the tetrahedral shape,and the active sites and metal ion binding sites were highly conserved with M42 family aminopeptidases.Through heterologous expression and affinity chromatography purification,halophlic aminopeptidases were obtained.The apparent molecular weight of CGA30₂942 was 65 kDa,and the apparent molecular weights of CGA73₁020,CGA73₂402 and CGA73₂818 were 58,55 and 60 kDa respectively.The aminopeptidase CGA73₂402 with the highest activity on lysine substrate was named NhTET,and its enzymatic properties were characterized.The enzyme has wide substrate specificity and the strongest hydrolysis ability of lysine.The optimal reaction temperature of this enzyme was 65?,the optimal concentration of NaCl was4.9 M,and that of KCl was 2.5 M.The optimal reaction pH was 8,which belonged to halophilic enzyme.NhTET can tolerate some organic solvents,its activity can be inhibited by EDTA,PMSF,DTT and?-mercaptoethanol,and the addition of Cu²⁺,Mn²⁺and other metal ions can enhance its activity.The discovery of these four novel enzymes would expand the resources of halophilic archaea aminopeptidases,they can be used in high-salt fermented foods?such as shrimp paste,fish paste?and can lay a foundation for the development and application of enzyme preparations for food industry.