Biodiversity And Extracellular Protease Of Cultured Halophilic Archaea Isolated From Saline Environment In Xinjiang

The halophilic archaea grow in a high salt environment and usually require 15%to 30%NaCl to maintain optimum growth conditions.Some halophilic archaea secrete proteases to degrade proteins in the environment to small molecules of peptides and amino acids involved in the metabolism.Present researches on halophilic archaea resources mainly focus on the investigation of halophilic archaea in natural salt lakes and saline lakes that are widespread in the western region of China.There are few investigations on the diversity of halophilic archaea in saline-alkali environments.The Tarim Basin located in the Xinjiang region is the largest inland basin in China,where the soil is extremely saline and alkaline.This environment is bound to contain abundant halophilic archaea.Therefore,this study focus on collecting the saline-alkaline soil in Tarim Basin,isolating halophilic archaea,clarifying the diversity of halophilic archaea,and describing the new taxa;then screening the strains producing extracellular proteases,tapping the genes coding extracellular protease,and revealing the function of the extracellular protease domain.A total of 68 strains of halophilic archaea were isolated from the saline-alkaline samples from the Tarim Basin using NOM medium.Based on the 16S rRNA gene sequence analysis and phylogenetic analysis,the 68 halophilic archaea belonged to 28species of 20 genera.The results of polyphasic taxonomy study showed that four strains of the 68 halophilic archaea represent four new taxa,Halobaculum rubrum NJ-3-1^T,Halogranum salinum SQT-7-1^T,Natrinema rubrum SQT-13-1^T,and Halovivax salinus SQT-29-1^T.Among 68 halophilic archaeal strains,ten strains were found to produce extracellular proteases and they belong to 5 species of 3 genera.Halococcus salifodinae EEST14 was found to produce high levels of extracellular protease and further used in identifying the genes encoding extracellular protease.The results showed that C450₁3147 was an extracellular protease of Halococcus salifodinae EEST14.Site-directed mutagenesis of the active site revealed that C450₁3147 is a serine protease and contains signal peptide,propeptide,catalytic domain and C-terminal extention?CTE?.The CTE can achieve functional interchange between similar extracellular proteases.The truncation experiment showed that the shortening of the CTE greatly decreased the activity of the extracellular protease.The presence of three cysteines in C450₁3147 plays an important role in enzyme activity.