Diversity And Extracellular Protease Of Cultured Halophilic Archaea Isolated From Salt Lakes In Qinghai And Inner Mongolia

Salt lake is one kind of extreme hypersaline environment which contains a large amount of halophilic archaea.It is the valuable resource for the exploitation and application of halophilic archaea.In this thesis,the diversity of the halophilic archaea from four salt lakes in Qinghai and Inner Mongolia,Qinghaihu salt lake,Chaerhan salt lake,Tarigen salt lake and Chaidamin salt lake,was characterized using culture dependent methods.Then the novel taxa were described by polyphasic taxonomic approaches.The strains isolated from two salt lakes in Inner Mongolia were screened for protease production.The enzymology properties of the crude enzyme from the strains showing higher protease activities were characterized.Phylogenetic analysis based on 16 S rRNA gene sequences revealed that,106 strains isolated from Qinghaihu salt lake belonged to 12 species in 10 genera,the dominant group was Halorubrum.38 strains isolated from Chaerhan salt lake belonged to 9 species in 4 genera,the dominant genera were Haloterrigena,Natrinema and Halalkalicoccus.72 strains isolated from Tarigen salt lake belonged to 10 species in 5 genera,the dominant genera were Natronorubrum and Halalkalicoccus.39 strains isolated from Chaidamin salt lake belonged to 5 species in 4 genera,the dominant genera were Natrinema,Natronorubrum and Halalkalicoccus.The study on the basis of the phylogenetic analysis and phenotypic distinctiveness also showed that strain CEH-5 represented a new species of Natrinema,while strain TRA-38 represented a novel species of a new genus.A total of 111 haloarchaeal strains isolated from the Tarigen and Chaidamin salt lakes were subjected to screening for extracellular protease production.The result showed that 10 halophilic archaeal strains possessed protease activities.Among them,strains Natrinema salaciae CDM-10 and Halalkalicoccus tibetensis CDM-12 produced a high level of proteases and were selected as tested strains.The proteases from strains CDM-10 and CDM-12 showed high stability.Compared with the protease from CDM-10,strain CDM-12 showed high tolerance towards a high concentration of NaCl.Some metal ions such as Mg2+ enchanced the extracellular protease activity of strain CDM-10,and Ca2+,K+ and Fe2+ inhibited the activity.The protease of strain CDM-12 was activated by Mn2+ but was inhibited by Mg2+,Zn2+,Fe2+ and Cu2+.The proteases from both strains CDM-10 and CDM-12 were inhibited by PMSF(a serine inhibitor)and HgBr2(a thiol inhibitor).The results indicated that similar type of proteases might be produced from strains CDM-10 and CDM-12.