Biodiversity And Extracellular Protease Of Cultured Halophilic Archaea Isolated From Saline Environments In Tibet Region

The special geography and climate of Tibet gave birth to the high salt environments different from other regions.These hypersaline environments harbour a large number of halophilic archaea.In this paper,the halophilic archaeal diversities of two hypersaline environments,Mangkang ancient solar saltern and Nieer salt lake were analyzed.The community structure was analyzed,and the taxonomic status of the unknown taxa was studied by polyphasic taxonomy.The mechanism of synthesis of haloarchaeal extracellular protease was studied and the protease was characterized.A totally of 191 haloarchaeal strains,133 strains are isolated from Mangkang ancient solar saltern,58 strains are isolated from Nieer salt lake.Phylogenetic analysis based on 16 S rRNA genes of all the strains indicated that the strains from Mangkang ancient solar saltern belonged to 17 species in 8 genera,the dominant group is Halolamina.And the strains from Nieer salt lake belong to 21 species in 13 genera,the dominant group is Halorubrum.Six strains isolated from Mangkang ancient solar salter may represent six novel species.These novel species were described And the resperentative type strains were Halobacterium tibetense XZYJT26 T,Halosimplex tibetense XZYJT29 T,Haladaptatus salinus XZYJT32 T,Halorussus litoreusXZYJT10 T,Halorussus tibetensis XZYJ18 T and Halorussus limi XZYJT40 T.A total of 133 strains of halophilic archaea were cultivated on NOM milk agar plate to test whether these strain can produce extracellular protease or not and a total of 11 strains of halophilic archaea were screened.Phylogenetic analysis showed that these 11 strains belonged to 4 different species.Strian XZYJ18 showed the highest protease activity,and has good ability to hydrolysis gelatin and milk.Strain XZYJ18 began to secrete a large number of proteinase at logarithmic phase transition to a plateau and the addition of yeast extract could inhibit the secretion of protease.Strain XZYJ18 can produce extracellular protease under starvation.The extracellular protease of strain XZYJ18 showed optimum activity at 55 °C,pH8.0 and 0 M NaCl.Interestingly,when the concentration of NaCl was up to 3 m,the activity of the enzyme was about 61% of the total enzyme activity.Mn2+ significantly enhanced the protease activity.In addition,the protease from strain XZYJ18 is a thiol-dependent serine protease,and its activity can be partially inhibited by both EDTA and pepstatin A.This suggested that the protease from strain XZYJ18 may be a novel protease.