Biodiversity And Extracellular Protease Of Haloarchaea Isolated From Dalian Marine Solar Saltern And Chaka Salt Lake

There are a large number of unique land salt lakes and marine solar salterns in China.These hypersaline environments harbor diverse halophilic archaea,many of them have not been systematically surveyed.Comparing to describe novel halophilic archaea species,the understanding of extracellular proteases of halophilic archaea are somewhat vacant.Therefore,the following works were focused.（1）The diversity of halophilic archaea in the Yingchengzi marine solar saltern in Dalian and Chaka salt lake by culture-dependent methods were surveyed.The 126 halophilic archaea isolated from Yingchengzi marine solar saltern and belong to 39 species in 16 genera Many strains may belong to novel species.Strains DL47 and DL50,two strains from the same species are very special,They all have multiple copies of the 16 S rRNA gene within each genome with the sequence difference greater than 9%.The polar lipid components of them do not contain phosphatidylglycerols.Based on these,a novel genus was proposed to accommodate the two strains.The 148 halophilic archaeal strains,representing 27 species in 8 genera,were isolated from Chaka salt lake.Strain C46 was determined to be a new species of the genus Halobaculum.（2）The extracellular protease producing strains from various salt environments were selected and their extracellular crude enzymes were studied.The diversity of extracellular proteases was revealed based on the species diversity,extracellular protease hydrolysis,substrate specificity and catalytic type.It was found that the diversity of extracellular protease in halophilic archaea was high.A halophilic archaeal strain DL61,producing high level enzyme and showing high enzyme activity,was screened from strains isolated frem Yingchengzi marine solar saltern.The gene coding extracellular protease C450₁3147 consisting of 526 amino acids was cloned.the heterologous expressed gene C450₁3147 showed extracellular protease activity.By using the heterologous expression system of Escherichia coli BL21 the maturation mechanism of protease C450₁3147 and the methods of increasing the refolding rate were preliminary discussed.It will be helpful for the theory and application research of extracellular protease of halophilic archaea（3）Haloferax volcanii H1424,heterologously expressing C450₁3147 was used as the starting strain for 30 days of fish sauce fermentation.A number of fish sauce fermentation indexes showed that C450₁3147 had obvious enzymatic hydrolysis advantage in high-salt food fermentation.This study laid a theoretical foundation for the practical application of extracellular protease of halophilic archaea.