Biodiversity And Extracellular Proteases Of Halophilic Archaea Isolated From Wulanhushao Salt Lake And Dongying Solar Saltern

China is rich in saline environment resources,and the abundant halophilic archaea in it remains to be discovered.In this research,culture dependent technology was used to study the haloarchaeal diversity in thalassohaline and athalassohaline saline environments,and to compare the differences of haloarchaeal groups between different environments.The search for haloarchaeal functional genes was based on genome sequence.A novel haloarchaeal extracelluar protease(halolysin)encoding gene was cloned,and the recombined protease was heterologously expressed,purified and renatued/refolded.The enzymatic characteristics of the novel halolysin were subsequently characterized.Hope to provide scientific basis for the research and development of salt-tolerant proteases.55 haloarchaea strains were isolated from athalassohaline saline environment Wulanhushao salt lake.They belonged to 17 species of 12 genus respectively,and genus Natronococcus was dominant genus.54 haloarchaea strains were isolated from thalassohaline saline environment Dongying solar saltern.They belonged to 16 species of 8 genus respectively,and genus Haloarcula was dominant genus.Haloarchaea in both two kinds of saline envrionments were abundant and different.Results of polyphasic taxonomy study showed WLHS5 T,WLHSJ27T and DYF38 isolated from these two environments represented one novel genus(Natronorussus halophilus gen.nov.,sp.nov.WLHS5T)and two novel species(Halovivax halophilus sp.nov.WLHSJ27 T,Salinigranum halophilum sp.nov.DYF38).A haloarchaea strain Haladaptatus sp.DYF46 was selected with the highest capacity of producing extacelluar protease among all isolates.5 potential halolysin genes were found in the genome of strain DYF46,and thses 5 genes were cloned and expressed in Haloferax volcanii H1424 individually.The gene hly was verified to encode a novel active halolysin Hly.Hly contained a signal peptide,propeptide and catalytic domain,without C-terminal extension(CTE)that is widely present in classical halolysins.The novel halolysin Hly could be recombined with other CTEs of the same kind of extracelluar proteases.Combination of CTE derived from neutral haloarchaea at the C-terminus of Hly had no effect on the catalytic activity of the recombinant protease.Recombinant halolysin Hly with high activity and purity was obtained by heterologous expression,Ni-sepharose purification,renaturation/refolding with high salt and gel filtration.Hly had high catalytic activity on both large protein substrate azocasein and small tetrapeptide substrate Suc-AAPF-p NA.Hly exhibited highest catalytic activity at low salt concentration with optimum at 0.5 M Na Cl although it was isolated from an extremely halophilic archaeon.Hly maintained considerable stability under high salt condition.Hly exhibited a broad temperature range from 30-60? with highest catalytic activity at 45?.Hly preferred alkalic conditions with optimal p H at 8.5/9.Hly was tolerant to a variety of metal ions,organic solvents and detergents.Hly was almost inactive with the serine-protease inhibitor PMSF,which indicated Hly is serine protease.In summary,a rich group of halophilic archaea were isolated from Wulanhushao salt lake and Dongying solar saltern,and polyphasic taxonomy study of three novel strains of halophilic archaea were completed in this research.A new type halophilic extracellular protease Hly with excellent characteristics was obtained,which laid a foundation for further application of it to the food industry.