Biodiversity And Extracellular Protease Of Cultured Halophilic Archaea Isolated From Salt Mine

Yipinglang salt mine is one of the largest salt mines in Yunnan province. The unique characteristics of halophilic archaea flora were determined by its special environment diversity as well as the novel taxa from this spot by the polyphasic taxonomical method. Meanwhile, halophilic archaeal strains that had a high extracellular protease production capacity were also studied in this paper.A total of 59 haloarchaeal strains were isolated from Yipinglang salt mine at Yunnan Province. Phylogenetic analysis based on 16 S rRNA genes of all the strains indicated that they belonged to 20 species in 14 genera. The dominant genera were Halorubrum, Natrinema and Halococcus. The polyphasic taxonomic study provided clear evidence that strain YPL13 represented a new species, while strain YPL18 represented a novel genus.The 59 haloarchaeal strains were screened for their ability to produce extracellular proteases. Strains YPL20 and YPL26 showed high protease production and therefore, selected for further analysis. 16 S rRNA gene and rpoB′ gene sequence analysis revealed that the two strains were similar strains of Halococcus salifodinae. The protease(s) from H. salifodinae YPL26 showed relatively high activity over a broad temperature range from 40 °C to 75 °C, a narrow range of pH from 7 to 9, and a wide salinity range from 0 to 3 M NaCl; and displayed optimum activity at 75 °C, pH 9 and 2.5 M NaCl. The protease(s) from H. salifodinae YPL20 were active over a broad temperature range from 45 °C to 75 °C, a wide range of pH from 7 to 10, and a wide salinity range from 0 to 2 M NaCl; and exhibited optimum activity at 50 °C, pH 9 and 0 M NaCl. The protease(s) from both H. salifodinae YPL20 and YPL26 strains exhibited good temperature and pH stability. Interestingly, Mn2+ significantly enhanced protease activity of strains YPL20 and YPL26, respectively. In addition, the protease activities of strains YPL20 and YPL26 were inhibited by the serine-protease inhibitor phenylmethylsulfonyl fluoride(PMSF) and p-chloromercuribenzoate(PCMB). The results showed that similar type of proteases might be produced from similar strains YPL20 and YPL26 of H. salifodinae, and further provided insight into the intraspecies diversity of haloarchaeal proteases. Additionally, their unusual properties of good temperature, pH, and salinity tolerance allowed them to be promising candidates for use in various industries.