A novel halolysin without C-terminal extension from an extremely halophilic archaeon.

Halolysins are extracellular proteases secreted by halophilic archaea for nutritional purposes. They bear great application potentials in various industries. Yet the diversity of halolysins remains underexplored. In this study, a halolysin from the extremely halophilic archaeon Haladaptatus sp. DYF46 (HlyHap) was identified to be a novel type of halolysin without C-terminal extension (CTE). Addition of the CTE of a halolysin from Halococcus salifodinae to HlyHap did not significantly affect its extracellular proteolytic activity. Mature HlyHap was generated from recombinant HlyHap precursor by high-affinity column refolding. HlyHap displayed optimal activity at 0.25-0.50 M NaCl, 45 °C and pH 8.5-9.0. Interestingly, HlyHap preferred a low salinity and was stable in a broad range of salinity, albeit from an extremely halophilic archaeon. Ca2+ and Mg2+ significantly promoted HlyHap activity. HlyHap activity was stable with organic solvents and detergents. The Km and Vmax values of HlyHap against azocasein were 0.018 mM and 7,179 U/mg, and those against succinyl-Ala-Ala-Pro-Phe-pNA were 0.32 mM and 3×106 μmol/min/μg, respectively. The unusual traits of HlyHap, a novel type of halolysin without CTE, may endow it with strong potential for various industrial uses, such as biocatalysis in fluctuating salinities and aqueous-organic solvent. KEY POINTS: • This is the first report of a novel type of halolysin without C-terminal extension • HlyHap was obtained by heterologous expression and high-affinity column refolding • HlyHap exhibited good salinity tolerance.