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 (Hly_Hap) 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 Hly_Hap did not significantly affect its extracellular proteolytic activity. Mature Hly_Hap was generated from recombinant Hly_Hap precursor by high-affinity column refolding. Hly_Hap displayed optimal activity at 0.25-0.50 M NaCl, 45 °C and pH 8.5-9.0. Interestingly, Hly_Hap preferred a low salinity and was stable in a broad range of salinity, albeit from an extremely halophilic archaeon. Ca²⁺ and Mg²⁺ significantly promoted Hly_Hap activity. Hly_Hap activity was stable with organic solvents and detergents. The K_m and V_max values of Hly_Hap 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 Hly_Hap, 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 • Hly_Hap was obtained by heterologous expression and high-affinity column refolding • Hly_Hap exhibited good salinity tolerance.