Identification and characterization of a novel halolysin from Halostella sp. PRR32 with two C-terminal extensions.

Extracellular proteases produced by haloarchaea, termed halolysins, possess potential applications in diverse fields including food fermentation and bio-remediation. In this study, an extracellular protease encoding gene, hly32^PRR32, from Halostella sp. PRR32 isolated from a salt mine in Anhui, China, was identified and expressed in Escherichia coli. The expressed protein MBP-Hly32 was purified and biochemically characterized. The results indicate that Hly32 belongs to the S8 family of serine proteases (halolysin). A BLAST search on NCBI reveals that Hly32 has an amino acid sequence identity of 68.87% with serine protease Hly176B from Haloarchaeobius sp. FL176. MBP-Hly32 contains a catalytic triad of Asp¹⁵⁹-His¹⁹⁸-Ser³⁵⁰ and two C-terminal extensions which are crucial for its activity. The optimal conditions for its enzyme activity are 50 °C, pH 8.0, and 4.0 M NaCl. Under these conditions, the K_m, V_max and K_cat for the MBP-Hly32 were determined to be 2.34 mM, 935.50 U·mg^-1 and 1472.40 s^-1, respectively. Metal ions and organic reagents affect its activity differently from the typical halolysins; for example, Ca²⁺, which enhances the activity of other halolysin enzymes, has no effect on MBP-Hly32. Furthermore, the activity of Hly32 was inhibited by the presence of PMSF, DTT, and EDTA. Furthermore, a three-dimensional structure prediction based on functional domains was obtained in this study which will facilitate modification and protein engineering halolysins to generate mutants with new physiological activities.