Purification and characterization of an intracellular heat-stable proteinase (pernilase) from the marine hyperthermophilic archaeon Aeropyrum pernix K1

Abstract

A novel intracellular serine proteinase from the marine aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) that we designated pernilase was purified by ammonium sulfate precipitation, anionic-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified enzyme was composed of a single polypeptide chain with a molecular mass of 50 kDa as determined by SDS-PAGE. The proteinase had a broad pH profile (pH 5–10) with an optimum pH of 9.0 for peptide hydrolysis. The optimum temperature for enzyme activity was 90°C. The enzyme was strongly inhibited by diisopropyl fluorophosphate (DFP) and phenylmethyl sulfonylfluoride (PMSF), suggesting that it corresponds to a serine proteinase. The enzyme was highly resistant to the reducing agents dithiothreitol and 2-mercaptoethanol but sensitive to the denaturing reagents guanidine-HCl and urea and also to the detergent sodium dodecyl sulfate (SDS). Pernilase showed high substrate specificity for Boc-Leu-Gly-Arg-MCA peptide. Thermostability of this enzyme showed half-lives of 85 min at 100°C and 12 min at 110°C.