Stability and Extrinsic Factors for Stability at above 100℃ of the Extracellular Proteinase Aeropyrolysin from the Hyperthermophilic Archaeon Aeropyrum pernix
Pamela CHAVEZ-CROOCKER*, Yoshihiko SAKO, and Aritsune UCHIDA
Laboratory of Marine Microbiology, Division of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Kyoto 606-01, Japan
Previously, we have reported on the purification and characterization of two extremely heat-stable proteases, Aeropyrolysin (1) and Pemilase (2), from Aeropyrum pernix, a hyperthermophilic archaeon isolated from a submarine hydrothermal vent located in Kodakara Island, Japan (3). Aeropyrolysin is a 52 kDa metallo-proteinase, highly resistant to denaturant agents and detergent. Its optimum temperature for activity is 100℃ in the absence of 1 mM Cac12 and 110℃ in the presence of 1 mM Cac12. Thermostability assays using 1 mM Cac12 at the optimum pH for enzyme activity (pH 8.0) showed half-lives of 2.5 h at 120℃ and 1.5 h at 125℃. The high stability of Aeropyrolysin at temperatures above 100℃ and studies on the intrinsic and extrinsic factors that affect it could give us interesting features on thermostabilization mechanisms.
In this study we report on the characterization and the attempts to further improve the thermostability of Aeropyrolysin through the study of its intrinsic stability and extrinsic stabilization factors. The native enzyme showed a pH dependent thermostability. The enzyme is rapidly inactivated at low pH but highly stable at alkaline pH (10.4). This thermal inactivation at 100℃ of the enzyme at pH 7 and 10.4 is not enzyme concentration dependent. In addition, some extrinsic factor for stability such as, calcium ion concentration, sodium and potassium salts, high and low molecular weight compounds and external hydrophobicity, were examined. The presence of calcium ion play an important role in the heat-stability of the enzyme. The half-life of the enzyme, in the presence of calcium ion and alkali pH, was improved to 3.7 h at 125℃ and 29 min at 130℃.
1. Sako, Y., Chavez-Croocker, P., and Ishida, Y. (1997). FEBS Letter, 415, 329-334. 2. Chavez-Croocker, P., Sako, Y., and Uchida, A. (1997). Extremophiles, submitted. 3. Sako, Y., Nomura, N., Uchida, A., Ishida,Y., Morii, H., Koga, Y., Hoaki, T., and Maruyama, T. (1996) Int. J. Syst. Bacteriol., 46, 1070-1077.
