This work reported a new extremely thermostable protease from a newly isolated hyper thermophilic Bacillus sp.MLA64 from “Dig Rostam” hot spring in Iran. The protease was purified with an 11.4-fold increase in specific activity and 28% recovery. The molecular weight of the purified enzyme was estimated to be 24 kDa. The enzyme was extremely stable and highly active in the temperature range from 40 to 100° C with an optimum temperature at 95°C. The enzyme was highly active over a wide range of pH from 8.0 to 12.5, with an optimum at pH 9.5. The thermostability of the enzyme was not enhanced in the presence of CaCl2, indicating that the enzyme is calcium independent. CD spectroscopy data revealed no conformational change occurred in the secondary structure of enzyme after 30 min incubation at 95°C. The enzyme showed high stability towards non-ionic surfactants and relative stability against anionic surfactant SDS. In addition, the enzyme was relatively stable towards oxidizing agents. The N-terminal amino acid sequence of the first 20 amino acids of the purified protease showed low homology with other bacterial peptidases, suggesting that the enzyme can be a new protease.

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