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Thermostable protease is very effective to improvethe industrial proceses in many fields. Two thermostableextracellular proteases from the culture supernatant of thethermophilic fungus Chaetomium thermophilum were purifiedto homogeneity by fractional ammonium sulfate precipitation,ion-exchange chromatography on DEAE-Sepharose, and Phenyl-Sepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular mas of the two purified enzymes wasestimated to be 3 kDa and 63 kDa, respectively. The twoproteases were found to be inhibited by PMSF, but not byexhibited maximal activity at pH 10.0 and the 63 kDa protease(PRO63) at pH 5.0. The optimum temperature for the twoproteases was 65oC. The PRO32 had a Km value of 6.6 mMand a Vmax value of 10.31mol/l/min, and PRO63 17.6 mMand 9.08mol/l/min, with casein as substrate. They werethermostable at 60oC. The protease activity of PRO33 andincubation at 70oC for 1 h. The thermal stability of the twoenzymes was significantly enhanced by Ca2+. The residualactivity of PRO33 and PRO63 at 70oC after 60 min wasapproximately 8.59% and 39.2%, respectively, when kept inthe bufer containing Ca2+. These properties make themaplicable for many biotechnological purposes.