Heat-stable extracellular proteolytic enzyme produced by Thermus caldophilus strain GK24, an extremely thermophilic bacterium.

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Proteolytic activity was detected in the culture supernatant of a newly isolated, extremely thermophilic bacterium belonging to the genus <i>Thermus</i>, and tentatively named <i>T. caldophilus</i> sp. n. strain GK24. The enzyme activity continued to increase for at least three days after cells reached the stationary phase of growth. Purification of the proteolytic enzyme was tried with ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. The most purified enzyme fraction thus obtained appeared to be homogeneous in a chromatographic analysis, but still had seven bands of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Treatment of the protease with denaturing reagents or organic solvents did not alter the chromatographic profile and the purified enzyme sample showed a large sedimentation coefficient of about I IS. The optimal pH of the hydrolytic activity of the enzyme was observed at around 7.8 for casein and 7.2 for <i>N</i>-carbobenzoxy-L-leucyl-L-tyrosinamide (Z-Leu-Tyr-NH<sub>2</sub>). The enzyme was stable in the pH range of 5 to 11 for 1 day at 4°C or for 1 h at 70°C. The enzyme sample showed a maximal activity at 90°C and had an extreme stability toward treatment by heat and denaturing reagents. The enzyme sample was inactivated almost completely by diisopropyl fluorophosphate (DFP), but not by ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis-(β-aminoethyl ether)<i>N</i>, <i>N</i>'-tetraacetic acid (EGTA). From these results, the enzyme seems to be a serine protease, and not to be a metallo-enzyme such as thermolysin. The enzyme also was hydrolytic active toward an ester compound, <i>N</i>-benzoyl-L-tyrosine ethyl ester (BTEE), but not toward <i>N</i>-benzoyl-L-arginine ethyl ester (BAEE).


  • J Biochem (Tokyo)

    J Biochem (Tokyo) 93(1), 7-13, 1983

    The Japanese Biochemical Society


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