An extremely heat-stable extracellular proteinase (aeropyrolysin) from the hyperthermophilic archaeon Aeropyrum pernix K1.
An extracellular metalloproteinase, which we had designated aeropyrolysin, from the aerobic marine hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820), was purified by ammonium sulfate precipitation, anionic exchange chromatography, and gel filtration chromatography. The purified enzyme was composed of a single polypeptide chain with a molecular mass of 52 kDa as determined by SDS-PAGE. The proteinase had a broad pH optimum (pH 5-9) with a maximal activity at pH 6-8 for azocasein hydrolysis. The optimum temperature for enzyme activity was 100 degrees C in the absence of 1 mM CaCl2 and 110 degrees C in the presence of 1 mM CaCl2. The enzyme activity was completely inhibited by EDTA and EGTA, indicating that it was a metalloproteinase. The enzyme was highly resistant to the denaturing reagents urea, guanidine-HCl, dithiothreitol, 2-mercaptoethanol and SDS. The enzyme also showed a high activity with the metalloproteinase specific substrate MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2. The enzyme was extremely thermostable showing half-lives of 2.5 h at 120 degrees C and 1.2 h at 125 degrees C in the presence of 1 mM CaCl2. These results indicate that this enzyme is one of the most thermostable extracellular proteinases reported to date.
Sako
Y
Y
Division of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Japan.
Croocker
P C
PC
Ishida
Y
Y
D002364:Caseins / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D019615:Crenarchaeota / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D011489:Protein Denaturation; D013379:Substrate Specificity
D002364:Caseins; D002614:Chelating Agents; D011480:Protease Inhibitors; C026018:azocasein; D008666:Metalloendopeptidases; C108911:aeropyrolysin