PURIFICATION AND CHARACTERIZATION OF ENDOPROTEASES FROM FRUIT BODY OF 'SHIMEJI'MUSHROOM, Lyophyllum aggregatum

Two proteolytic enzymes, L.a. protease I and II, were purified from the fruit body of 'shimeji'mushroom, Lyophyllum aggregatum, by ammonium sulfate precipitation, gel filtration, hydrophobic chromatography and ion-exchange chromatography. The enzymes were assayed using t-butyloxycarbonyl-Ala-Ala-Pro-Phe p-nitroanilide as a substrate. Each of the final enzyme preparations was homogeneous on polyacrylamide gel electrophoresis. The molecular weights of the enzymes were estimated as 44,000 and 46,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzymes had the same optimal pH range of 7–8. Protease I preferentially hydrolyzed peptide bonds of the carboxyl-terminal sides of phenylalanine and leucine, and slowly hydrolyzed the peptide bonds of alanine, threonine and asparagine. On the other hand, protease II showed broader substrate specificity. Both enzymes were almost completely inactivated by diisopropyl phosphofluoridate, and partially inhibited by chymostatin. Protease I was also inhibited weakly by o-phenanthroline. These unusual proteases may have potential for specific food treatment applications.