Purification and Characterization of an Extracellular Aspartate Protease fromPhycomyces blakesleeanus

Abstract

De Vicente, J. I., De Arriaga, D., Del Valle, P., Soler, J., and Eslava, A. P. 1996. Purification and Characterization of an Extracellular Aspartate Protease fromPhycomyces blakesleeanus. Fungal Genetics and Biology20,115–124. An acid protease has been found in the culture broth ofPhycomyces blakesleeanusgrowing under standard conditions. It has been induced up to 70-fold with several complex growth media and the enzyme has been purified to homogeneity and characterized. The molecular mass of the native enzyme was estimated by gel filtration to be 40 kDa. The acid protease ofPhycomycesmigrated as a single band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, corresponding to a molecular mass of 35 kDa. The glycoprotein nature for the acid protease was deduced from its binding to a concanavalin A–Sepharose 4B column. The carbohydrate moiety is composed of mannose and rhamnose. Its amino acid composition was determined, and its isoelectric point was estimated to be 4.2, the optimum pH was 2.5 to 3, and the optimum temperature was 70°C, using hemoglobin as a substrate. The enzyme showed thermal stability between 37 and 50°C. The thermodynamic parameters for hemoglobin hydrolysis and thermal inactivation were calculated. With Lys-Pro-Ile-Glu-Phe-Phe(4-NO2)-Arg-Leu as the substrate, theKm,kcat, andVmaxvalues were 8.78 μM, 1.25 s−1, and 2.12 μmol min−1mg−1, respectively. The protease was insensitive to phenylmethylsulfonyl fluoride,O-phenanthroline,N-ethylmaleimide, iodoacetamide, ethylenediaminetetraacetate, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, and trypsin inhibitor. However, pepstatin A established a strong competitive inhibition against it, with aKivalue of 1.33 nM.The data suggest that this protease has properties of an aspartate-type proteinase.