Purification and Characterization of Extracellular Proteinases of Aspergillus oryzae

ABSTRACT

The extracellular proteinases of Aspergillus oryzae EI 212 were separated into two active fractions by (NH₄)₂SO₄ and ethanol fractionation followed by diethyl-aminoethyl-Sephadex A-50 and hydroxyapatite chromatography. The molecular weight was estimated by gel filtration to be about 70,000 and 35,000 for proteinases I and II, respectively. Optimum pH for casein and hemoglobin hydrolysis was 6.5 at 60 C for proteinase I and 10.0 at 45 C for proteinase II, and for gelatin hydrolysis it was 6.5 at 45 C for both enzymes. The enzymes were stable over the pH range 6 to 8 at 30 C for 60 min. The enzyme activity for both the proteinases was accelerated by Cu²⁺ and inhibited by Fe²⁺, Fe³⁺, Hg²⁺, and Ag⁺. Halogenators (e.g., N-chlorosuccinimide) and diisopropyl fluorophosphate inhibited proteinase II. Sulfhydryl reagents such as p-chloromercuribenzoate and iodoacetate inhibited proteinase I. Sulfhydryl compounds accelerated the action of both enzymes.