Extracellular Protease of Penicillium roqueforti. II. Characterization of a Purified Enzyme Preparation

Abstract

The extracellular protease (E.C. 3.4.4.99) of Penicillium roqueforti BP-13 was isolated from concentrated cell-free extract by fractionation on Sephadex. Molecular weights of 49,000 and 45,000 daltons were established from gel filtration and gel electrophoretic techniques. The protease exhibited an activation energy of 8,000 cal/mol for the hydrolysis of casein. Above 45 C the enzyme was subject to irreversible, first order thermal inactivation. Neither serine protease inhibitor, oxalate, or calcium-(II) had significant effect on enzymatic activity. Modification of the carboxyl groups with diazoacetoglycine methyl ester decreased activity by 90% indicating aspartic or glutamic acid residues at the active site. Of 15 peptides and amino acid esters evaluated as substrates none were hydrolyzed detectably. Casein components, αs- and β-casein, were extensively hydrolyzed after 20 h incubation with the enzyme. κ-casein appeared to be unaffected. The rapid release of trichloroacetic acid-soluble nitrogen from a 1% casein suspension indicated that the fungal protease was a nonspecific proteolytic enzyme. Milk clotting studies revealed that the enzyme was 14 times more proteolytic than calf rennet for identical clotting times. However, because of its general proteolytic characteristic, the fungal protease was not an acceptable substitute for calf rennet in small cheese-making trials. © 1974, American Dairy Science Association. All rights reserved.