Extracellular Protease of Penicillium roqueforti. I. Production and Characteristics of Crude Enzyme Preparation

Abstract

The proteolytic activity of various strains of Penicillium roqueforti (ATCC 10110, ATCC 6987, Japan BP-13) was qualitatively assayed by streaking a suspension of spores on the surface of Czapek-Dox agar containing 1.0% sodium caseinate and 20 mM calcium chloride. Extracellular proteolytic activity was based on the precipitation of the calcium-sensitive caseins in the agar medium. Protease was produced by shake-culturing (225 rpm) the various strains of fungi in Czapek-Dox broth containing .5% Proteose-Peptone No. 3 at 25 to 27 C for 72 to 78 h. A cell-free extract of the strain releasing the largest quantities of extracellular protease (BP-13) was prepared by centrifugation of the vegetative growth, followed by filtering the supernatant through Millipore filters. The assay for proteolytic activity was based on the amount of trichloroacetic acid-soluble nitrogen released after 9 min incubation at 30 C with 1.0% Hammersten casein at pH 5.75. The extracellular protease had pH optima of 3.0 and 5.5 for bovine serum albumin and casein. Maximum stability to pH occurred in the range of 3.0 to 6.0. The optimum temperature for proteolytic activity was 46 C; thermal inactivation commenced at 48 C. © 1974, American Dairy Science Association. All rights reserved.