The fungus Tritirachium album Limber was grown by submerged fermentation, investigating the conditions for maximal secretion of proteases into the medium. Enzyme secretion starts when the stationary phase of growth is reached, and when the culture medium is depleted of glucose and amino acids, suggesting a catabolite repression of the enzyme.

The main proteolytic enzyme was named proteinase with respect to its keratin hydrolyzing activity. It was isolated from the culture medium and purified by crystallization and chromatography on DEAE-Sephadex and Sephadex G-75. Homogeneity was ascertained by disc gel electrophoresis and by isoelectric focusing. The molecular weight of proteinase K was determined by gel filtration to be 18500 ± 500, the isoelectrice point was pH 8.9. The enzyme was shown to be a serine protease by inactivation with diisopropyl phosphofluoridate and phenylmethylsulfonyl fluoride. It displayed strong proteolytic activity on denatured but also on native proteins as demonstrated by the rapid inactivation of bovine ribonuclease. The pH-optimum was in the range between pH 7.5–12.0. A specificity of the enzyme for peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids was observed.