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 f 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.Screening for microorganisms with proteolytic activity towards native proteins we found a strain of the mold Tritirachium album which secrets a highly active mixture of proteases along with an aminopeptidase into the culture medium. The isolation of proteases from this organism seemed of interest to us because the organism can grow on native keratin as sole carbohydrate and nitrogen source.In this paper the cultivation of Tritirachium album and the purification and some properties of the main protease will be described. This enzyme was named "proteinase K" with respect to its keratin hydrolyzing activity. A characteristic property of the enzyme is its ability to hydrolyze native proteins. This is the first Abbreviations. Ac, acetyl-; Ac-Tyr-OEt, acetyl-Ltyrosine ethyl ester; Bz-Arg-OEt, benzoyl-L-arginine ethyl ester; 2' : 3'-CMP, 2' : 3'-cyclic cytidine monophosphate; EDTA, ethylenediamine tetraacetate; Tos-Lys-CHzC1, l-chloro-3-tosylamido-7-amino-2-heptanone (tosyl lysylchloromethyl ketone); Tos-Phe-CHzC1, L-( I -tosylamido-2-pheny1)ethyl chloromethyl ketone; Z-, benzyloxycarbonyl-; -Nan, 4-nitroanilide.Enzymes. Creatine kinase (EC 2.7.3.2); lactate dehydrogenase (EC 1.1.1.27); malate dehydrogenase (EC 1.1.1.37); ribonuclease (EC 3.1.4.22).Definition. One Anson-U is defined as that amount of enzyme which, under standard conditions, liberates one millimole of Folin-positive amino acids (calculated as tyrosine) per min.report on the purification of a fungal protease from the genus Tritirachium. MATERIALS AND METHODSAll reagents, enzymes and enzyme substrates were products of E. Merck (Darmstadt). OrganismTritirachium album Limber (Merck strain No. 2429) was isolated from soil manured with horn chips. The strain is deposited at the Centraalbureau voor Schimmelcultures at Baarn (CBS no. 747.69). C...

No abstract

No abstract