Production of extracellular alkaline and neutral proteases of Ustilago maydis

Abstract

Protease production of Ustilago maydis in mineral salts medium is influenced by the type and concentration of the nitrogen source. Culture studies point to a coordinated derepression and inducer regulation. Glucose as carbon source promotes the rate of cell division as well as the overall protease activity. The enzymes from culture supernatants were investigated by sodium dodecyl sulfate-gelatin-polyacrylamide gel electrophoresis. Due to the specific effect of several tested inhibitors, two enzyme groups (P1 and P2) can be distinguished, each with multiple bands of activity. The P1 enzymes, sensitive to phenylmethyl-sulfonyl fluoride but not affected by the cysteine protease inhibitor iodoacetamide are probably serine proteases, while inhibition of P2 enzymes by 1,10-phenanthroline suggested that these are metalloproteases. P2 enzymes require Ca 2+ for their activity in the substrate gel. All proteases demonstrate nonspecific endoprotease activities and hydrolyze gelatin in the alkaline and neutral pH range; the P1 enzymes are active even at slightly acidic values. The pattern of enzyme activity as detected on the substrate gels varied during growth of U. maydis and was influenced by altering test conditions.