Purification and partial characterization of two acidic proteases from the white-rot fungus Sporotrichum pulverulentum

Abstract

Two acidic proteases from the white-rot fungus Sporotrichum pulverulentum have been purified and partially characterized. The enzymes were purified in four steps, protease I 152-fold and protease II 127-fold. The purity of the enzymes was investigated by analytical isoelectric focusing and by dodecylsulfate/polyacrylamide gel electrophoresis. The isoelectric points of protease I and protease II were at pH 4.7 and 4.2 respectively. The molecular weights were 28,000 and 26,000 and the pH optima 5.0 and 5.2. Both enzymes were inhibited by heavy metal ions such as Ag+, Hg2+ and to some extent also by Cu2+. Partial inhibition was also observed with EDTA and alpha, alpha'-dipyridyl. The specificities of protease I and II were investigated using human fibrinopeptide A as substrate. Protease I splits off the arginine in the C-terminal position, while protease II splits at the carboxyl side of both Phe-8 and Leu-9 in fibrinopeptide A. A physiological effect of the two proteases has also been demonstrated. Thus, if a culture solution from S. pulverulentum grown on cellulose was treated with the individual proteases or a mixture of the enzymes a considerable increase in endo-1,4-beta-glucanase activity was obtained. It may be that the endo-glucanases are produced in a zymogenic form and activated by the proteases. However, other explanations for the phenomenon are also possible.