ABSTRACT
The presence of proteins in the culture liquid of filamentous fungi under study was found to induce the secretion of proteinases. The inhibitory analysis of the major extracellular proteinases of the saprotrophic fungus Trichoderma harzianum and the phytopathogenic fungus Alternaria alternata showed that they both belong to the group of serine proteinases. The substrate specificity of these proteinases and their sensitivity to inhibitors suggest that the enzyme of T. harzianum is a subtilisin-like proteinase and the enzyme of A. alternata is a trypsin-like proteinase. This difference between the proteinases may reflect the physiological difference between their producers (saprotroph and phytopathogen).
Subject(s)
Alternaria/metabolism , Fungal Proteins/metabolism , Subtilisin/metabolism , Trichoderma/metabolism , Trypsin/metabolism , Alternaria/growth & development , Culture Media, Conditioned/metabolism , Plant Diseases/microbiology , Substrate Specificity , Trichoderma/growth & developmentABSTRACT
Extracellular proteases secreted by the filamentous fungus Trichoderma harzianum have been identified. A proteinase active towards Z-Ala-Ala-Leu-pNa--the substrate of subtilisin-like proteases--dominated in the culture medium. This proteinase is synthesized de novo in response to addition of a protein substrate to the medium. Changing the carbohydrate in the culture medium changed the quantitative and qualitative spectrum of secreted enzymes. The most active extracellular proteinase of Trichoderma harzianum was purified 322-foldfrom the culture medium and obtained with a yield of 7.2%. The molecular mass of this proteinase is 73 kD and its pI is 5.35. The isolated enzyme has two distinct activity maxima, at pH 7.5 and 10.0, and is stable in the pH range 6.0-11.0. The temperature optimum for enzyme activity is 40 degrees C at pH 8. 0. The proteinase is stable up to 45-50 degrees C (depending on the substrate used). Calcium ions stabilized the enzyme at 55-60 degrees C. According to data on the study of functional groups of the active center and substrate specificity, the enzyme isolated from the culture medium of Trichoderma harzianum is a subtilisin-like serine proteinase.
