Enzymes secreted by filamentous fungi: regulation of secretion and purification of extracellular protease of Trichoderma harzianum

摘要

Extracellular proteases secreted by the filamentous fungus Trichoderma harzianum have been identified. A proteinase active towards Z-ala-ala-leu-pNa, a substrate of subtilisin-like proteases, predominated in the culture medium. This proteinase was synthesized de novo in response to addition of a protein substrate to the medium. A change in the carbohydrate in the culture medium resulted in quantitative and qualitative changes in the spectrum of secreted enzymes. The most active, an extracellular proteinase of Trichoderma harzianum, was purified 322-fold from the culture medium and obtained with a yield of 7.2%. The molecular mass of the studied proteinase was 73 kDa and its pI was 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 of the enzyme is 40 deg C at pH 8.0. The proteinase is stable up to 45-50 deg C (depending on the used substrate). Ca~2+ stabilizes the enzyme at 55-60 deg C. According to data obtained in the study of functional groups of the active center and substrate specificity, we conclude that the enzyme isolated from the culture medium of Trichoderma harzianum is a subtilisin-like serine proteinase.