Enzymes Secreted by Filamentous Fungi: Regulation of Secretion and Purification of an Extracellular Protease of Trichoderma harzianum

Y. E. Dunaevsky^1*, T. N. Gruban², G. A. Beliakova², and M. A. Belozersky¹

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (7-095) 939-3181; E-mail: dun@funbio.genebee.msu.su

²School of Biology, Lomonosov Moscow State University, Moscow, 119899 Russia

^* To whom correspondence should be addressed.

Received September 21, 1999; Revision received October 29, 1999

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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°C at pH 8.0. The proteinase is stable up to 45-50°C (depending on the substrate used). Calcium ions stabilized the enzyme at 55-60°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.

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KEY WORDS: protease, filamentous fungi, secretion

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