Aspartyl protease from Trichoderma harzianum CECT 2413: cloning and characterization

Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Apartado 1095, E-41080 Sevilla, Spain 1 Author for correspondence: Tahía Benítez. Tel: +34 95 4557109. Fax: +34 95 4557104. e-mail: tahia{at}us.es A gene that encodes an extracellular aspartyl protease from Trichoderma harzianum CECT 2413, papA , has been isolated and characterized. Based on several conserved regions of other fungal acid proteases, primers were designed to amplify a probe that was used to isolate the papA gene from a genomic library of T. harzianum. papA was an intronless ORF which encoded a polypeptide of 404 aa, including a prepropeptide at the N-terminal region formed by one putative signal peptide, a second peptide which could be cleaved to activate the enzyme and the active protease of calculated 36·7 kDa and pI 4·35. Northern experiments indicated that papA gene was pH regulated, repressed by ammonium, glucose and glycerol, and induced by organic nitrogen sources. The promoter possessed potential AreA, PacC and MYC sites for nitrogen, pH and mycoparasitism regulation respectively, but lacked potential CreA sites for carbon regulation. IEF and zymograms indicated that PAPA was a pepstatin-sensitive aspartyl protease of pI 4·5. Transformants from T. harzianum CECT 2413 cultivated in yeast extract-supplemented medium overexpressed papA and had a fourfold increase in protease activity compared to the wild-type, while transformants that overexpressed the ß-1,6-glucanase gene bgn16 . 2 and papA had an additional 30% increase in ß-1,6-glucanase activity compared to bgn16.2 single transformants. Overexpression of both genes in ammonium-supplemented medium did not result in higher levels of PAPA and/or BGN16.2 proteins. These results indicated that both PAPA and ß-1,6-glucanase undergo proteolysis in ammonium-supplemented medium but PAPA is not responsible for ß-1,6-glucanase degradation. Keywords: fungal proteases, nitrogen carbon and pH regulation, protein overproduction, proteolysis The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AJ276388 .