Aspartyl protease from Trichoderma harzianum CECT 2413: cloning and characterization

Area of Science:

• * Molecular Biology
• * Mycology
• * Biochemistry

Background:

• * Trichoderma harzianum is a fungus known for its biocontrol properties.
• * Fungal proteases play crucial roles in nutrient acquisition and pathogenicity.
• * Understanding gene regulation in T. harzianum is vital for optimizing its industrial applications.

Purpose of the Study:

• * To isolate and characterize the papA gene encoding an extracellular aspartyl protease from T. harzianum.
• * To investigate the transcriptional regulation of the papA gene.
• * To explore the functional role of the PAPA protease and its interaction with beta-1,6-glucanase.

Main Methods:

• * Gene isolation using PCR and genomic library screening.
• * Sequence analysis of the papA gene and its predicted protein product.
• * Northern blot analysis for gene expression studies.
• * Isoelectric focusing (IEF) and zymography for protease characterization.
• * Genetic transformation of T. harzianum for gene overexpression studies.

Main Results:

• * The papA gene was isolated, encoding an intronless 404-amino acid polypeptide with a prepropeptide.
• * PAPA was identified as a pepstatin-sensitive aspartyl protease (pI 4.5).
• * papA expression is pH-regulated, repressed by ammonium and carbon sources, and induced by organic nitrogen.
• * Overexpression of papA in T. harzianum increased protease activity fourfold.
• * Co-overexpression of papA and bgn16.2 enhanced beta-1,6-glucanase activity, but PAPA did not degrade beta-1,6-glucanase in ammonium-supplemented medium.

Conclusions:

• * The papA gene encodes a regulated extracellular aspartyl protease in T. harzianum.
• * PAPA protease activity is influenced by nutrient availability and pH.
• * PAPA does not appear to be responsible for the proteolysis of beta-1,6-glucanase observed in ammonium-supplemented medium.