Cloning and heterologous expression of aspartic protease SA76 related to biocontrol in Trichoderma harzianum

Abstract

Trichoderma harzianum is a soil-borne filamentous fungus that exhibits biological control properties because it parasitizes a large variety of phytopathogenic fungi. The production of hydrolytic enzymes appears to be a key element in the parasitic process. Among the enzymes released by Trichoderma, the aspartic proteases play a major role. A gene (SA76) encoding an aspartic protease was cloned by 3′ rapid amplification of cDNA ends from T. harzianum T88. The coding region of the gene is 1593 bp long, encoding a polypeptide of 530 amino acids with a predicted molecular mass 55 kDa and a pI of 4.5. The catalytic aspartic residues characteristic of aspartic proteases are conserved with an active-site motif (DSG); however, the DSG in the N-terminal lobe is unusual in that Ser replaced Thr. Northern blot analysis indicated that SA76 was induced in response to different fungal cell walls. Aspartic protease SA76 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. The enzyme activity culminates (10.5 U mL-1) 72 h after induction with galactose. The temperature optimum of the enzyme was 45°C and its pH optimum was 3.5. The culture supernatant of the S. cerevisiae strain that expressed the aspartic protease SA76 was able to inhibit the growth of five phytopathogenic fungi. The inhibition of mycelial growth varied between 7% and 38%. © 2007 Harbin Institute of Technology.