Blue native-PAGE analysis of Trichoderma harzianum secretome reveals cellulases and hemicellulases working as multienzymatic complexes.

ABSTRACT

Plant cell wall-degrading enzymes produced by microorganisms possess important biotechnological applications, including biofuel production. Some anaerobic bacteria are able to produce multienzymatic complexes called cellulosomes while filamentous fungi normally secrete individual hydrolytic enzymes that act synergistically for polysaccharide degradation. Here, we present evidence that the fungus Trichoderma harzianum, cultivated in medium containing the agricultural residue sugarcane bagasse, is able to secrete multienzymatic complexes. The T. harzianum secretome was firstly analyzed by 1D-BN (blue native)-PAGE that revealed several putative complexes. The three most intense 1D-BN-PAGE bands, named complexes [I], [II], and [III], were subsequently subjected to tricine SDS-PAGE that demonstrated that they were composed of smaller subunits. Zymographic assays were performed using 1D-BN-PAGE and 2D-BN/BN-PAGE demonstrating that the complexes bore cellulolytic and xylanolytic activities. The complexes [I], [II], and [III] were then trypsin digested and analyzed separately by LC-MS/MS that revealed their protein composition. Since T. harzianum has an unsequenced genome, a homology-driven proteomics approach provided a higher number of identified proteins than a conventional peptide-spectrum matching strategy. The results indicate that the complexes are formed by cellulolytic and hemicellulolytic enzymes and other proteins such as chitinase, cutinase, and swollenin, which may act synergistically to degrade plant cell wall components.