TRICHODERMA HARZIANUM 354.33 SPENT MEDIUM: CHARACTERIZATION AND APPLICATIONS

Abstract

Fungal genetic engineering is difficulted due to the presence of the fungal cell wall, which represents an impediment for exogenous genetic material to be introduced. Trichoderma harzianum is a saprophytic fungus which has the capability of secreting fungal cell wall degrading enzymes into the medium. These enzymes are of great interest in industry due to their potential uses in fungal genetic engineering and as biocontrol agents. In this study, enzyme mixes obtained from a Trichoderma harzianum culture were characterized in order to see if routine treatments affected their activity and what their composition is. Moreover, the enzyme mix was tested to see if protoplasts were generated when incubating them in an isosmotic medium and whether cell wall removal could prevent mycelium growth from spores. Results show that treatments such as dialysis or freezing can signify a slight decrease in the overall enzymatic activity and that lyophilization allows enzyme storage at room temperature for at least 6 months. The composition of the enzyme mix depends on the producing strain and on the media it is incubated on, but proteins are secreted into the medium and some of them are thought to be chitinases and glucanases. Furthermore, a novel quality control system of the enzyme cocktail activity was developed based on an agar frotis on an agar-cell wall suspension, which allows production of new batches to be standardized in terms of enzyme activity. Finally, the enzyme mix is unable inhibit the growth of mycelium from spores, meaning that it cannot be used as an antifungal for crops or other food substrates, yet protoplast production from different species yielded in better results. Multiple enzyme mixes from different producing organisms were tested on different model organisms, and the cocktail containing enzymes from Trichoderma harzianum and Paenibacillus pabuli equally show promising production yields in different organisms.