Fusão de protoplastos entre Penicillium echinulatum e Trichoderma harzianum para obtenção de variabilidade visando a produção de celulases

Abstract

The study of cellulolytic fungi has proved to be important, considering economic interest of the cellulase complex, especially in the textile industry and, more recently, for energy purposes. In this work, the protoplast fusion was used to combine genotypes of mutants partially non repressed for cellulases production of Penicillium echinulatum (9A02S1B9) and Trichoderma harzianum (AS5CH3) using the technique dead donor, intending to obtain recombinants with higher cellulases production. In this strategy, both strains had their mycelium treated with Glucanex  0,01 g/mL, to lyse the cell wall. The protoplast obtained from the benomyl-resistant (9A02S1B9) were heat-inactivated (technique of dead donor) at 60ºC, before the step of fusion, induced by PEG4000 and Ca2+, with protoplast of the sensitive-benomyl strain (AS5CH3). Twenty four sub-clones were selected from one fusion product, after stabilization and selection strategies for precocity and efficiency in the formation clearing zones of by cellulose hydrolysis in Petri plates. The fusion products showed similar morphology and sporulation to one of parents, thirteen similar to Penicillium, nine similar to Trichoderma and two showed altered forms. The fusion products which segregate to the morphology of T. harzianum resistance to benomyl, being able to grow and sporulate in media containing up to 100 μg/mL of this inhibitor. The morphology, the profile of bands, obtained by RAPD, and the pattern of cellulase secretion by fusion products were ever more similar to one of parents. The fusants presented variation in the halo of cellulose hydrolysis in Petri plates, and in the activity on filter paper (FPAases), - glicosidase or endoglicanase, when grown submerged cultivation or solid state. From this variability, significant improvement was verified for some of the parental strains. The application of the protoplast fusion methodology to obtain recombinant between P. echinulatum and T. harzianum, using the technique of dead donor, has proved to be adequate to generate variability in the production of cellulases.