Secretômica e atividades enzimáticas da linhagem selvagem 2HH e do mutante S1M29 de Penicillium echinulatum

Abstract

The use of lignocellulolytic enzymes secreting by microorganisms for the production of second-generation ethanol has motivated research in the field of fermentation processes engineering, genomics and secretomics. Among the microorganisms with the potential for cellulases production are genetic variants of the filamentous fungus Penicillium echinulatum characterized by produce high enzymatic titers. In this work, it was studied the secretome of the wild type 2HH and mutant S1M29 of P. echinulatum in submerged cultivation on different carbon sources: glucose, glycerol, cellulose and sugar cane bagasse pretreated by steam explosion). Enzymatic analysis allowed verifying that P. echinulatum produces cellulases, hemicellulases, esterases, and minor proportion, pectinases and amylases. Furthermore, the major enzymes titers for most enzymes dosed were verified in the mutant strain. It was verified in the media formulated with sugar cane bagasse or cellulose the induction of the highest enzyme production for both strains. The analysis of secretome by 1DPAGE followed by LC-MS/MS, of samples collected at 96 hours of cultivation, showed that in both strains there is a predominance of CAZy enzymes, being cellulases, hemicellulases and fungal cell wall degrading enzymes the most prevalent. Cellobiohydrolases, endoglucanases, β-glucosidase, xylanase, endoxylanase, β-mannanases and xylosidases were identified and, in smaller amounts, ligninases, pectinases, amylases, esterases and swollenin, among other proteins (adhesion, chaperones, oxidoreductases, proteases, peptidases, lipases, glutaminases and hypothetical). The media elaborated with glucose or glycerol were used for producing of amylases, ligninases and fungal cell wall degrading enzymes. Highlights the secretion of 2-3 times more cellulases by the mutant, being the medium prepared with sugar cane bagasse afforded the secretion of large cellulases quantities for the mutant. In this condition, the cellulolytic complex of S1M29 strain consists of 55% cellobiohydrolases, 38% endoglucanases and 1% β-glucosidase. These data suggest that during the genetic improvement of the fungus changes occurred, although not directional, possibly at the level of regulation of gene expression, post-translational modifications and changes in the ability to secrete extracellular proteins, that have made the mutant S1M29 a potential strain to be employed in hydrolysis of lignocellulose.