Secreted protein extract analyses present the plant pathogen Alternaria alternata as a suitable industrial enzyme toolbox.

Lignocellulosic plant biomass is the most abundant carbon source in the planet, which makes it a potential substrate for biorefinery. It consists of polysaccharides and other molecules with applications in pharmaceutical, food and feed, cosmetics, paper and textile industries. The exploitation of these resources requires the hydrolysis of the plant cell wall, which is a complex process. Aiming to discover novel fungal natural isolates with lignocellulolytic capacities, a screening for feruloyl esterase activity was performed in samples taken from different metal surfaces. An extracellular enzyme extract from the most promising candidate, the natural isolate Alternaria alternata PDA1, was analyzed. The feruloyl esterase activity of the enzyme extract was characterized, determining the pH and temperature optima (pH 5.0 and 55-60 °C, respectively), thermal stability and kinetic parameters, among others. Proteomic analyses derived from two-dimensional gels allowed the identification and classification of 97 protein spots from the extracellular proteome. Most of the identified proteins belonged to the carbohydrates metabolism group, particularly plant cell wall degradation. Enzymatic activities of the identified proteins (ß-glucosidase, cellobiohydrolase, endoglucanase, ß-xylosidase and xylanase) of the extract were also measured. These findings confirm A. alternata PDA1 as a promising lignocellulolytic enzyme producer. SIGNIFICANCE: Although plant biomass is an abundant material that can be potentially utilized by several industries, the effective hydrolysis of the recalcitrant plant cell wall is not a straightforward process. As this hydrolysis occurs in nature relying almost solely on microbial enzymatic systems, it is reasonable to infer that further studies on lignocellulolytic enzymes will discover new sustainable industrial solutions. The results included in this paper provide a promising fungal candidate for biotechnological processes to obtain added value from plant byproducts and analogous substrates. Moreover, the proteomic analysis of the secretome of a natural isolate of Alternaria sp. grown in the presence of one of the most used vegetal substrates on the biofuels industry (sugar beet pulp) sheds light on the extracellular enzymatic machinery of this fungal plant pathogen, and can be potentially applied to developing new industrial enzymatic tools. This work is, to our knowledge, the first to analyze in depth the secreted enzyme extract of the plant pathogen Alternaria when grown on a lignocellulosic substrate, identifying its proteins by means of MALDI-TOF/TOF mass spectrometry and characterizing its feruloyl esterase, cellulase and xylanolytic activities.