TEMPORAL ANALYSIS OF SECRETED PROTEINS FROM TRICHODERMA HARZIANUM GROWN ON SUGARCANE BAGASSE AS CARBON SOURCE

Pedro Alves Martins (UnB); Ana Carolline Ribeiro de Toledo Pinto (UnB); Diana Paola Gómez Mendoza (UnB); Marcelo Valle de Sousa (UnB); Edivaldo Ximenes Ferreira Filho (UnB); Carlos André Ornelas Ricart (UnB)

Trichoderma harzianum is a mesophilic and asexual filamentous fungi found on several soils and habitats. T. harzianum possesses great metabolic versatility and therefore is able to produce several extracelular enzymes, such as cellulases and xylanases, which can be commercially aplied to industrial processes. The present work aimed at analyzing the secretome of T. harzianum during the initial hours of culture in liquid medium containing sugarcane bagasse as carbon source. T. harzianum T₄ spores were obtained from culture oon Petri dishes containing synthetic medium supplemented with 1% sugarcane bagasse. The culture was maintained at 28ºC for approximately ten days, then spores were collected using 0.9% NaCl solution and the resulting suspension (1x10⁸ ml^-1) was used to inoculate three flasks with 500mL of synthetic medium broth with sugarcane bagasse. The culture was maintained at 28ºC for 48h under agitation. Samples were collected at 0, 6, 12, 24, 30, 36 and 48h of growth and centrifuged at 14000rpm/10min. The resulting supernatants were subjected to enzymatic assays for cellulases (CMCases and FPases), hemicellulases (xylanases), mananases and pectinases. Protein patterns were analyzed using electrophoretic techniques such as SDS-PAGE and 2D-PAGE. As expected, the first hours of culture presented low enzymatic activity, which increased during time.  For most of the enzyme groups analyzed, activity began approximately at the end of 24h of culture.  For xylanases and pectinases, initial activity began at 12h of culture. At 48h of growth, the highest enzyme activities were detected: xylanases (3.92 UI/mL), CMCases (0.91 UI/mL), FPases (0.89 UI/mL), mananases (0.72 UI/mL), pectinases (1.50 UI/mL). SDS-PAGE analysis showed many different protein patterns along time. As some bands seemed to continuosly increase along time, others simply disappeared at some determined time. Interestingly, some bands seemed to reach a maximum and then decreased on expression. Samples are presently being submitted to 2D-PAGE for comparison of proteome maps. We expect that the identification by MALDI-TOF-TOF Mass Spectrometry (MS) of the proteins differentially expressed during culture time may lead to a better understanding of the processes involved in sugarcane bagasse degradation by T. harzianum and contribute to an optimization of enzymatic treatments of lignocellulosic biomass.