Banca de QUALIFICAÇÃO: EDUARDO DA SILVA FRANÇA

Production, characterization, purification and application of laccase using coffee grounds by filamentous fungi.

Production, Lacase, Filamentous fungi, Agro-industrial residues

Laccases are versatile enzymes that can catalyze the oxidation of a wide range of phenolic and non-phenolic substances. The aim of this study was to investigate the biotechnological potential of filamentous fungi for the production of laccase, using coffee grounds as substrate, as well as the removal of textile dyes. Thus, the characterization of the coffee grounds by FTIR and SEM was carried out before and after the submerged and solid-state fermentation processes. A selection was made with 24 samples of filamentous fungi and two types of fermentation to evaluate the production of laccase in coffee grounds. In addition, a factorial design 2² with 4 central points was performed to evaluate the influence of the independent humidity and temperature variables on the laccase production response variable. Each independent variable was investigated at three levels: minimum (temperature 25 °C and humidity 40%), central (temperature 31 °C and humidity 50%) and maximum (temperature 37 °C and humidity 60%). In turn, enzymatic characterization was also performed in crude extract to evaluate the effect of temperature and pH on lacquer activity. As well as the determination of the total phenolic compounds and total protein contents of the coffee grounds extract before and after the fermentation process in solid state. Discoloration assays of the dyes Yellow Remazol, Black Remazol and Red Remazol were also carried out, following the variation of pH, discoloration and laccase activity in 14 days of submerged fermentation by Lentinus sp. UCP 1206. The results obtained in FTIR and SEM showed a better use of coffee grounds by Lentinus sp. UCP 1206 in solid-state fermentation with a maximum laccase yield of 2.254 U/mL. The factorial design showed that the higher the humidity, the higher the enzyme production, and the higher the temperature, the lower the enzyme production. The crude extract laccase showed the highest activity at 40 °C and pH 4.0 with residual enzymatic activity of 84.51% and remaining stable for 60 minutes at 40 °C, with residual enzymatic activity of 74%. The total phenolic compounds were 66.55 mg GAE/g for coffee grounds and 24.69 mg GAE/g for coffee grounds after solid fermentation. The total protein content was 4.74 μg/mL for coffee grounds and 9.48 μg/mL for solid brew coffee grounds. Regarding the discoloration process, it was observed that the highest percentage of discoloration occurred on the 14th day with 94.24%, 84.63% and 88.24% for Yellow Remazol, Black Remazol and Red Remazol, respectively. In turn, the enzyme production during decolorization was 1.593 U/mL, 1.495 U/mL and 1.477 U/mL in Remazol Yellow, Remazol Black and Remazol Red, respectively. The results obtained in this study show that coffee grounds proved to be a promising substrate for the production of laccase in solid state fermentation as an alternative to reduce the costs of the process. The fungus Lentinus sp. UCP 1206 has shown promise for the production and decolorization of textile dyes.