| Congresso Brasileiro de Microbiologia 2023 | Resumo: 364-1 | ||||
Resumo:The growing concern with the preservation of aquatic ecosystems and the imminent risk of contamination of potable water by pharmaceutical residues have driven intense research into the development of efficient and sustainable processes for the degradation of these contaminants. This is due to the recognition that conventional methods are inadequate to deal effectively with this problem. Thus, bioremediation emerged as an approach to reduce the effects arising from the presence of these compounds on environment. Among the organisms used, fungi and their enzymes have played a fundamental role in the degradation of substances such as lignocellulose, as well as in the mineralization and/or transformation of recalcitrant compounds (including pharmaceuticals). Therefore, this work’s objective was to identify the enzymatic potential of five filamentous fungi from Arroio Dourado stream sediments (FAR3, FAR5, FAR12, FAR14, FAR18) for laccase and peroxidases production. To assess the enzymatic activity of Laccase ABTS 1.8 mM (Sigma Aldrich), Manganese Peroxidase (MnP) and Lignin Peroxidase (LiP), solid state fermentation was utilized for fungal growth, using rice straw as substrate and incubated in BOD at 28 ºC for 4 periods, 24 hours (t1), 7 days (t2), 15 days (t3), 30 days (t4). Enzymatic extract was obtained washing the fermented with phosphate buffer (pH 7,4) and centrifuged at 4000 rpm to 4 cycles of 10 minutes. The extract was frozen and lyophilized. A 2 g.L-1 solution of the extract was prepared to evaluate the enzymes. The assay was performed on 96-well microplate. Laccase reading was performed at 420; MnP at 280 nm and LiP, 320 nm. Reading was performed on Flashscan530 microreader for 6 minutes at 22 ºC. To calculate U (μmol.min-1.L-1), the following equation was applied: U.L-1 = (∆A x Vt x 106) / (ɛ x Vs x d x t), where ɛ was molar absorptivity coefficient (mol. L-1); Vs (mL), sample amount; Vt (mL), total plate volume; t (min.) time performed and 106, the correction factor. In this case, the Vs used to ABTS was 40µL, while others were 20µL. Laccase results showed low activity compared to literature. The best result was put by FAR18 in t3 = 13.54 U.L-1. However, the results to LiP were more promising, due to FAR14 t3 = 179.98 U. L-1, FAR18 t4 = 135.84 U.L-1 and FAR3 t4 = 112.24 U.L-1. The MnP highlights were FAR14 t2 = 40.26 U.L-1, FAR18 t2 = 52.26 U.L-1 and FAR3 t1 = 52.84 U.L-1. Results revealed that the best incubation time is variable to the enzyme type and fungus metabolism. Overall, FAR18 was the most promising to continue the study due to its activity in all 3 enzymes on the trial (and high values to LiP and MnP). The enzymatic profile, with a predominance of the production of an enzyme, corroborates findings in the literature, and the propensity for one or another enzyme is influenced by the substrate. In this case, to better evaluate the FAR18 profile, substrate variation is indicated. About low production of Laccase, temperatures could have been a component, since this enzyme seems to be more active at temperatures above 40 ºC with copper as an inducer, compared to studies that did not use it. The results are promising and these factors should be evaluated in future stages. Therefore, it can be inferred that the analyzed fungi have the potential to be applied in bioremediation, especially FAR18, while optimized enzyme production and its identification are in progress. Palavras-chave: Bioremediation, extracellular enzymes, oxidases, enzymatic kinetics | |||||