Navegando por Palavras-chave "Lignin Peroxidase"

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    Potencial ligninolítico de fungos de três ambientes brasileiros: Manguezal, Cerrado e Caatinga
    (Universidade Federal de São Paulo (UNIFESP), 2021) Martinho, Vivian [UNIFESP]; Vasconcellos, Suzan Pantaroto de [UNIFESP]; Universidade Federal de São Paulo
    Filamentous fungi have abilities to produce a wide range of bioactive compounds, including enzymes with multiple biotechnological applications. In this context, ligninases stand out, which could be explored richly in terms of application in industries that use plant biomass as a raw material, such as biofuels production, reducing the greenhouse gas emission and climate changes. This study was performed in bioprospecting by fungal isolates from mangrove, cerrado and caatinga collections with potential ligninolytic activity, thinking about the search for an optimized and sustainable production of biofuels, using viable technologies and reuse of vegetable biomass. Thus, the filamentous fungi were quantitatively related to ligninase activities, with an emphasis on Lignin Peroxidases (LiP), Manganese Peroxidases (MnP) and Lacases (Lac), making use spectrophotometric techniques. Statistical analyzes were used to compare the average activities in relation to each studied environment. LiP activities were highlighted in relation to the other enzymes, and the results obtained were selected to the performed of the Design of Experiments (DOE) on three different levels of the factors: sodium chloride concentration (NaCl), pH and temperature. Therefore, the isolates 63.1 – Fusarium sp. (mangrove), LRA 9 (cerrado), and FE 120 (caatinga), were selected for the analysis of the optimization of the reaction variables LiP activities employing the DOE, using the software R. Analysis of Variance (ANOVA) was applied to identify the effects of factors on LiP activities. The correlation of the predicted and observed data, as well as the analysis of residues, were performed to check the adequacy of the models. Moreover, response surfaces and contour profiles (2D) were generated. The largest activities of LiP and Lac were from the isolate 63.1 – Fusarium sp., with 101.9 and 41.5 U/L, respectively. And the largest activity of MnP was 11.4 U/L, of the isolate LFO 3 – Phlebiopsis sp. Analyzing the results of Design of Experiments, it was observed that all models showed a good quality of fit and a good correlation between the data predicted by models and observed experimentally. The coefficient of determination (R2 ) found were: 0.9471 (63.1 – Fusarium sp.), 0.8792 (LRA 9) and 0.9674 (FE 120). In addition, it was possible to verify that, at the significance level of 0.05, all factors had a significant influence on the LiP activities, for the mangrove and caatinga isolates, the NaCl concentration did not show any xii significant influence for the cerrado isolate. It is concluded that the present study brought relevant data regarding the selection of filamentous fungi, from different environments, with ligninolytic enzymes abilities comparable to the reference literature, whose results could be optimized by statistical analyses that will significantly contribute to the possible future application of such strains in the optimization of the vegetable biomass processing chain to increase the production of biofuels.
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    Seleção de fungos filamentosos como eficientes fontes de lignina-peroxidases
    (Universidade Federal de São Paulo (UNIFESP), 2020-08-21) Lima, Lidiane Maria dos Santos [UNIFESP]; Vasconcellos, Suzan Pantaroto de [UNIFESP]; Universidade Federal de São Paulo
    Selection of Filamentous Fungi as Efficient Sources of Lignin-Peroxidases Lignin peroxidases (LiP) are ligninolytic enzymes normally produced by biodegradable fungi of lignocellulose in nature. Currently, interest in such enzymes has increased, due to their high potential for biotechnological application. In this sense, the search for microbial cultures, of different habitats, with different environmental conditions, exponentially increases the acquisition of notable enzymes for the most diverse purposes. An example is the application of biorefineries of cellulosic ethanol, which aims at the efficient transformation of lignocellulosic biomass into bioproducts, such as biofuels and energy, thus being considered a sustainable alternative to petroleum derivatives. However, one of the obstacles to the lignocellulose valorization on an industrial scale is the complexity and recalcitrance of biomass, especially lignin. It is in this context that the development and optimization of enzymatic preparations based on lignin-peroxidases are required, in order to enable the cleavage and digestibility of lignocellulosic fiber. Thus, the present study was initiated by the screening of environmental filamentous fungi with respect to the effectiveness of lignin-peroxidases, using dye degradation tests of aromatic molecular structures, such as remazol brilliant blue R (RBBR) and methylene blue (AM), as compounds for detecting fungal ligninolytic activity. Thus, six (6) filamentous fungi FPZSP3_01, FPZSP3_05, FPZSP3_47, FPZSP3_91, FPZSP1_129 and FPZSP1_141 isolated from soil increased by filter cake from sugar and alcohol refineries, were selected. Thus, such microorganisms were subjected to in-depth analysis about the enzymatic kinetics of their lignin-peroxidases, analyzing the crude enzymatic extract (microbial supernatant) from the following strains: FPZSP3_47 (Mucor sp.), FPZSP1_129 (Byssochlamys nivea) and FPZSP1 (Paecilomyces saturatus). The apparent kinetic constants KM, Vmax and kcat were determined in two conditions, pH 3.0 at room temperature (20 ºC±2), and pH 9.0, NaCl 4M, at 65 ºC, in addition to the influence of different temperatures, pH ranges and salinities in the activities enzyme detected. The filamentous fungus Mucor sp. (FPZSP3_47) belonging to the phylum Mucoromycota was selected for protein characterization studies. The apparent kinetic constants KM (mM) 55.65 ± 6.56, Vmax (mmol min-1) 414.75 ± 6.37 and kcat (min-1) 7.45 were higher at pH 3.0 at room temperature for Mucor sp. FPZSP3_47. The lignin-peroxidases of Mucor sp., Byssochlamys nivea and Paecilomyces saturatus exhibited activity and stability for 30 hours at all temperatures evaluated (4 ºC, ambient, 30 ºC and 65 ºC), with two optimal temperatures (30 ºC and 65 ºC) being selected. ºC). Such conditions were selected to analyze the activity dependence and enzymatic stability at pH. All ligninases of Mucor sp., Byssochlamys nivea and Paecilomyces saturatus were active and stable over a wide pH range (5.0 to 9.0). The pH 9.0 and the temperature of 65 ºC were selected to investigate the effect of NaCl (0.1 to 4 M) on the stability of LiP, and the results revealed wide tolerance to NaCl, for 24 hours. The LiP of the isolate Mucor sp. was purified by ion-exchange chromatography and analyzed on SDS-PAGE 12%, featuring a protein with a molecular mass estimated at 60 kDa and specific activity of 0.013 U/mg. It is worth mentioning that this is the first description of ligninases of a fungus of the genus Mucor sp. in the literature, therefore, a potential candidate for the optimization of processes associated with the production of ethanol from vegetable biomass.