RESUMO
Marine-derived fungi proved to be a rich source of biologically active compounds. The genus Penicillium has been extensively studied regarding their secondary metabolites and biological applications. However, the photoprotective effects of these metabolites remain underexplored. Herein, the photoprotective potential of Penicillium echinulatum, an Antarctic alga-associated fungus, was assessed by UV absorption, photostability study, and protection from UVA-induced ROS generation assay on human immortalized keratinocytes (HaCaT) and reconstructed human skin (RHS). The photosafety was evaluated by the photoreactivity (OECD TG 495) and phototoxicity assays, performed by 3T3 neutral red uptake (3T3 NRU PT, OECD TG 432) and by the RHS model. Through a bio-guided purification approach, four known alkaloids, (-)-cyclopenin (1), dehydrocyclopeptine (2), viridicatin (3), and viridicatol (4), were isolated. Compounds 3 and 4 presented absorption in UVB and UVA-II regions and were considered photostable after UVA irradiation. Despite compounds 3 and 4 showed phototoxic potential in 3T3 NRU PT, no phototoxicity was observed in the RHS model (reduction of cell viability < 30%), which indicates their very low acute photoirritation and high photosafety potential in humans. Viridicatin was considered weakly photoreactive, while viridicatol showed no photoreactivity; both compounds inhibited UVA-induced ROS generation in HaCaT cells, although viridicatol was not able to protect the RHS model against UVA-induced ROS production. Thus, the results highlighted the photoprotective and antioxidant potential of metabolites produced by P. echinulatum which can be considered a new class of molecules for photoprotection, since their photosafety and non-cytotoxicity were predicted using recommended in vitro methods for topical use.
Assuntos
Alcaloides/química , Penicillium/química , Pele/efeitos da radiação , Raios Ultravioleta , Células 3T3 , Alcaloides/toxicidade , Animais , Antioxidantes , Dermatite Fototóxica , Células HaCaT , Humanos , Camundongos , Vermelho Neutro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Protetores SolaresRESUMO
This study evaluated the potential use of elephant grass biomass, a highly productive species, for cellulase and xylanase production by the cellulolytic mutant Penicillium echinulatum 9A02S1 in submerged cultivation, using untreated biomass, biomass pretreated with different concentrations of NaOH, H2SO4 or NH4OH, or biomass pretreated with H2O at 121 °C. For filter paper activity, all cultivation carried out with pretreated elephant grass under the evaluated conditions showed superior activity when compared with the control (untreated elephant grass). The activities of endoglucanases and ß-glucosidases were higher in the cultivation prepared from pretreated samples than the control made with cellulose (Celuflok(®)). Without pretreatment, elephant grass can be used for xylanase production, enabling similar activities to those obtained in the cultivation with cellulose, reducing the enzyme production cost. These results indicate that the pretreatment of elephant grass, especially when pretreated with H2SO4, may be used as a partial or total replacement for cellulose to cellulase production, and untreated elephant grass may be used for xylanase production.
Assuntos
Celulase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Poaceae/metabolismo , Biomassa , Meios de Cultura , Especificidade por SubstratoRESUMO
The enzymatic hydrolysis of Pennisetum purpureum (elephant grass) was evaluated at high total solid levels (from 4% to 20% (w/v)) in a concomitant ball milling treatment in a rotating hydrolysis reactor (RHR). The greatest glucose yield was 20.17% when 4% (w/v) untreated biomass was employed. When sugars obtained from enzymatic hydrolysis were submitted to fermentation with Saccharomyces cerevisiae, the greatest ethanol yield was 22.61% when 4% (w/v) untreated biomass was employed; however, the highest glucose concentration (12.47g/L) was obtaining using 20% (w/v) solids and highest ethanol concentration (6.1g/L) was obtained using 16% (w/v) solids. When elephant grass was hydrolyzed in the rotating hydrolysis reactor, ethanol production was about double that was produced when the biomass was hydrolyzed in a static reactor (SR). These data indicate that it is possible to produce ethanol from elephant grass when milling treatment and enzymatic hydrolysis are performed at the same time.
Assuntos
Biocombustíveis , Biomassa , Etanol , Pennisetum , Etanol/análise , Etanol/metabolismo , Fermentação , Hidrólise , Pennisetum/química , Pennisetum/metabolismo , Saccharomyces cerevisiaeRESUMO
The enhancement of enzyme complex produced by Penicillium echinulatum grown in several culture media components (bagasse sugarcane pretreated by various methods, soybean meal, wheat bran, sucrose, and yeast extract) was studied to increment FPase, xylanase, pectinase, and ß-glucosidase enzyme activities. The present results indicated that culture media composed with 10 g/L of the various bagasse pretreatment methods did not have any substantial influence with respect to the FPase, xylanase, and ß-glucosidase attained maximum values of, respectively, 2.68 FPU/mL, 2.04, and 115.4 IU/mL. On the other hand, proposed culture media to enhance ß-glucosidase production composed of 10 g/L steam-exploded bagasse supplemented with soybean flour 5.0 g/L, yeast extract 1.0 g/L, and sucrose 10.0 g/L attained, respectively, 3.19 FPU/mL and 3.06 IU/mL while xylanase was maintained at the same level. The proteomes obtained from the optimized culture media for enhanced FPase, xylanase, pectinase, and ß-glucosidase production were analyzed using mass spectrometry and a panel of GH enzyme activities against 16 different substrates. Culture medium designed to enhance ß-glucosidase activity achieved higher enzymatic activities values (13 measured activities), compared to the culture media for FPase/pectinase (9 measured activities) and xylanase (7 measured activities), when tested against the 16 substrates. Mass spectrometry analyses of secretome showed a consistent result and the greatest number of spectral counts of Cazy family enzymes was found in designed ß-glucosidase culture medium, followed by FPase/pectinase and xylanase. Most of the Cazy identified protein was cellobiohydrolase (GH6 and GH7), endoglucanase (GH5), and endo-1,4-ß-xylanase (GH10). Enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse performed with ß-glucosidase enhanced cocktail achieved 51.4 % glucose yield with 10 % w/v insoluble solids at enzyme load of 15 FPU/g material. Collectively the results demonstrated that it was possible to rationally modulate the GH activity of the enzymatic complex secreted by P. echinulatum using adjustment of the culture medium composition. The proposed strategy may contribute to increase enzymatic hydrolysis of lignocellulosic materials.
Assuntos
Meios de Cultura/farmacologia , Glicosídeo Hidrolases/metabolismo , Complexos Multienzimáticos/metabolismo , Penicillium/efeitos dos fármacos , Penicillium/enzimologia , Celulase/metabolismo , Celulose , Meios de Cultura/química , Meios de Cultura/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Glicosídeo Hidrolases/biossíntese , Hidrólise/efeitos dos fármacos , Complexos Multienzimáticos/química , Penicillium/metabolismo , Poligalacturonase/metabolismo , Saccharum , beta-Glucosidase/biossíntese , beta-Glucosidase/metabolismoRESUMO
The development of more productive strains of microorganisms and processes that increase enzyme levels can contribute to the economically efficient production of second generation ethanol. To this end, cellulases and xylanases were produced with the S1M29 mutant strain of Penicillium echinulatum, using different concentrations of cellulose (20, 40, and 60 g L(-1)) in batch and fed-batch processes. The highest activities of FPase (8.3 U mL(-1)), endoglucanases (37.3 U mL(-1)), and xylanases (177 U mL(-1)) were obtained in fed-batch cultivation with 40 g L(-1) of cellulose. The P. echinulatum enzymatic broth and the commercial enzyme Cellic CTec2 were tested for hydrolysis of pretreated sugar cane bagasse. Maximum concentrations of glucose and xylose were achieved after 72 h of hydrolysis. Glucose yields of 28.0% and 27.0% were obtained using the P. echinulatum enzymatic extract and Cellic CTec2, respectively.