RESUMO
Laccases (EC 1.10.3.2) are oxidoreductases that belong to the multicopper oxidase subfamily and are classified as yellow/white or blue according to their absorption spectrum. Yellow laccases are more useful for industrial processes since they oxidize nonphenolic compounds in the absence of a redox mediator and stand out for being more stable and functional under extreme conditions. This study aimed to characterize a new laccase that was predicted to be present in the genome of Chitinophaga sp. CB10 - Lac_CB10. Lac_CB10, with a molecular mass of 100.06 kDa, was purified and characterized via biochemical assays using guaiacol as a substrate. The enzyme demonstrated extremophilic characteristics, exhibiting relative activity under alkaline conditions (CAPS buffer pH 10.5) and thermophilic conditions (80-90°C), as well as maintaining its activity above 50% for 5 h at 80°C and 90°C. Furthermore, Lac_CB10 presented a spectral profile typical of yellow laccases, exhibiting only one absorbance peak at 300 nm (at the T2/T3 site) and no peak at 600 nm (at the T1 site). When lignin was degraded using copper as an inducer, 52.27% of the material was degraded within 32 h. These results highlight the potential of this enzyme, which is a novel yellow laccase with thermophilic and alkaline activity and the ability to act on lignin. This enzyme could be a valuable addition to the biorefinery process. In addition, this approach has high potential for industrial application and in the bioremediation of contaminated environments since these processes often occur at extreme temperatures and pH values. IMPORTANCE: The characterization of the novel yellow laccase, Lac_CB10, derived from Chitinophaga sp. CB10, represents a significant advancement with broad implications. This enzyme displays exceptional stability and functionality under extreme conditions, operating effectively under both alkaline (pH 10.5) and thermophilic (80-90°C) environments. Its capability to maintain considerable activity over extended periods, even at high temperatures, showcases its potential for various industrial applications. Moreover, its distinctive ability to efficiently degrade lignin-demonstrated by a significant 52.27% degradation within 32 h-signifies a promising avenue for biorefinery processes. This newfound laccase's characteristics position it as a crucial asset in the realm of bioremediation, particularly in scenarios involving contamination at extreme pH and temperature levels. The study's findings highlight the enzyme's capacity to address challenges in industrial processes and environmental cleanup, signifying its vital role in advancing biotechnological solutions.
Assuntos
Estabilidade Enzimática , Lacase , Lignina , Lacase/metabolismo , Lacase/genética , Lacase/isolamento & purificação , Lacase/química , Lignina/metabolismo , Concentração de Íons de Hidrogênio , Bacteroidetes/enzimologia , Bacteroidetes/genética , Especificidade por Substrato , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Temperatura , Biodegradação Ambiental , Guaiacol/metabolismo , Cobre/metabolismoRESUMO
Alicyclobacillus spp. is the cause of great concern for the food industry due to their spores' resistance (thermal and chemical) and the spoilage potential of some species. Despite this, not all Alicyclobacillus strains can spoil fruit juices. Thus, this study aimed to identify Alicyclobacillus spp. strains isolated from fruit-based products produced in Argentina, Brazil, and Italy by DNA sequencing. All Alicyclobacillus isolates were tested for guaiacol production by the peroxidase method. Positive strains for guaiacol production were individually inoculated at concentration of 103 CFU/mL in 10 mL of orange (pH 3.90) and apple (pH 3.50) juices adjusted to 11°Brix, following incubation at 45 °C for at least 5 days to induce the production of the following spoilage compounds: Guaiacol, 2,6-dichlorophenol (2,6-DCP) and 2,6-dibromophenol (2,6-DBP). The techniques of micro-solid phase extraction by headspace (HS-SPME) and gas-chromatography with mass spectrometry (GC-MS) were used to identify and quantify the spoilage compounds. All GC-MS data was analyzed by principal component analysis (PCA). The effects of different thermal shock conditions on the recovery of Alicyclobacillus spores inoculated in orange and apple juice (11°Brix) were also tested. A total of 484 strains were isolated from 48 brands, and the species A. acidocaldarius and A. acidoterrestris were the most found among all samples analyzed. In some samples from Argentina, the species A. vulcanalis and A. mali were also identified. The incidence of these two main species of Alicyclobacillus in this study was mainly in products from pear (n = 108; 22.3 %), peach (n = 99; 20.5 %), apple (n = 86; 17.8 %), and tomato (n = 63; 13 %). The results indicated that from the total isolates from Argentina (n = 414), Brazil (n = 54) and Italy (n = 16) were able to produce guaiacol: 107 (25.8 %), 33 (61.1 %) and 13 (81.2 %) isolates from each country, respectively. The PCA score plot indicated that the Argentina and Brazil isolates correlate with higher production of guaiacol and 2,6-DCP/2,6-DBP, respectively. Heatmaps of cell survival after heat shock demonstrated that strains with different levels of guaiacol production present different resistances according to spoilage ability. None of the Alicyclobacillus isolates survived heat shocks at 120 °C for 3 min. This work provides insights into the incidence, spoilage potential, and thermal shock resistance of Alicyclobacillus strains isolated from fruit-based products.
Assuntos
Alicyclobacillus , Sucos de Frutas e Vegetais , Frutas , Cromatografia Gasosa-Espectrometria de Massas , Guaiacol , Esporos Bacterianos , Alicyclobacillus/isolamento & purificação , Alicyclobacillus/genética , Alicyclobacillus/classificação , Alicyclobacillus/crescimento & desenvolvimento , Sucos de Frutas e Vegetais/microbiologia , Guaiacol/análogos & derivados , Guaiacol/metabolismo , Guaiacol/farmacologia , Frutas/microbiologia , Esporos Bacterianos/crescimento & desenvolvimento , Esporos Bacterianos/isolamento & purificação , Microbiologia de Alimentos , Contaminação de Alimentos/análise , Brasil , Microextração em Fase Sólida , Argentina , Malus/microbiologia , Itália , Temperatura Alta , Citrus sinensis/microbiologiaRESUMO
Nitroimidazole compounds are well-known bioactive substances, and the structural activity relationship has been reported whereby the position of the nitro group within the imidazole ring has a large influence on the activity. This study focuses on synthesising new trypanocidal agents from the hybridisation of metronidazole with different natural phenols (eugenol, dihydroeugenol and guaiacol). Two different coupling methodologies have been explored in order to analyse the influence of the connector on bioactivity: i) classic direct esterification (AD compounds) and ii) "click" chemistry using a triazole connector (AC compounds). The in vitro trypanocidal tests show good results for both AC and AD hybrid compounds against both epimastigote and trypomastigote forms of T. cruzi. In silico studies showed positive data for most of the synthesised compounds and, in general present low toxicological risks. The AC compounds present lower ClogP (lipophilicity) values than those found for the AD series and higher TPSA (topological polar surface area) values, suggesting lower lipophilicity may be related to the presence of the triazole connector. The AD series compounds have higher Drug Score values than the AC series derivatives, suggesting better general properties for a pharmacological action.
Assuntos
Doença de Chagas , Tripanossomicidas , Trypanosoma cruzi , Humanos , Doença de Chagas/tratamento farmacológico , Eugenol , Metronidazol/farmacologia , Metronidazol/uso terapêutico , Relação Estrutura-Atividade , Triazóis/uso terapêutico , Tripanossomicidas/química , Guaiacol/síntese química , Guaiacol/química , Guaiacol/farmacologiaRESUMO
Herbicides are agrochemicals applied in the control of weeds. With the frequent and repetitive use of these substances, serious problems have been reported. Compounds of natural origin and their derivatives are attractive options to obtain new compounds with herbicidal properties. By aiming to develop compounds with potentiated herbicidal activity, phenoxyacetic acids were synthesized from eugenol and guaiacol. The synthesized compounds were characterized and the herbicidal potential of phenoxyacetic acids and precursors was evaluated through bioassays regarding the germination and initial development of Lactuca sativa and Sorghum bicolor seedlings, with the induction of DNA damage. The induction of changes in the mitotic cycle of meristematic cells of roots of L. sativa was also analyzed. At the concentration of 3 mmol L-1, phenols and their respective phenoxyacetic acids presented phytotoxic and cytotoxic activities in L. sativa and S. bicolor. Eugenol and guaiacol also presented genotoxic action in L. sativa. The toxic effect of eugenoxyacetic acid was more pronounced in L. sativa than in S. bicolor, similar to the commercial 2,4-D herbicide. Molecular properties of the phenols and their derivatives phenoxyacetic acids were compared with the ones obtained for the herbicide 2,4-D, where it was found a correlation between their molecular properties and bioactivity.
Assuntos
Herbicidas , Ácido 2,4-Diclorofenoxiacético/toxicidade , Eugenol/toxicidade , Germinação , Guaiacol , Herbicidas/toxicidadeRESUMO
Nine yeast strains isolated from Latin American biodiversity were screened for ferulic acid (FA) consumption and conversion into aroma compounds such as vanillin, vanillic acid (VA), and 4-vinylguaiacol (VG). Selected strains (Rhodotorula mucilaginosa UFMG-CM-Y3647, UFMG-CM-Y2190, UFMG-CM-Y665) were evaluated in flask experiments to investigate the influence of the pH media on bioconversion and a two-step process was conducted to maximize the metabolites production. The effect of pH was found to be significantly important for FA bioconversion, as acidic conditions (pH < 6.0) improved VA accumulation, with highest production of 1.14 ± 0.02 and 1.25 ± 0.03 g/L shown by UFMG-CM-Y3647 and UFMG-CM-Y2190, respectively. The two-step process favored 4-VG production for most strains, being UFMG-CM-Y2190 the best producer, its cultures reaching 1.63 ± 0.09 g/L after 55 hr, showing a productivity of 29.59 ± 1.55 mg/(L·hr), as glucose affected the metabolites pool and redirected yeast metabolism. R mucilaginosa UFMG-CM-Y3647 was selected for scaled-up cultivations in a 2-L bioreactor, where pH-controlled pH 5.5 and aeration of 2.5 vvm was found to be the best condition to improve VA productivity, attaining final concentrations of 1.20 ± 0.02 g/L-1 (78% molar yield) and a productivity of 40.82 ± 0.57 mg/(L·hr).
Assuntos
Benzaldeídos/metabolismo , Ácidos Cumáricos/metabolismo , Guaiacol/análogos & derivados , Odorantes/análise , Saccharomyces cerevisiae/metabolismo , Biodiversidade , Biotecnologia , Biotransformação , Guaiacol/metabolismo , América Latina , Saccharomyces cerevisiae/classificaçãoRESUMO
Alicyclobacillus acidoterrestris is a cause of major concern for the orange juice industry due to its thermal and chemical resistance, as well as its spoilage potential. A. acidoterrestris spoilage of orange juice is due to off-flavor taints from guaiacol production and some halophenols. The present study aimed to evaluate the effectiveness of antimicrobial Photodynamic Treatment (aPDT) as an emerging technology to inactivate the spores of A. acidoterrestris. The aPDT efficiency towards A. acidoterrestris was evaluated using as photosensitizers the tetracationic porphyrin (Tetra-Py+-Me) and the phenothiazinium dye new methylene blue (NMB) in combination with white light-emitting diode (LED; 400-740 nm; 65-140 mW/cm2). The spores of A. acidoterrestris were cultured on YSG agar plates (pH 3.7 ± 0.1) at 45 °C for 28 days and submitted to the aPDT with Tetra-Py+-Me and NMB at 10 µM in phosphate-buffered saline (PBS) in combination with white light (140 mW/cm2). The use of Tetra-Py+-Me at 10 µM resulted in a 7.3 ± 0.04 log reduction of the viability of A. acidoterrestris spores. No reductions in the viability of this bacterium were observed with NMB at 10 µM. Then, the aPDT with Tetra-Py+-Me and NMB at 10 µM in orange juice (UHT; pH 3.9; 11°Brix) alone and combined with potassium iodide (KI) was evaluated. The presence of KI was able to potentiate the aPDT process in orange juice, promoting the inactivation of 5 log CFU/mL of A. acidoterrestris spores after 10 h of white light exposition (140 mW/cm2). However, in the absence of KI, both photosensitizers did not promote a significant reduction in the spore viability. The inactivation of A. acidoterrestris spores artificially inoculated in orange peels (105 spores/mL) was also assessed using Tetra-Py+-Me at 10 and 50 µM in the presence and absence of KI in combination with white light (65 mW/cm2). No significant reductions were observed (p < .05) when Tetra-Py+-Me was used at 10 µM, however at the highest concentration (50 µM) a significant spore reduction (≈ 2.8 log CFU/mL reductions) in orange peels was observed after 6 h of sunlight exposition (65 mW/cm2). Although the color, total phenolic content (TPC), and antioxidant capacity of orange juice and peel (only color evaluation) seem to have been affected by light exposition, the impact on the visual and nutritional characteristics of the products remains inconclusive so far. Besides that, the results found suggest that aPDT can be a potential method for the reduction of A. acidoterrestris spores on orange groves.
Assuntos
Alicyclobacillus/efeitos da radiação , Citrus sinensis/microbiologia , Sucos de Frutas e Vegetais/microbiologia , Luz , Fármacos Fotossensibilizantes/farmacologia , Antibacterianos/farmacologia , Guaiacol , Azul de Metileno/análogos & derivados , Azul de Metileno/farmacologia , Porfirinas/farmacologia , Esporos Bacterianos/efeitos da radiaçãoRESUMO
The MeOH extract from leaves of Saururus cernuus L. (Saururaceae) displayed in vitro activity against trypomastigote forms of T. cruzi (100% of parasite death at 200⯵g/mL), suggesting the presence of bioactive compounds. Thus, the bioactivity-guided fractionation was carried out, leading to the isolation of three related neolignan derivatives, identified as threo-austrobailignan-5 (1), threo-austrobailignan-6 (2), and threo-dihydroguaiaretic acid (3). Anti-T. cruzi activity of compounds 1-3 was performed against cell-derived trypomastigotes and intracellular amastigotes. Additionally, the mammalian cytotoxicity was investigated using NCTC cells. Compound 2 was the most effective against extracellular trypomastigotes with IC50 of 3.7⯵M, while compound 3 showed activity in both clinically relevant forms of the parasite, trypomastigotes and amastigotes, with IC50 values of 7.0 and 16.2⯵M, respectively. However, the structurally related compound 1 was inactive. Based on these results, compounds 2 and 3 were selected to evaluate the mechanism of cellular death. Compound 2 induced alteration in the plasma membrane permeability and consequently in the ROS levels after 120â¯min of incubation. By using flow cytometry and fluorescence microscopy, compound 3 showed alterations in the mitochondrial membrane potential (ΔΨm) of trypomastigotes. Considering the promising chemical and biological properties of neolignans 2 and 3, these compounds could be used as starting points to develop new lead compounds for Chagas disease.
Assuntos
Lignanas/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Saururaceae/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Brasil , Células Cultivadas , Guaiacol/análogos & derivados , Lignanas/isolamento & purificação , Macrófagos Peritoneais/parasitologia , Camundongos Endogâmicos BALB C , Estrutura Molecular , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Folhas de Planta/química , Espécies Reativas de Oxigênio/metabolismo , Tripanossomicidas/isolamento & purificaçãoRESUMO
Nejayote is an alkaline wastewater generated during the nixtamalization process. Nejayote contains high-value compounds such as ferulic acid (FA), which is widely employed as a substrate for the biotechnological production of flavors and aromas. In the present study, the isolation, identification, and characterization of a native strain of Bacillus megaterium were performed, and its capacity to produce 4-vinylguaiacol (4VG) from ferulic acid was evaluated by employing growing cell and resting cell systems. Growing cells of native B. megaterium biotransformed 6 mM crude FA in nejayote into 2.1 mM 4VG, reaching a productivity of 0.21 mM h-1 4VG, while nejayote enriched with FA at 10, 15, and 25 mM resulted in the formation of 2.4, 3.8, and 6.2 mM 4VG and productivities of 0.24, 0.38, and 0.51 mM h-1 4VG, respectively. In the resting cell system, from 6 and 25 mM pure FA, 3.5 mM 4VG was produced (0.18 mM h-1 4VG), while at 10 and 15 mM FA, 4.6 and 5.1 mM 4VG (average of 0.24 mM h-1 4VG) were obtained, respectively. The native B. megaterium strain, isolated from nejayote, showed great biotechnological potential to produce 4VG from crude FA contained in this wastewater, in which other Bacillus species, such as B. licheniformis and B. cereus, were unable to grow and biotransform FA into 4VG.
Assuntos
Bacillus megaterium/classificação , Bacillus megaterium/metabolismo , Ácidos Cumáricos/metabolismo , Águas Residuárias/microbiologia , Zea mays , Bacillus megaterium/genética , Bacillus megaterium/crescimento & desenvolvimento , Biomassa , Biotransformação , Ácidos Cumáricos/química , Guaiacol/análogos & derivados , Guaiacol/metabolismo , Filogenia , Águas Residuárias/químicaRESUMO
PURPOSE: Dry socket (DS) is one the most common and symptomatic post-extraction complications; however, no consensus on its treatment has been reached. This study aimed to develop a novel dressing material for DS containing the phenolic agent guaiacol and evaluate its biological properties. METHODS: An inclusion complex of guaiacol and ß-cyclodextrin (Gu/ßcd) was prepared by freeze-drying. Its antibacterial activity over six oral bacteria was analyzed using the microdilution method, and its cytotoxicity in osteoblasts was assessed with the MTT assay. The alveolar healing process induced by Gu/ßcd was evaluated histologically after the treatment of DS in rats. RESULTS: ßcd complexation potentiated Gu's antibacterial effect and reduced its cytotoxicity in osteoblasts. Bone trabeculae were formed in the alveolar apices of rats treated with Gu/ßcd by day 7. On day 14, woven bone occupied the apical and middle thirds of the sockets; on day 21, the entire alveolus was filled by newly formed bone, which was in a more advanced stage of repair than the positive control (Alvogyl™). CONCLUSION: The improvement in Gu's biological properties in vitro and the rapid alveolar repair in comparison with Alvogyl™ in vivo demonstrated the benefits of the Gu/ßcd complex as a future alternative for the treatment of DS.
Assuntos
Antibacterianos/uso terapêutico , Alvéolo Seco/tratamento farmacológico , Guaiacol/uso terapêutico , Osteoblastos/efeitos dos fármacos , Infecção da Ferida Cirúrgica/prevenção & controle , beta-Ciclodextrinas/uso terapêutico , Processo Alveolar/patologia , Animais , Antibacterianos/administração & dosagem , Bandagens , Sobrevivência Celular/efeitos dos fármacos , Alvéolo Seco/complicações , Alvéolo Seco/diagnóstico por imagem , Alvéolo Seco/patologia , Guaiacol/administração & dosagem , Espectroscopia de Ressonância Magnética , Masculino , Ratos , Ratos Wistar , beta-Ciclodextrinas/administração & dosagemRESUMO
Different oak species or origins have been studied to find an alternative to traditional ones and Quercus humboldtii from Colombia is a candidate. Some studies analyse the composition of this oak, but no work has been found to study its use for wine treatment. The aim was to evaluate volatile composition of toasted Q. humboldtii oak chips and the wines treated with these chips. To evaluate its oenological potential Q. alba and Q. petraea have been included in this trial. The wines were subjected to a sensory analysis. Results indicated that oak chips and wines macerated for 90â¯days with toasted Colombian chips exhibited higher 5-methylfurfural, guaiacol, trans-isoeugenol and syringol concentrations than others, while the furfural and cis-ß-methyl-γ-octalactone content was lower. Sensory analysis showed that the wines treated with Colombian oak presented more notes of smoked and less of coconut. Therefore, this oak could probably be considered suitable for ageing.