RESUMO
Papain-like cysteine proteases (PLCPs) in plants are essential to prevent phytopathogen invasion. In order to search for cysteine protease inhibitors and to investigate compounds that could be associated to pineapple Fusarium disease, a chemistry investigation was performed on Fusarium proliferatum isolated from Ananas comosus (pineapple) and cultivated in Czapek medium. From F. proliferatum extracts, nine secondary metabolites were isolated and characterized by nuclear magnetic resonance spectroscopy and mass spectrometry experiments: beauvericin (1), fusaric acid (2), N-ethyl-3-phenylacetamide (3), N-acetyltryptamine (4), cyclo(L-Val-L-Pro) cyclodipeptide (5), cyclo(L-Leu-L-Pro) cyclodipeptide (6), cyclo(L-Leu-L-Pro) diketopiperazine (7), 2,4-dihydroxypyrimidine (8), and 1H-indole-3-carbaldehyde (9). Compounds 1, 3, and 6 showed significant inhibition of papain, with IC50 values of 25.3 ± 1.9, 39.4 ± 2.5, and 7.4 ± 0.5 µM, respectively. Compound 1 also showed significant inhibition against human cathepsins V and B with IC50 of 46.0 ± 3.0 and 6.8 ± 0.7 µM, respectively. The inhibition of papain by mycotoxins (fusaric acid and beauvericin) may indicate a mechanism of Fusarium in the roles of infection process.
Assuntos
Ananas/enzimologia , Cisteína Proteases/química , Inibidores de Cisteína Proteinase/química , Fusarium/química , Micotoxinas/química , Proteínas de Plantas/química , Ananas/química , Ananas/microbiologia , Inibidores de Cisteína Proteinase/metabolismo , Fusarium/metabolismo , Cinética , Espectrometria de Massas , Micotoxinas/metabolismo , Metabolismo SecundárioRESUMO
Alicyclobacillus spp. are spore forming bacteria that are often related to the deterioration of acidic products such as beverages and citrus juices. After the process of industrial pasteurization, the spore produced by the bacteria can germinate and the microorganism can grow, causing sensory abnormalities in the product. Alternative biopreservatives, such as the antimicrobial compounds, are of considerable importance to the food industry. Papain and bromelain are proteolytic enzymes derived frompapaya and pineapple, respectively. These enzymes are widely used in medicine and in the pharmaceutical and food industries, but while some studies have described their antibacterial action, no studies of the Alicyclobacillus spp. exist. The aimof this studywas to analyze the antibacterial effect of papain and bromelain on Alicyclobacillus spp. through 1) determining minimum inhibitory and bactericidal concentration (MIC and MBC); 2) determining the death time curve of the micro-organism in the presence and absence of enzymes; and 3) investigating the enzymatic mechanism on the microorganism. The antibacterial activity of enzymes in combination with nisin was also evaluated. The results showed that for the Alicyclobacillus acidoterrestris strain, the MIC of papain was 0.98 µg/mL and the MBC was 3.91 µg/mL, while theMIC of bromelain was 62.5 µg/mL and the MBCwas 250 µg/mL. The concentration of 4 ×MIC for both the enzymes was sufficient to eliminate 4 logs of the micro-organism after 24 h of incubation. Through the use of enzyme inhibitors specific for cysteine proteases, it was found that the antibacterial activity of papain and bromelain is not related to its proteolytic activity, butmay be related to other activities, such as amidse and esterase. The synergistic activity of the enzymes revealed a fractional inhibitory concentration (FIC) level of 0.16. Combination with nisin revealed an FIC of 0.25 for papain and 0.19 for bromelain, indicating synergism between both compounds. The application of enzymes in reconstituted orange juice contaminated with A. acidoterrestris was found to be effective, as after 48 h of incubation, at three different temperatures, the initial microbial population was eliminated. This study showed that the enzymes papain and bromelain have an antibacterial effect on A. acidoterrestris.
Assuntos
Alicyclobacillus/efeitos dos fármacos , Antibacterianos/farmacologia , Bromelaínas/farmacologia , Nisina/farmacologia , Papaína/farmacologia , Amidoidrolases/metabolismo , Ananas/enzimologia , Bebidas/microbiologia , Carica/enzimologia , Citrus sinensis/microbiologia , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/química , Sinergismo Farmacológico , Esterases/metabolismo , Testes de Sensibilidade Microbiana , PasteurizaçãoRESUMO
Bromelain is a set of proteolytic enzymes found in pineapple (Ananas comosus) tissues such as stem, fruit and leaves. Because of its proteolytic activity, bromelain has potential applications in the cosmetic, pharmaceutical, and food industries. The present study focused on the recovery of bromelain from pineapple peel by liquid-liquid extraction in aqueous two-phase micellar systems (ATPMS), using Triton X-114 (TX-114) and McIlvaine buffer, in the absence and presence of electrolytes CaCl2 and KI; the cloud points of the generated extraction systems were studied by plotting binodal curves. Based on the cloud points, three temperatures were selected for extraction: 30, 33, and 36°C for systems in the absence of salts; 40, 43, and 46°C in the presence of KI; 24, 27, and 30°C in the presence of CaCl2 . Total protein and enzymatic activities were analyzed to monitor bromelain. Employing the ATPMS chosen for extraction (0.5 M KI with 3% TX-114, at pH 6.0, at 40°C), the bromelain extract stability was assessed after incorporation into three cosmetic bases: an anhydrous gel, a cream, and a cream-gel formulation. The cream-gel formulation presented as the most appropriate base to convey bromelain, and its optimal storage conditions were found to be 4.0 ± 0.5°C. The selected ATPMS enabled the extraction of a biomolecule with high added value from waste lined-up in a cosmetic formulation, allowing for exploration of further cosmetic potential.
Assuntos
Ananas/enzimologia , Bromelaínas/química , Bromelaínas/isolamento & purificação , Fracionamento Químico/métodos , Cosméticos/química , Micelas , BiotecnologiaRESUMO
Bromelain is a mixture of proteolytic enzymes present in all tissues of the pineapple (Ananas comosus Merr.), and it is known for its clinical therapeutic applications, food processing, and as a dietary supplement. The use of pineapple waste for bromelain extraction is interesting from both an environmental and a commercial point of view, because the protease has relevant clinical potential. We aimed to study the optimization of bromelain extraction from pineapple waste, using the aqueous two-phase system formed by polyethylene glycol (PEG) and poly(acrylic acid). In this work, bromelain partitioned preferentially to the top/PEG-rich phase and, in the best condition, achieved a yield of 335.27% with a purification factor of 25.78. The statistical analysis showed that all variables analyzed were significant to the process.
Assuntos
Resinas Acrílicas/química , Ananas/enzimologia , Bromelaínas/isolamento & purificação , Resíduos Industriais , Polietilenoglicóis/química , Bromelaínas/química , Eliminação de ResíduosRESUMO
This paper focuses on the feasibility of unconventional aqueous two-phase systems for bromelain purification from pineapple processing waste. The main difference in comparison with conventional systems is the integration of the liquid-liquid extraction technique with fractional precipitation, which can decrease the protein content with no loss of biological activity by removing of unwanted molecules. The analysis of the results was based on the response surface methodology and revealed that the use of the desirability optimisation methodology (DOM) was necessary to achieve higher purification factor values and greater bromelain recovery. The use of DOM yielded an 11.80-fold purification factor and 66.38 % biological activity recovery using poly(ethylene glycol) (PEG) with a molar mass of 4,000, 10.86 % PEG concentration (m/m) and 36.21 % saturation of ammonium sulphate.
Assuntos
Sulfato de Amônio/química , Ananas/enzimologia , Bromelaínas/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Polietilenoglicóis/química , Bromelaínas/química , Proteínas de Plantas/químicaRESUMO
The aim of this work was to evaluate the activity of bromelain in pineapple plants (Ananas comosus var. Comosus), Pérola cultivar, produced in vitro in different culture conditions. This enzyme, besides its pharmacological effects, is also employed in food industries, such as breweries and meat processing. In this work, the enzymatic activity was evaluated in the tissues of leaves and stems of plants grown in culture medium without plant growth regulator. The most significant levels of bromelain were observed in leaf tissue after 4 months of culture in vitro in medium with a filter paper bridge, followed by medium gelled by the agar. The results of this study, regarding the different structures of the pineapple (leaves and stems) in vitro showed that the activity of bromelain varied depending on the culture conditions, the time and structure of which was quantified, ensuring a viable strategy in the production of seedlings with high levels of bromelain in subsequent phases of micropropagation.
Assuntos
Ananas/enzimologia , Ananas/crescimento & desenvolvimento , Bromelaínas/metabolismo , Técnicas de Cultura/métodos , Ensaios Enzimáticos/métodos , Ananas/metabolismo , Bromelaínas/biossíntese , Bromelaínas/isolamento & purificação , Meios de Cultura/química , Folhas de Planta/enzimologia , Caules de Planta/enzimologia , ProteóliseRESUMO
Two phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) isoforms of 74 and 65 kDa were found to coexist in vivo in pineapple leaves, a constitutive Crassulacean Acid Metabolism plant. The 65 kDa form was not the result of proteolytic cleavage of the larger form since extraction methods reported to prevent PEPCK proteolysis in other plant tissues failed to yield a single immunoreactive PEPCK polypeptide in leaf extracts. In this work, the smaller form of 65 kDa was purified to homogeneity and physically and kinetically characterized and showed parameters compatible with a fully active enzyme. The specific activity was nearly twice higher for decarboxylation of oxaloacetate when compared to carboxylation of phosphoenolpyruvate. Kinetic parameters fell within the range of those estimated for other plant PEPCKs. Its activity was affected by several metabolites, as shown by inhibition by 3-phosphoglycerate, citrate, malate, fructose-1,6-bisphosphate, l-asparagine and activation of the decarboxylating activity by succinate. A break in the Arrhenius plot at about 30°C indicates that PEPCK structure is responsive to changes in temperature. The results indicate that pineapple leaves contain two PEPCK forms. The biochemical characterization of the smaller isoform performed in this work suggests that it could participate in both carbon and nitrogen metabolism in vivo by acting as a decarboxylase.
Assuntos
Ananas/enzimologia , Fosfoenolpiruvato Carboxilase/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Descarboxilação , Ácido Oxaloacético/metabolismo , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxilase/química , Fotossíntese/fisiologia , Folhas de Planta/química , Proteínas de Plantas/química , Isoformas de Proteínas , TemperaturaRESUMO
Pineapple internal browning (IB) is a chilling injury that produces enzymatic browning associated with flesh translucency. Pineapple biodiversity allowed the investigation of how polyphenol oxidase (PPO) and peroxidase (POD) activities with their different isoforms are involved in the IB mechanism. Fruits of four varieties that expressed IB symptoms differently, Smooth Cayenne (SCay) and the hybrids MD2, Flhoran 41 (Flh 41), and Flhoran 53 (Flh 53), were stressed by cold. The susceptible varieties showed classical brown spots but different patterns of IB, whereas MD2 and controls showed no IB. Enzymatic activities were measured on fruit protein extracts and PPO and POD isoforms separated on mini-gels (PhastSystem). Only PPO activity was significantly enhanced in the presence of IB. Up to six PPO isoforms were identified in the susceptible varieties. PPO was barely detectable in the nonsusceptible variety MD2 and in controls. The number of PPO isoforms and the total PPO activity after chilling are varietal characteristics.
Assuntos
Ananas/enzimologia , Catecol Oxidase/genética , Regulação Enzimológica da Expressão Gênica , Peroxidase/genética , Proteínas de Plantas/genética , Ananas/genética , Catecol Oxidase/metabolismo , Temperatura Baixa , Frutas/enzimologia , Frutas/genética , Peroxidase/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Nitrate reductase (NR, EC 1.6.6.1) activity in higher plants is regulated by a variety of environmental factors and oscillates with a characteristic diurnal rhythm. In this study, we have demonstrated that the diurnal cycle of NR expression and activity in pineapple (Ananas comosus, cv. Smooth Cayenne) can be strongly modified by changes in the day/night temperature regime. Plants grown under constant temperature (28 degrees C light/dark) showed a marked increase in the shoot NR activity (NRA) during the first half of the light period, whereas under thermoperiodic conditions (28 degrees C light/15 degrees C dark) significant elevations in the NRA were detected only in the root tissues at night. Under both conditions, increases in NR transcript levels occurred synchronically about 4 h prior to the corresponding elevation of the NRA. Diurnal analysis of endogenous cytokinins indicated that transitory increases in the levels of zeatin, zeatin riboside and isopentenyladenine riboside coincided with the accumulation of NR transcripts and preceded the rise of NRA in the shoot during the day and in the root at night, suggesting these hormones as mediators of the temperature-induced modifications of the NR cycle. Moreover, these cytokinins also induced NRA in pineapple when applied exogenously. Altogether, these results provide evidence that thermoperiodism can modify the diurnal cycle of NR expression and activity in pineapple both temporally and spatially, possibly by modulating the day/night changes in the cytokinin levels. A potential relationship between the day/night NR cycle and the photosynthetic pathway performed by the pineapple plants (C(3) or CAM) is also discussed.