Search results for: Pth enzymes

Authors: Uc-Cayetano E. G., Ake-Uh O. E., Villanueva-Mena I. E., Ordonez L. C.

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Carbon nanotubes (CNTs) and other graphitic nanostructures are materials with extraordinary physical, physicochemical and electrochemical properties which are being aggressively investigated for a variety of sensing applications. Thus, sensing of biological molecules such as proteins, DNA, glucose and other enzymes using either single wall or multiwall carbon nanotubes (MWCNTs) has been widely reported. Despite the current progress in this area, the electrochemical response of CNTs used in a variety of sensing arrangements still needs to be improved. An alternative towards the enhancement of this CNTs' electrochemical response is to chemically (or physically) modify its surface. The influence of the decoration with iron oxide nanoparticles in different types of MWCNTs on the amperometric sensing of glucose, urea, and cholesterol in solution is investigated. Commercial MWCNTs were oxidized in acid media and subsequently decorated with iron oxide nanoparticles; finally, the enzymes glucose oxidase, urease, and cholesterol oxidase are chemically immobilized to oxidized and decorated MWCNTs for glucose, urease, and cholesterol electrochemical sensing. The results of the electrochemical characterizations consistently show that the presence of iron oxide nanoparticles decorating the surface of MWCNTs enhance the amperometric response and the sensitivity to increments in glucose, urease, and cholesterol concentration when compared to non-decorated MWCNTs.

Keywords: WCNTs, enzymes, oxidation, decoration

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Authors: Shilpa Mukundaraj, Nagaraju Shivaiah

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Amyloidogenic conditions, driven by protein aggregation into insoluble fibrils, which pose significant challenges in the clinical condition of diabetes management, particularly through the amyloidogenic LVEALYL sequence in insulin B-chain. This study explores a dual therapeutic strategy involving cysteine protease enzymes such as papain and ficin and inhibitory peptides to target insulin amyloidosis. Combining in silico, in vitro, and in vivo methodologies, the research aims to inhibit amyloid formation and degrade preformed fibrils. Inhibitory peptides were designed using structure-guided approaches in Rosetta to specifically target the LVEALYL sequence. Concurrently, cysteine protease enzymes, including papain and ficin, were evaluated for their fibril disassembly potential. In vitro experiments, utilizing SDS- PAGE and spectroscopic techniques, confirmed dose-dependent degradation 50 to 300ug in vitro and 60mg/kg in vivo of amyloid aggregates by these enzymes, with significant disaggregation observed at higher concentrations 20mg. Peptide inhibitors effectively reduced fibril formation, as evidenced by reduced Thioflavin T fluorescence and circular dichroism spectroscopy. Complementary in silico analyses, including molecular docking and dynamic simulations, provided structural insights into enzyme binding interactions with amyloidogenic regions. Key residues involved in substrate recognition and cleavage were identified, with computational findings aligning strongly with experimental data. These insights confirmed the specificity of papain and ficin in targeting insulin fibrils. For translational potential, an in vivo rat model was developed, involving subcutaneous insulin amyloid injections to induce localized amyloid deposits. Over six days of enzyme treatment, a marked reduction in amyloid burden was observed through histological findings and biochemical assay superoxide dismutase can provide insights into oxidative damage due to amyloid deposition. Furthermore, inflammatory markers IL-6, TNFα were significantly attenuated in treated groups, emphasizing the dual role of enzymes in amyloid clearance and inflammation modulation. This integrative study highlights the promise of cysteine protease enzymes and inhibitory peptides as complementary therapeutic strategies for managing insulin amyloidosis. By targeting both the formation and persistence of amyloid fibrils, this dual approach offers a novel and effective avenue for amyloidosis treatment.

Keywords: insulin amyloidosis, peptide inhibitors, cysteine protease enzymes, amyloid degradation

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Authors: Saroj Poudel, Joshua Mancini, Douglas Pike, Jennifer Timm, Alexei Tyryshkin, Vikas Nanda, Paul Falkowski

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On the early Earth, protein-metal complexes likely harvested energy from a reduced environment. These complexes would have been precursors to the metabolic enzymes of ancient organisms. Hydrogenase is an essential enzyme in most anaerobic organisms for the reduction and oxidation of hydrogen in the environment and is likely one of the earliest evolved enzymes. To attempt to reinvent a precursor to modern hydrogenase, we computationally designed a short thirteen amino acid peptide that binds the often-required catalytic transition metal Nickel in hydrogenase. This simple complex can achieve hundreds of hydrogen evolution cycles using light energy in a broad range of temperature and pH. Biophysical and structural investigations strongly indicate the peptide forms a di-nickel active site analogous to Acetyl-CoA synthase, an ancient protein central to carbon reduction in the Wood-Ljungdahl pathway and capable of hydrogen evolution. This work demonstrates that prior to the complex evolution of multidomain enzymes, early peptide-metal complexes could have catalyzed energy transfer from the environment on the early Earth and enabled the evolution of modern metabolism

Keywords: hydrogenase, prebiotic enzyme, metalloenzyme, computational design

Procedia PDF Downloads 218

Authors: Mostafa A. Amer, I. A. El-Samra, I. I. Abou-ElSeoud, S. M. El-Abd, N. K. Shawertamimi

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Tomato Fusarium wilt disease caused by Fusarium oxysporum f. sp. Lycopercisi (FOL) is considered one of the most destructive diseases in Egypt. Effect of some biotic inducers such as Bacillus megaterium var. phosphaticum, Glomus intraradices and Glomus macrocarpum at seven different mixed treatments, was tested for their ability to induce resistance in tomato plants against the disease. According to pathogenicity tests, all the tested isolates of FOL showed wilt symptoms on both of the tested cultivars; however, they considerably varied in percentages of disease incidence (DI) and disease severity (DS). Castle Rock was more susceptible than Peto 86, which was relatively resistant. Pretreatment of both cultivars, under greenhouse conditions, with the tested biotic inducers alone or in combination with each other's, significantly increased the induction of chitinase, β-1,3-glucanase, peroxidase, and polyphenoloxidase and reduced disease incidence and severity, compared with untreated noninoculated (C1) and untreated inoculated (C2) controls. Application of a combination of BMP, with GI and GM was the most effective in increasing the induction rated of the tested enzymes, compared with the other treatments. Induction of enzymes in most of the tested bioinducers treatments gradually increased, attaining maximum values after 48 or/and 72 hrs after challenging with FOL, then gradually declined. GI was the least effective bioinducer.

Keywords: F. oxysporum f. sp. lycopersici, defense enzymes, induced systemic resistance, ISR, B. megaterium var. phosphaticum, G. macrocarpum, G. intraradices

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Authors: Wayde Veldman, Ozlem T. Bishop, Igor Polikarpov

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In bacteria, mono and disaccharides are phosphorylated during uptake into the cell via the widely used phosphoenolpyruvate (PEP)-dependent phosphotransferase transport system. As an initial step in the phosphorylated disaccharide metabolism pathway, certain glycoside hydrolase family 1 (GH1) enzymes play a crucial role in releasing phosphorylated and non-phosphorylated monosaccharides. However, structural determinants for the specificity of these enzymes still need to be clarified. GH1 enzymes are known to have a wide array of functions. According to the CAZy database, there are twenty-one different enzymatic activities in the GH1 family. Here, the structure and substrate specificity of a GH1 enzyme from Bacillus licheniformis, hereafter known as BlBglH, was investigated. The sequence of the enzyme BlBglH was compared to the sequences of other characterized GH1 enzymes using sequence alignment, sequence identity calculations, phylogenetic analysis, and motif discovery. Through these various analyses, BlBglH was found to have sequence features characteristic of the 6-phospho-β-glucosidase activity enzymes. Additionally, motif and structure comparisons of the three most commonly studied GH1 enzyme-activities revealed a shared loop amongst the different structures that consist of different sequence motifs – this loop is thought to guide specific substrates (depending on activity) towards the active-site. To further affirm BlBglH enzyme activity, molecular docking and molecular dynamics simulations were performed. Docking was carried out using 6-phospho-β-glucosidase enzyme-activity positive (p-Nitrophenyl-beta-D-glucoside-6-phosphate) and negative (p-Nitrophenyl-beta-D-galactoside-6-phosphate) control ligands, followed by 400 ns molecular dynamics simulations. The positive-control ligand maintained favourable interactions within the active site until the end of the simulation. The negative-control ligand was observed exiting the enzyme at 287 ns. Binding free energy calculations showed that the positive-control complex had a substantially more favourable binding energy compared to the negative-control complex. Jointly, the findings of this study suggest that the BlBglH enzyme possesses 6-phospho-β-glucosidase enzymatic activity.

Keywords: 6-P-β-glucosidase, glycoside hydrolase 1, molecular dynamics, sequence analysis, substrate specificity

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Authors: Ghada Hasabo, Mahmoud Saber Elbasiouny, Mervat Abdelsalam, Sherin Ghaleb, Niveen Eldessouky

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In the near future, nanotechnology is envisaged for large scale use. Hence health and safety issues of nanoparticles should be promptly addressed. In the present study the acute hepatoxicity assessment due to high single oral dose of nano iron and micro iron particles were studied. The normal daily activities, biochemical alterations, blood coagulation, histopathological changes in Wister rats were the aspect of the toxicological assessment.This work found that significant alterations in biochemical enzymes (serum iron level, liver enzymes, albumin, and bilirubin levels), blood coagulation (PT, PC, INR), and histopathological changes occurred more prominently in the nano iron particle treated group.

Keywords: nanobiotechnology, nanosystems, nanomaterials, nanotechnology

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Authors: Pinar Ercan, Sedef N. El

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The goals of this study were to determine the total anthocyanin and procyanidin contents and their in vitro bioaccessibilities of apple, red grape and cinnamon by a static in vitro digestion method reported by the COST FA1005 Action INFOGEST, as well as in vitro inhibitory effects of these food samples on starch and lipid digestive enzymes. While the highest total anthocyanin content was found in red grape (164.76 ± 2.51 mg/100 g), the highest procyanidin content was found in cinnamon (6432.54±177.31 mg/100 g) among the selected food samples (p<0.05). The anthocyanin bioaccessibilities were found as 10.23±1 %, 8.23±0.64 %, and 8.73±0.70 % in apple, red grape, and cinnamon, respectively. The procyanidin bioaccessibilities of apple, red grape, and cinnamon were found as 17.57±0.71 %, 14.08±0.74 % and 18.75±1.49 %, respectively. The analyzed apple, red grape and cinnamon showed the inhibitory activity against α-glucosidase (IC50 544.27±21.94, 445.63±15.67, 1592±17.58 μg/mL, respectively), α-amylase (IC50 38.41±7.26, 56.12±3.60, 3.54±0.86 μg/mL, respectively), and lipase (IC50 52.65±2.05, 581.70±54.14, 49.63±2.72 μg/mL, respectively). Red grape sample showed the highest inhibitory activity against α-glucosidase, cinnamon showed the highest inhibitory activity against α-amylase and lipase according to IC50 (concentration of inhibitor required to produce a 50% inhibition of the initial rate of reaction) and Catechin equivalent inhibition capacity (CEIC50) values. This study reported that apple, grape and cinnamon samples can inhibit the activity of digestive enzymes in vitro. The consumption of these samples would be used in conjunction with a low-calorie diet for body weight management.

Keywords: anthocyanin, α-amylase, α-glucosidase, lipase, procyanidin

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Authors: Mannar R. Maurya, Bithika Sarkar, Fernando Avecilla

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Peroxidase activity is possibly successfully used for different industrial processes in medicine, chemical industry, food processing and agriculture. However, they bear some intrinsic drawback associated with denaturation by proteases, their special storage requisite and cost factor also. Now a day’s artificial enzyme mimics are becoming a research interest because of their significant applications over conventional organic enzymes for ease of their preparation, low price and good stability in activity and overcome the drawbacks of natural enzymes e.g serine proteases. At present, a large number of artificial enzymes have been synthesized by assimilating a catalytic center into a variety of schiff base complexes, ligand-anchoring, supramolecular complexes, hematin, porphyrin, nanoparticles to mimic natural enzymes. Although in recent years a several number of vanadium complexes have been reported by a continuing increase in interest in bioinorganic chemistry. To our best of knowledge, the investigation of artificial enzyme mimics of vanadium complexes is very less explored. Recently, our group has reported synthetic vanadium schiff base complexes capable of mimicking peroxidases. Herein, we have synthesized monoidovanadium(IV) and dioxidovanadium(V) complexes of pyrazoleone derivateis ( extensively studied on account of their broad range of pharmacological appication). All these complexes are characterized by various spectroscopic techniques like FT-IR, UV-Visible, NMR (1H, 13C and 51V), Elemental analysis, thermal studies and single crystal analysis. The peroxidase mimic activity has been studied towards oxidation of pyrogallol to purpurogallin with hydrogen peroxide at pH 7 followed by measuring kinetic parameters. The Michaelis-Menten behavior shows an excellent catalytic activity over its natural counterparts, e.g. V-HPO and HRP. The obtained kinetic parameters (Vmax, Kcat) were also compared with peroxidase and haloperoxidase enzymes making it a promising mimic of peroxidase catalyst. Also, the catalytic activity has been studied towards the oxidation of 1-phenylethanol in presence of H2O2 as an oxidant. Various parameters such as amount of catalyst and oxidant, reaction time, reaction temperature and solvent have been taken into consideration to get maximum oxidative products of 1-phenylethanol.

Keywords: oxovanadium(IV)/dioxidovanadium(V) complexes, NMR spectroscopy, Crystal structure, peroxidase mimic activity towards oxidation of pyrogallol, Oxidation of 1-phenylethanol

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Authors: Maryam Monazzah, Ronak Samadpour, Mehdi Nasr-esfahani, Fatemeh Qalavand, Marziye Motamedi

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Bipolaris sorokiniana, and Heterodera filipjevi, are important wheat diseases that lead to yield losses worldwide. Identifying novel resistant sources helps us combat these devastating diseases. In this study, we studied the role of Cre3 gene and antioxidant enzymes in the immune responses of wheat genotypes to H. filipjevi and B. sorokiniana. Therefore, real-time PCR analysis using Cre3 gene marker, a resistant gene to cereal cyst nematodes, was conducted on leaves and roots, along with changes ‎in the activity of antioxidant enzymes, peroxidase, and catalase. Enzyme activity assay was performed on roots attacked by nematode and in leaves infected with Bipolaris. Wheat accessions including “Bam” (resistant), “Parsi” (moderately-resistant), “Azar2”, “Ohadi”, “Homa” (highly-susceptible) were previously screened against both stresses under greenhouse and field conditions. Results showed that Cre3 expression against cyst nematodes was significantly higher in resistant cultivars compared to susceptible cultivars. Cre3 was used in marker-assisted selection programs to identify genotypes carrying resistant genes to cyst nematodes. Interestingly, Cre3 was also up-regulated in both tissues of resistant cultivars to B. sorokiniana. Therefore, Cre3 in wheat similarly modulates immunity against B. sorokiniana and might be one of the central components of the induced immune system in wheat. The activity of antioxidant enzymes also indicated the highest increase in resistant genotypes upon both stresses that subsequently neutralize oxidative stress in tissues and decrease damage. Further studies on these resistance components may help us gain insight into the molecular basis of resistance and shed new light on the interaction and overlap between different forms of stress.

Keywords: Bipolaris sorokiniana, Heterodera filipjevi, resistant gene expression, wheat

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Authors: Paula K. Novelli, Margarida M. Barros, Luciana F. Flueri

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Utilization of agricultural by-products, wheat ban and soybean bran, as substrate for solid state fermentation (SSF) was studied, aiming the achievement of different enzymes from Aspergillus sp. with distinct biological characteristics and its application and improvement on animal nutrition. Aspergillus niger and Aspergillus oryzea were studied as they showed very high yield of phytase and protease production, respectively. Phytase activity was measure using p-nitrophenilphosphate as substrate and a standard curve of p-nitrophenol, as the enzymatic activity unit was the quantity of enzyme necessary to release one μmol of p-nitrophenol. Protease activity was measure using azocasein as substrate. Activity for phytase and protease substantially increased when the different biochemical characteristics were considered in the study. Optimum pH and stability of the phytase produced by A. niger with wheat bran as substrate was between 4.0 - 5.0 and optimum temperature of activity was 37oC. Phytase fermented in soybean bran showed constant values at all pHs studied, for optimal and stability, but low production. Phytase with both substrates showed stable activity for temperatures higher than 80oC. Protease from A. niger showed very distinct behavior of optimum pH, acid for wheat bran and basic for soybean bran, respectively and optimal values of temperature and stability at 50oC. Phytase produced by A. oryzae in wheat bran had optimum pH and temperature of 9 and 37oC, respectively, but it was very unstable. On the other hand, proteases were stable at high temperatures, all pH’s studied and showed very high yield when fermented in wheat bran, however when it was fermented in soybean bran the production was very low. Subsequently the upscale production of phytase from A. niger and proteases from A. oryzae were applied as an enzyme additive in fish fed for digestibility studies. Phytases and proteases were produced with stable enzyme activity of 7,000 U.g-1 and 2,500 U.g-1, respectively. When those enzymes were applied in a plant protein based fish diet for digestibility studies, they increased protein, mineral, energy and lipids availability, showing that these new enzymes can improve animal production and performance. In conclusion, the substrate, as well as, the microorganism species can affect the biochemical character of the enzyme produced. Moreover, the production of these enzymes by SSF can be up to 90% cheaper than commercial ones produced with the same fungi species but submerged fermentation. Add to that these cheap enzymes can be easily applied as animal diet additives to improve production and performance.

Keywords: agricultural by-products, animal nutrition, enzymes production, solid state fermentation

Procedia PDF Downloads 326

Authors: Jaturong Matidtor, Krisna R. Torrissen, Saengtong Pongjareankit, Sudaporn Tongsiri, Jiraporn Rojtinnakorn

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Low survival rate has being particular problem in nursery of marble goby juvenile. The aim of this study was to investigate effect of garlic extract on protein digestive pancreatic enzymes, trypsin (T) and chymotrypsin (C). The marble goby were reared with commercial feed mixed garlic extract at concentration of 0 (control), 0.3, 0.5, 1.0, 3.0 and 5.0% (w/w) for 6 weeks. Analysis of the digestive enzymes at 2 and 6 weeks was performed. Growth parameters; weight gain (WG), specific growth rate (SGR) and feed efficiency (FE), were identified. For T, C and T/C at 2 weeks, values of T and T/C ratio of 0.3% (w/w) group showed significant difference (p < 0.05) with the highest values of 17685.64± 11981.77 U/mg protein and of 51.64 ± 27.46 U/mg protein, respectively. For C at 2 weeks, 0% (w/w) group showed the highest values of 16191.76± 2225.56 U/mg protein. Whereas value of T, C and T/C ratio at 6 weeks, there was no significant difference (p > 0.05). For growth performance, it significantly increased in all garlic extract fed groups (0.3-5.0%, w/w), both at 2 and 6 weeks. At 2 weeks, values of WG and SGR of 0.5% (w/w) group showed the highest values of 71.51 ± 1.60%, and 3.85 ± 0.07%, respectively. For FE, 0.3% (w/w) group showed the highest value of 60.21 ± 6.51%. At 6 weeks, it illustrated that all growth parameters of 5.0% (w/w) group were the highest values; WG = 35.06 ± 5.66%, SGR = 2.14 ± 0.30%, and FE = 5.86 ± 0.68%. We suggested that garlic extract could be available for protein digestive enzyme and growth enhancement in marble goby nursery with artificial feed. This result will be high benefit for commercial aquaculture of marble goby.

Keywords: marble goby, nursery, garlic extract, digestive enzyme, growth

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Authors: Ana Tsokilauri

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The uppermost factor in the regulation of lignin-cellulose activity of decaying white rot or free rot are the substances serving as carbon and nitrogen nutrition of microorganisms and are considered as the most important factor of generative activity of white rot. The research object was Basidiomycete Fungi, peculiar and common in Georgia, and the separation of 10 of them as pure crops. The unidentified pure crops have tasted in order to be determined the potential of synthesis of lignin-degrading enzymes and the substrate of optimal lignocellulose growth. One of the most important aspects of the research conducted on Basidiomycetes was the use of specific lignocellulosic residues for selecting Fungi as a substrate of their growth. In order to increase lignocellulose with the help of substrate, crops were selected from the screening stage that showed good laccase activity. (Dusheti 1; Dusheti 10; Fshavi 5; Fshavi1; Fshavi 8; Fshavi 32; Manglisi 26; Sabaduri20; Dusheti 7; Sabaduri 1 ), Among the selected cultures, the crops with good laccase activity against the following substances, in particular: Dusheti 1- in this case, the rate of enzymatic activity on bran substrate was -105,6 u/ml, mandarin-96,4 u/ml. In case of corn - 102,9 u/ml. In case of Dusheti 7- the indicators were as follows: bananas-121,7 u/ml, mandarin-125,4 u/ml, corn - 117,1 u/ml. In the case of Sanaduri 32, the laccase activity was as follows: pomegranate- 101,2 u/ml. As a result of conducted experiments, the synthesis and activity rates of enzymes depending on plant substrates varied within a fairly wide range, which is still being under research.

Keywords: Lignocellulosic substrate, Basidiomycetes, white-rot basidiomycetes, Laccase

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Authors: Hassan Nour, Nouh Mounadi, Oussama Abchir, Belaidi Salah, Samir Chtita

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Cholinesterase enzymes are biological catalysts essential for the transformation of acetylcholine, which is a neurotransmitter implicated in memory and learning, into acetic acid and choline, altering the neurotransmission process in Alzheimer’s disease patients. Therefore, inhibition of cholinesterase enzymes is a relevant strategy for the symptomatic treatment of Alzheimer’s disease. The current investigation aims to explore potential Cholinesterase (ChE) inhibitors through a comprehensive computational approach. Forty-nine phytoconstituents extracted from Cannabis sativa L were in-silico screened using molecular docking, pharmacokinetic and toxicological analysis to evaluate their possible inhibitory effect towards the cholinesterase enzymes. Two phytoconstituents belonging to cannabinoid derivatives were revealed to be promising candidates for Alzheimer therapy by acting as cholinesterase inhibitors. They have exhibited high binding affinities towards the cholinesterase enzymes and showed their ability to interact with key residues involved in cholinesterase enzymatic activity. In addition, they presented good ADMET profiles allowing them to be promising oral drug candidates. Furthermore, molecular dynamics (MD) simulations were executed to explore their interactions stability under mimetic biological conditions and thus support our findings. To corroborate the docking results, the binding free energy corresponding to the more stable ligand-ChE complexes was re-estimated by applying the MM-PBSA method. MD and MM-PBSA studies affirmed that the ligand-ChE recognition is spontaneous reaction leading to stable complexes. The conducted investigations have led to great findings that would strongly guide the pharmaceutical industries towards the rational development of potent anti-Alzheimer agents.

Keywords: alzheimer’s disease, molecular docking, cannabis sativa l, cholinesterase inhibitors

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Authors: Emad Hafez, Mahmoud Seleiman

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Two field experiments were carried out in order to investigate the effect of chemical inducers [benzothiadiazole 0.9 mM L-1, oxalic acid 1.0 mM L-1, salicylic acid 0.2 mM L-1] on physiological and technological traits as well as on yields and antioxidant enzyme activities of barley grown under abiotic stress (i.e. water surplus and deficit conditions). Results showed that relative water content, leaf area, chlorophyll and yield as well as technological properties of barley were improved with chemical inducers application under water surplus and water-stress conditions. Antioxidant enzymes activity (i.e. catalase and peroxidase) were significantly increased in barley grown under water-stress and treated with chemical inducers. Yield and related parameters of barley presented also significant decrease under water-stress treatment, while chemical inducers application enhanced the yield-related traits. Starch and protein contents were higher in plants treated with salicylic acid than in untreated plants when water-stress was applied. In conclusion, results show that chemical inducers application have a positive interaction and synergetic influence and should be suggested to improve plant growth, yield and technological properties of water stressed barley. Salicylic acid application was better than oxalic acid and benzothiadiazole in terms of plant growth and yield improvement.

Keywords: antioxidant enzymes, drought stress, Hordeum vulgare L., quality, yield

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Authors: Aneta Maćczak, Bożena Bukowska, Jaromir Michałowicz

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Bisphenols are one of the most widely used chemical compounds worldwide. They are used in the manufacturing of polycarbonates, epoxy resins and thermal paper which are applied in plastic containers, bottles, cans, newspapers, receipt and other products. Among these compounds, bisphenol A (BPA) is produced in the highest amounts. There are concerns about endocrine impact of BPA and its other toxic effects including hepatotoxicity, neurotoxicity and carcinogenicity on human organism. Moreover, BPA is supposed to increase the incidence the obesity, diabetes and heart disease. For this reason the use of BPA in the production of plastic infant feeding bottles and some other consumers products has been restricted in the European Union and the United States. Nowadays, BPA analogues like bisphenol F (BPF) and bisphenol S (BPS) have been developed as alternative compounds. The replacement of BPA with other bisphenols contributed to the increase of the exposure of human population to these substances. Toxicological studies have mainly focused on BPA. In opposite, a small number of studies concerning toxic effects of BPA analogues have been realized, which makes impossible to state whether those substituents are safe for human health. Up to now, the mechanism of bisphenols action on the erythrocytes has not been elucidated. That is why, the aim of this study was to assess the effect of BPA and its selected analogues such as BPF and BPS on the activity of antioxidant enzymes, i.e. catalase (EC 1.11.1.6.), glutathione peroxidase (E.C.1.11.1.9) and superoxide dismutase (EC.1.15.1.1) in human erythrocytes. Red blood cells in respect to their function (transport of oxygen) and very well developed enzymatic and non-enzymatic antioxidative system, are useful cellular model to assess changes in redox balance. Erythrocytes were incubated with BPA, BPF and BPS in the concentration ranging from 0.5 to 100 µg/ml for 24 h. The activity of catalase was determined by the method of Aebi (1984). The activity of glutathione peroxidase was measured according to the method described by Rice-Evans et al. (1991), while the activity of superoxide dismutase (EC.1.15.1.1) was determined by the method of Misra and Fridovich (1972). The results showed that BPA and BPF caused changes in the antioxidative enzymes activities. BPA decreased the activity of examined enzymes in the concentration of 100 µg/ml. We also noted that BPF decreased the activity of catalase (5-100 µg/ml), glutathione peroxidase (50-100 µg/ml) and superoxide dismutase (25-100 µg/ml), while BPS did not cause statistically significant changes in investigated parameters. The obtained results suggest that BPA and BPF disrupt redox balance in human erythrocytes but the observed changes may occur in human organism only during occupational or subacute exposure to these substances.

Keywords: antioxidant enzymes, bisphenol A, bisphenol a analogues, human erythrocytes

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Authors: J. M. Kim, Y. W. Jung, E. Lee, Y. K. Kwack, , S. K. Sim*

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Cyanobacterial blooms in lakes, reservoirs, and rivers have been environmental and social issues due to its toxicity, odor, etc. Among the cyanotoxins, microcystins exist mostly within the cyanobacterial cells, and they are released from the cells. Therefore, an innovative technology is needed to detoxify the harvested microalgae for environment-friendly utilization of the harvested microalgae. This study develops detoxification method of microcystins in the harvested microalgae and recycling harvested microalgae with food waste using salt-tolerant mushroom strains and natural ecosystem decomposer. During this eco-friendly organic waste recycling process, diverse bacteria or various enzymes of the salt-tolerant mushroom strains decompose the microystins and cyclic peptides. Using PHLC/Mass analysis, it was verified that 99.8% of the microcystins of the harvested microalgae was detoxified in the harvested mushroom as well as in the recycled organic biomass. Further study is planned to verify the decomposition mechanisms of the microcystins by the bacteria or enzymes. In this study, the harvested microalgae is mixed with the food waste, and then the mixed toxic organic waste is used as mushroom compost by adjusting the water content of about 70% using cellulose such as sawdust cocopeats and cottonseeds. The mushroom compost is bottled, sterilized, and salt-tolerant mushroom spawn is inoculated. The mushroom is then cultured and growing in the temperature, humidity, and CO2 controlled environment. During the cultivation and growing process of the mushroom, microcystins are decomposed into non-toxic organic or inorganic compounds by diverse bacteria or various enzymes of the mushroom strains. Various enzymes of the mushroom strains decompose organics of the mixed organic waste and produce nutritious and antibiotic mushrooms. Cultured biomass compost after mushroom harvest can be used for organic fertilizer, functional bio-feed, and RE-100 biomass renewable energy source. In this eco-friendly organic waste recycling process, no toxic material, wastewater, nor sludge is generated; thus, sustainable with the circular economy.

Keywords: microalgae, microcystin, food waste, salt-tolerant mushroom strains, sustainability, circular economy

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Authors: Hassan Nour, Nouh Mounadi, Oussama Abchir, Belaidi Salah, Samir Chtita

Abstract:

Cholinesterase enzymes are biological catalysts essential for the transformation of acetylcholine, which is a neurotransmitter implicated in memory and learning, into acetic acid and choline, altering the neurotransmission process in Alzheimer’s disease patients. Therefore, inhibition of cholinesterase enzymes is a relevant strategy for the symptomatic treatment of Alzheimer’s disease. The current investigation aims to explore potential cholinesterase (ChE) inhibitors through a comprehensive computational approach. Forty-nine phytoconstituents extracted from Cannabis sativa L. were in-silico screened using molecular docking and pharmacokinetic and toxicological analysis to evaluate their possible inhibitory effect on the cholinesterase enzymes. Two phytoconstituents belonging to cannabinoid derivatives were revealed to be promising candidates for Alzheimer's therapy by acting as cholinesterase inhibitors. They have exhibited high binding affinities towards the cholinesterase enzymes and showed their ability to interact with key residues involved in cholinesterase enzymatic activity. In addition, they presented good ADMET profiles allowing them to be promising oral drug candidates. Furthermore, molecular dynamics (MD) simulations were executed to explore their interaction stability under mimetic biological conditions and thus support our findings. To corroborate the docking results, the binding free energy corresponding to the more stable ligand-ChE complexes was re-estimated by applying the MM-PBSA method. MD and MM-PBSA studies affirmed that the ligand-ChE recognition is a spontaneous reaction leading to stable complexes. The conducted investigations have led to great findings that would strongly guide the pharmaceutical industries toward the rational development of potent anti-Alzheimer agents.

Keywords: Alzheimer’s disease, molecular docking, Cannabis sativa L., cholinesterase inhibitors, molecular dynamics, ADMET, MM-PBSA

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Authors: Ayuko Itsuki, Sachiyo Aburatani

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Soil enzyme activities in Kasuga-yama Hill Primeval Forest (Nara, Japan) were examined to determine levels of mineralization and metabolism. Samples were selected from the soil surrounding laurel-leaved (B_B-1) and Carpinus japonica (B_B-2 and P_w) trees for analysis. Cellulase, β-xylosidase, and protease activities were higher in B_B-1 samples those in B_B-2 samples. These activity levels corresponded to the distribution of cellulose and hemicellulose in the soil horizons. Cellulase, β-xylosidase, and chymotrypsin activities were higher in soil from the P_w forest than in that from the B_B-2 forest. The relationships between the soil enzymes calculated by Spearman’s rank correlation indicate that the interactions between enzymes in B_B-2 samples were more complex than those in P_w samples.

Keywords: comparative analysis, enzyme activities, forest soil, Spearman's rank correlation

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Authors: Shiva Rezaei, Seyed Jalal Hosseinimehr, Abbasali Karimpour Malekshah, Mansooreh Mirzaei, Fereshteh Talebpour Amiri, Mehryar Zargari

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Objective(s): Cyclophosphamide (CP), as an antineoplastic drug, is widely used in cancer patients, and liver toxicity is one of its complications. Sinapic acid (SA), as a natural phenylpropanoid, has antioxidant, anti-inflammatory, and anti-cancer properties. Materials and Methods: The purpose of the current study was to determine the protective effect of SA versus CP-induced liver toxicity. In this research, BALB/c mice were treated with SA (5 and 10 mg/kg) orally for one week, and CP (200 mg/kg) was injected on day 3 of the study. Oxidative stress markers, serum liver-specific enzymes, histopathological features, caspase-3, and nuclear factor kappa-B cells were then checked. Results: CP induced hepatotoxicity in mice and showed structural changes in liver tissue. CP significantly increased liver enzymes and lipid peroxidation and decreased glutathione. The immunoreactivity of caspase-3 and nuclear factor kappa-B cells was significantly increased. Administration of SA significantly maintained histochemical parameters and liver function enzymes in mice treated with CP. Immunohistochemical examination showed SA reduced apoptosis and inflammation. Conclusion: The data confirmed that SA with anti-apoptotic, anti-oxidative, and anti-inflammatory activities was able to preserve CP-induced liver injury in mice.

Keywords: apoptosis, cyclophosphamide, liver injury, inflammation, oxidative stress, sinapic acid

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Authors: Mohammad Yusuf, Qazi Fariduddin

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The main aim of this study was to explore the different cultivars of Vigna radiata on basis of photosynthesis, antioxidants and proline to assess Ni-sensitive and Ni-tolerant cultivar. Seeds of five different cultivars were sown in soil amended with different levels of Ni (0, 50, 100, or 150 mg kg 1). At 30 d stage, plants were harvested to assess the various parameters. The Ni treatment diminished growth, leaf water potential, chlorophyll content and net photosynthesis along with nitrate reductase and carbonic anhydrase activities in the concentration dependent manner whereas, it enhanced proline content and various antioxidant enzymes. The varieties T-44 found least affected, whereas PDM-139 experienced maximum damage at 150 mg kg-1 of Ni. Moreover, T-44 possessed maximum activity of antioxidant enzymes and proline content at all the levels of metal whereas PDM-139 possessed minimum values. Therefore, T-44 and PDM-139 were established as the most resistant and sensitive varieties, respectively.

Keywords: Vigna radiata, antioxidants, nickel, photosynthesis, proline

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Authors: Li Na Zhao, Arieh Warshel

Abstract:

The design of enzymes is an extremely challenging task, and this is also true for metalloenzymes. In the case of naturally evolved enzymes, one may consider the active site residues as the musicians in the enzyme orchestra, while the metal can be considered as their concertmaster. Together they catalyze reactions as if they performed a masterpiece written by nature. The Lactonase can be thought as a member of the amidohydrolase family, with two concertmasters, Fe and Zn, at its active site. It catalyzes the quorum sensing signal- N-acyl homoserine lactones (AHLs or N-AHLs)- by hydrolyzing the lactone ring. This process, known as quorum quenching, provides a strategy in the treatment of infectious diseases without introducing selection pressure. However, the activity of lactonase is metal-dependent, and this dependence hampers the clinic usage. In our study, we use the empirical valence bond (EVB) approach to evaluate the catalytic contributions decomposing them to electrostatic and other components.

Keywords: enzyme redesign, empirical valence bond, lactonase, quorum quenching

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Authors: Charline Monnier, Rudolf Andrys, Irene Castellino, Lucie Zemanova

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Due to their inherent sustainability and compatibility with green chemistry principles, enzymes are attracting increasing attention for various applications like bioremediation or biocatalysis. These natural catalysts boast remarkable substrate specificity and operate under mild biological conditions. However, their intrinsic limitations, such as instability at high temperatures or in organic solvents, impede their wider applicability. Enzyme immobilization on supportive matrices emerges as a promising strategy to address these challenges. This approach not only facilitates enzyme reusability but also offers the potential to modulate their stability, activity, and selectivity. The present study investigates the immobilization and application of two distinct groups of hydrolases on supportive matrices: PETases, naturally capable of PolyEthylene Terephthalate (PET) degradation, and cholinesterases (ChEs), key enzymes in neurotransmitter regulation. All tested enzymes will be immobilized on porous and non-porous particles using both covalent and non-covalent methods. Additionally, the stability of PETases and cholinesterases will be explored, followed by exposure to denaturing conditions to assess their resilience under harsh conditions. Furthermore, due to the exceptional catalytic efficiency and selectivity, their biocatalytic efficiency will be tested using xenobiotic substrates, aiming to establish them as replacements for conventional chemical catalysts in environmentally friendly processes. By exploiting the power of enzyme immobilization, this research strives to unlock the full potential of these biocatalysts for sustainable and efficient technological advancements.

Keywords: biocatalysis, bioremediation, enzyme efficiency, enzyme immobilization, green chemistry

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Authors: Gajanan V. Mane, Rashmi More, Mahesh S. Sonawane, Tushar Lodha, Rohit Sharma

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The diversity of fungi isolated from two different termite species viz., Odontoterms assmuthi and O. abesus was investigated by dilution- plate method, combined with morphological characteristics and sequencing of internal transcribed spacer region. In total, ninety-six fungi were isolated and purified, out of which 69 isolates were obtained from O. assmuthi belonging to 18 genera and 31 species, whereas 27 isolates were obtained from O. abesus belonging to 15 genera and 17 species. The fungal strains were screened for laccase, amylase, cellulase and pectinase enzymes production. Twenty-seven strains were positive for laccase, 59 strains were positive for amylase, 71 strains were positive for cellulase and 72 strains were positive for pectinase enzymes. The antimicrobial activities of the isolated fungi were tested by the dual plate culture method against standard pathogens. Bioactive secondary metabolites were identified by HPLC and LCMS. Four isolates viz., Penicillium goetzii MG 57, Epicoccum sp. MG 39, Penicillium tanzanicum MG 30, Aspergillus polyporicola MG 54, showed positive antimicrobial activity against standard pathogens, Streptococcus pneumonia MCC 2425, Staphylococcus aureus MCC 2408, Pseudomonas aeruginosa MCC 2080, Escherichia coli MCC 2412, Enterococcus faecalis MCC 2409, Klebsiella pneumonia MCC 2451, Micrococcus luteus MCC 2155 and Candida albicans MCC 1151. In conclusion, the study showed that the insect gut harbor fungal diversity, which is futuristic with biotechnological potential and could be a good source of enzymes and antibiotics.

Keywords: termites, fungi, its, enzyme, antimicrobial activity

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Authors: Deepak Kumar Mittal, Alok Kumar Jena, Deepmala Joshi

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The present study was carried out to observe the hepatoprotective effect and antioxidant activity of the aqueous extract of the roots of Polygonum bistorta (PB) (200 mg/kg) and Zingiber roseum (ZR) (250 mg/kg) in rats treated with carbon tetrachloride (0.15 ml/kg, i.p.). Extract of PB and ZR at the tested doses restored the levels of liver homogenate enzymes, glutathione peroxidase, glutathione-S-transferase, superoxide dismutase and catalase enzymes, significantly. The activities of MTT assay significantly recovered the damage and supported the biochemical observations. This study suggests that Zingiber roseum has a higher protective effect on liver, compared to Polygonum bistorta, against carbon tetrachloride-induced hepatotoxicity and possesses antioxidant activities. Also, extracts exhibited moderate anticancer activity towards cell viability at higher concentration.

Keywords: Polygonum bistorta, Zingiber roseum, hepatoprotective effect, carbon tetrachloride, anti-cancerous

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Authors: T. Bettaieb, S. Soufi, S. Arbaoui

Abstract:

Stevia rebaudiana Bert. is a natural sweet plant. The leaves contain diterpene glycosides stevioside, rebaudiosides A-F, steviolbioside and dulcoside, which are responsible for its sweet taste and have commercial value all over the world as sugar substitute in foods and medicines. Stevia rebaudiana Bert. is sensitive temperature lower than 9°C. The possibility of its outdoor culture in Tunisian conditions demand genotypes tolerant to low temperatures. In order to evaluate the low temperature tolerance of eight genotypes of Stevia rebaudiana, the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalases (CAT) were measured. Before carrying out the analyses, three genotypes of Stevia were exposed for 1 month at a temperature regime of 18°C during the day and 7°C at night similar to winter conditions in Tunisia. In response to the stress generated by low temperature, antioxidant enzymes activity revealed on native gel and quantified by spectrophotometry showed variable levels according to their degree of tolerance to low temperatures.

Keywords: chilling tolerance, enzymatic activity, stevia rebaudiana bert, low thermal stress

Procedia PDF Downloads 444

Authors: Asma Mosbah, Hanane Khither, Kamelia Mosbah, Noreddine Kacem Chaouche, Mustapha Benboubetra

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In the present investigation, total oil (TO) and its neutral lipid fraction (NLF) extracted from the seed of the well know studied medicinal plant Nigella sativa were tested for their therapeutically effect on alcohol-induced liver injury in rat model. Male Albino rats were divided into five groups of eight animals each and fed a Lieber–DeCarli liquid diet containing 5% ethanol for experimental groups and dextran for control group, for a period of six weeks. Afterwards, rats received, orally, treatments with Nigella sativa extracts (TO, NLF) and N- acetylcysteine (NAC) as a positive control for four weeks. Activities of antioxidant enzymes; superoxide dismutase (SOD) and catalase (CAT), as well as malondialdehyde (MDA) and reduced glutathione (GSH). Biochemical parameters for kidney and liver functions, in treated and non treated rats, were evaluated throughout the time course of an experiment. Liver histological changes were taken into account. Enzymatic activities of both SOD and CAT increased significantly in rats treated with NLF and TO. While MDA level decreased in TO and NLF treated rats, GSH level increased significantly in TO and NLF treated rats. We noted equally a decrease in liver enzymes AST, ALT, and ALP. Microscopic observation of slides from the liver of ethanol treated rats showed a severe hepatotoxicity with lesions. Treatment with fractions leads to an improvement in liver lesions and a marked reduction in necrosis and infiltration. As a conclusion, both extracts of Nigella sativa seeds, TO and NLF, possess an important therapeutic protective potential against ethanol-induced hepatotoxicity in rats.

Keywords: alcohol-induced hepatotoxicity, antioxidant enzymes, Nigella sativa seeds, oil fractions

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Authors: Nompumelelo P. Mathebula, Roger A. Sheldon, Daniel P. Pienaar, Moira L. Bode

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The preparation of enantiopure Morita-Baylis-Hillman (MBH) adducts remains a challenge in organic chemistry. MBH adducts are highly functionalised compounds which act as key intermediates in the preparation of compounds of medicinal importance. MBH adducts are prepared in racemic form by reacting various aldehydes and activated alkenes in the presence of DABCO. Enantiopure MBH adducts can be obtained by employing Enzymatic kinetic resolution (EKR). This technique has been successfully demonstrated in our group, amongst others, using lipases in either hydrolysis or transesterification reactions. As these methods only allow 50% of each enantiomer to be obtained, our interest grew in exploring other enzymatic methods for the synthesis of enantiopure MBH adducts where, theoretically, 100% of the desired enantiomer could be obtained.Dehydrogenase enzymes can be employed on prochiral substrates to obtain optically pure compounds by reducing carbon-carbon double bonds or carbonyl groups of ketones. Ketoreductases have been used historically to obtain enantiopure secondary alcohols on an industrial scale. Ketoreductases are NAD(P)H-dependent enzymes and thus require nicotinamide as a cofactor. This project focuses on employing ketoreductase enzymes to selectively reduce ketones derived from Morita-Baylis-Hillman (MBH) adducts in order to obtain these adducts in enantiopure form.Results obtained from this study will be reported. Good enantioselectivity was observed using a range of different ketoreductases, however, reactions were complicated by the formation of an unexpected by-product, which was characterised employing single crystal x-ray crystallography techniques. Methods to minimise by-product formation are currently being investigated.

Keywords: ketoreductase, morita-baylis-hillman, selective reduction, x-ray crystallography

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Authors: Omar Ali, Adesh Ramsubhag, Jayaraj Jayaraman

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Seaweed extracts have been reported as plant biostimulants which could be a safer, organic alternative to harsh pesticides. The incentive to use seaweed-based biostimulants is becoming paramount in sustainable agriculture. The current study, therefore, screened a commercial extract of A. nodosum in tomatoes, cultivated in Trinidad to showcase the multiple beneficial effects. Foliar treatment with an A. nodosum commercial extract led to significant increases in fruit yield and a significant reduction of incidence of bacterial spots and early blight diseases under both greenhouse and field conditions. Investigations were carried out to reveal the possible mechanisms of action of this biostimulant through defense enzyme assays and transcriptome profiling via RNA sequencing of tomato. Studies into disease control mechanisms by A. nodosum showed that the extract stimulated the activity of enzymes such as peroxidase, phenylalanine ammonia-lyase, chitinase, polyphenol oxidase, and β-1,3-glucanase. Additionally, the transcriptome survey revealed the upregulation and enrichment of genes responsible for the biosynthesis of growth hormones, defense enzymes, PR proteins and defense-related secondary metabolites, as well as genes involved in the nutrient mobilization, photosynthesis and primary and secondary metabolic pathways. The results of the transcriptome study also demonstrated the cross-talks between growth and defense responses, confirming the bioelicitor and biostimulant value of seaweed extracts in plants. These effects could potentially implicate the benefits of seaweed extract and validate its usage in sustainable crop production.

Keywords: A. nodosum, biostimulants, elicitor, enzymes, growth responses, seaweeds, tomato, transcriptome analysis

Procedia PDF Downloads 162

Authors: Ludmila A. Gavriliuc, Jerry McLarty, Heather E. Kleiner, J. Michael Mathis

Abstract:

Abstract -- Background: The citrus coumarine Auraptene (Aur) is an effective chemopreventive agent, as manifested in many models of diseases and cancer. Nuclear factor erythroid 2-related factor (Nrf2) is an important regulator of genes induced by oxidative stress, such as glutathione S-transferases, heme oxygenase-1, and peroxiredoxin 1, by activating the antioxidant response element (ARE). Genetic and biochemical evidence has demonstrated that glutathione (GSH) and glutathione-dependent enzymes, glutathione reductase (GR), glutathione peroxidases (GPs), glutathione S-transferases (GSTs) are responsible for the control of intracellular reduction-oxidation status and participate in cellular adaptation to oxidative stress. The effect of Aur on the activity of GR, GPs (Se-GP and Se-iGP), and content of GSH in the liver, kidney, and spleen is insufficiently explored. Aim: Our goal was the examination of the Aur influence on the redox-system of GSH in Nrf2 wild type and Nrf2 knockout mice via activation of Nrf2 and ARE. Methods: Twenty female mice, 10 Nrf2 wild-type (WT) and 10 Nrf2 (-/-) knockout (KO), were bred and genotyped for our study. The activity of GR, Se-GP, Se-iGP, GST, G6PD, CytP450 reductase, catalase (Cat), and content of GSH were analyzed in the liver, kidney, and spleen using Spectrophotometry methods. The results of the specific activity of enzymes and the amount of GSH were analyzed with ANOVA and Spearman statistical methods. Results: Aur (200 mg/kg) treatment induced hepatic GST, GR, Se-GP activity and inhibited their activity in the spleen of mice, most likely via activation of the ARE through Nrf2. Activation in kidney Se-GP and G6PD by Aur is also controlled, apparently through Nrf2. Results of the non-parametric Spearman correlation analysis indicated the strong positive correlation between GR and G6PD only in the liver in WT control mice (r=+0.972; p < 0.005) and in the kidney KO control mice (r=+0.958; p < 0.005). The observed low content of GSH in the liver of KO mice indicated an increase in its participation in the neutralization of toxic substances with the absence of induction of GSH-dependent enzymes, such as GST, GR, Se-GP, and Se-iGP. Activation of CytP450 in kidney and spleen and Cat in the liver in KO mice probably revealed another regulatory mechanism for these enzymes. Conclusion: Thereby, obtained results testify that Aur can modulate the activity of genes and antioxidant enzymatic redox-system of GSH, responsible for the control of intracellular reduction-oxidation status.

Keywords: auraptene, glutathione, GST, Nrf2

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Authors: Miguel Pereira, Lígia Pimentel, Manuela Pintado

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Animal blood is a major by-product of slaughterhouses and still represents a cost and environmental problem in some countries. To be eliminated, blood should be stabilised by cooking and afterwards the slaughterhouses must have to pay for its incineration. In order to reduce the elimination costs and valorise the high protein content the aim of this study was the optimization of hydrolysis conditions, in terms of enzyme ratio and time, in order to obtain hydrolysates with biological activity. Two enzymes were tested in this assay: pepsin and proteases from Cynara cardunculus (cardosins). The latter has the advantage to be largely used in the Portuguese Dairy Industry and has a low price. The screening assays were carried out in a range of time between 0 and 10 h and using a ratio of enzyme/reaction volume between 0 and 5%. The assays were performed at the optimal conditions of pH and temperature for each enzyme: 55 °C at pH 5.2 for cardosins and 37 °C at pH 2.0 for pepsin. After reaction, the hydrolysates were evaluated by FPLC (Fast Protein Liquid Chromatography) and tested for their antioxidant activity by ABTS method. FPLC chromatograms showed different profiles when comparing the enzymatic reactions with the control (no enzyme added). The chromatogram exhibited new peaks with lower MW that were not present in control samples, demonstrating the hydrolysis by both enzymes. Regarding to the antioxidant activity, the best results for both enzymes were obtained using a ratio enzyme/reactional volume of 5% during 5 h of hydrolysis. However, the extension of reaction did not affect significantly the antioxidant activity. This has an industrial relevant aspect in what concerns to the process cost. In conclusion, the enzymatic blood hydrolysis can be a better alternative to the current elimination process allowing to the industry the reuse of an ingredient with biological properties and economic value.

Keywords: antioxidant activity, blood, by-products, enzymatic hydrolysis

Procedia PDF Downloads 510