Acetylcholinesterase purification from human erythrocytes using magnetic nanoparticles containing procainamide.

This work presents a magnetic purification method of human erythrocyte Acetylcholinesterase (EC 3.1.1.7; AChE) based on affinity binding to procainamide (Proca) as ligand. Acetylcholinesterase is an acetylcholine-regulating enzyme found in different areas of the body and associated with various neurological disorders, such as Parkinson, Alzheymer and Amyotrophic Lateral Sclerosis. AChE from human erythrocyte purification has been attempted in recent years with low degree of purity. Here, magnetic nanoparticles (MNP) were synthesized and coated with polyaniline (PANI) and procainamide (PROCA) was covalently linked to the PANI. The extracted human erythrocyte AChE formed a complex with the MNP@PANI-PROCA and an external magnet separated it from the undesired proteins. Finally, the enzyme was collected by increasing the ionic strength. Experimental Box-Behnken design was developed to optimize this process of human erythrocyte AChE purification protocol. The enzyme was purified in all fifteen experiments. However, the best AChE purification result was achieved, about 2000 times purified, when 100 mg of MNP@PANI-PROCA was incubated for one hour with 4 ml hemolysate extract. The SDS-PAGE of this preparation presented a molecular weight of approximately 70 kDa, corroborating with few previous studies of AChE from erythrocyte purification.

Shrimp laccase degrades polycyclic aromatic hydrocarbons from an oil spill disaster in Brazil: A tool for marine environmental bioremediation.

Our work aims to purify, characterize and evaluate a laccase from by-products of the shrimp farming industry (Litopenaeus vannamei) for the degradation of Polycyclic Aromatic Hydrocarbons (PAHs) from 2019 oil spill in Brazilian coast. The enzyme was purified by affinity chromatography and characterized as thermostable, with activity above 90 °C and at alkaline pH. In addition, the laccase was also tolerant to copper, lead, cadmium, zinc, arsenic, hexane and methanol, with significant enzymatic activation in acetone and 10 mM mercury. Concerning PAHs' degradation, the enzyme degraded 42.40 % of the total compounds, degrading >50 % of fluorene, C4-naphthalenes, C3-naphthalenes, C2-naphthalenes, anthracene, acenaphthene, 1-methylnaphthalene and 2-methylnaphthalene. Thus, this laccase demonstrated important characteristics for bioremediation of marine environments contaminated by crude oil spills, representing a viable and ecological alternative for these purposes.

Acetylcholinesterase from the charru mussel Mytella charruana: kinetic characterization, physicochemical properties and potential as in vitro biomarker in environmental monitoring of mollusk extraction areas.

Acetylcholinesterase (AChE; EC 3.1.1.7) from aquatic organisms have been used to evaluate the exposure of specimens to pesticides and heavy metals at sublethal levels in environmental samples. AChE of Mytella charruana was extracted to characterize its physicochemical and kinetic properties as well as the effect of organophosphate (dichlorvos, diazinon, chlorpyrifos, methyl-parathion and temephos), carbamates (carbaryl, carbofuran and aldicarb), benzoylureas (diflubenzuron and novaluron), pyrethroid (cypermethrin) and juvenile hormone analog - JHA (pyriproxyfen) and the effect of metal ions: Hg2+, Cd2+, Pb2+, As3+, Cu2+ and Zn2+, in order to evaluate the potential of the enzyme as biomarker. The optimum pH of M. charruana AChE was 8.5 and the maximum activity peak occurred at 48 °C, being highly thermostable maintaining 97.8% of its activity after incubation at 60 °C. The Michaelis-Menten constants (km) for the substrates acetylthiocholine and propionylthiocholine were 2.8 ± 1.26 and 4.94 ± 6.9 mmol·L-1, respectively. The Vmax values for the same substrates were 22.6 ± 0.90 and 10.2 ± 4.94 mU·mg-1, respectively. Specific inhibition results suggest an AChE presenting active site with dimensions between those of AChE and butyrylcholinesterase (BChE). The IC20 values related to the effect of the pesticides on the enzyme showed higher inhibitory power of temephos (0.17 µmol·L-1), followed by aldicarb (0.19 µmol·L-1) and diflubenzuron (0.23 µmol·L-1). Metal ions inhibited M. charruana enzyme in the following order: Hg2+ > Pb2+ > Cd2+ > As3+ > Cu2+ > Zn2+. These data suggest that the enzyme showed potential as in vitro biomarker of the exposure to temephos, mercury, zinc and copper.

Chemical composition, antimicrobial activity and synergistic effects with conventional antibiotics under clinical isolates by essential oil of Hymenaea rubriflora Ducke (FABACEAE).

This work aimed to investigate the chemical composition, antimicrobial activity, synergistic effect, and structure changes of the essential oil of Hymenaea rubriflora (EOHr). Forty-five constituents were identified in the essential oil, corresponding to 94.43% of the compounds present, being the main components E-Caryophyllene (36.72 ± 1.05%), Germacrene D (16.13 ± 0.31%), α-Humulene (6.06 ± 0.16%), ß-elemene (5.61 ± 0.14%) and δ-Cadinene (3.76 ± 0.07%). Antimicrobial activity was evaluated, presenting antibacterial and antifungal activity with MIC ranging from 0.62 to 40 µL/mL. The essential oil had a synergistic effect when combined with gentamicin and fluconazole. Structural changes were also evaluated and it was possible to observe that EOHr action was related to changes in membrane permeability. The findings obtained here suggest that the use of the essential oil of H. rubriflora in the treatment of infectious diseases presents a potential for the future development of pharmaceutical products.

Chemical and biological activities of faveleira (Cnidoscolus quercifolius Pohl) seed oil for potential health applications.

Faveleira (Cnidoscolus quercifolius) is an emerging Brazilian plant, with seeds rich in edible oil. This study investigates physicochemical properties, chemical composition, thermal and oxidative stability, in vitro and in vivo toxicity, antioxidant, antinociceptive and anti-inflammatory activities of faveleira seed oil. It was observed that the oil has low acidity, value of peroxide, chlorophyll, carotenoids, ß-carotene and high concentrations of unsaturated fatty acids. In addition to presenting thermal and oxidative stability and high total phenolic content, with vanillin, eugenol and quercetin were predominating. The oil showed no toxicity in vitro and in vivo, and presented antioxidant, anti-inflammatory and antinociceptive activities. These findings provide relevant and appropriate conditions for processing of faveleira seed oil as functional food.

Brain acetylcholinesterase of three perciformes: From the characterization to the in vitro effect of metal ions and pesticides.

Estuarine environments gather pollution from large regions including urban and industrial zones. The monitoring of environmental quality in these areas constitutes a real requirement for global sustainable development. Therefore, the aim of this study was to characterize the physicochemical and kinetic parameters of brain acetylcholinesterase (AChE) in the species Centropomus undecimalis, Diapterus auratus and Diapterus rhombeus and to assess the effects (in vitro) of pesticides and metal ions on their respective activities in order to investigate them as potential biomarkers. Physicochemical properties such as thermostability, optimal pH and temperature, as well as kinetic parameters were investigated. AChE was pointed as the predominant cholinesterase (ChE) in the brains of the species under study. The highest optimum pH value was observed for C. undecimalis (8.0), and the lowest for D. rhombeus and D. auratus, with 7.2 and 7.0, respectively. The optimal temperature was 35 °C for the three species. The AChEs of the three species presented moderate thermostability, since they retained 61%, 72% and 67% of the activity up to 45 °C (C. undecimalis, D. auratus and D. rhombeus, respectively). The carbamate carbofuran showed to be the strongest inhibitor even at very low concentrations (IC50: 0.182, 0.174 and 0.203 µmol/L - C. undecimalis, D. auratus and D. rhombeus, respectively), followed by dichlorvos and carbaryl. According to the findings, the AChE of these species may be proposed as in vitro biomarker of exposure to carbofuran and dichlorvos (all three species) and carbaryl (D. auratus and D. Rhombeus), as well as for exceeding limit concentrations of Hg2+ (D. rhombeus) and As3+ (D. auratus) in biomonitoring programs located or not at estuarine environments.

Recovery of fibrinolytic and collagenolytic enzymes from fish and shrimp byproducts: potential source for biomedical applications / Recuperação de enzimas fibrinolíticas e colagenolitícas de resíduos de peixes e camarões: fonte potencial para aplicações biomédicas

Fish and shrimp industries generate a significant amount of by-products. These by-products can be used for the extraction of enzymes of biomedical interest, such as fibrinolytic and collagenolytic. Thus, this work aimed to perform a screening of fish and shrimp byproducts as sources of enzymes with fibrinolytic and collagenolytic activities and to characterize the biochemical properties of crude extracts with collagenolytic activity from Cichla ocellaris residues. Fibrinolytic enzymes were recovered with activities between 5.51 ± 0.02 U.mL-1 (Caranx crysos) and 56.16 ± 0.42 U.mL-1 (Litopenaeus vannamei), while collagenolytic enzymes were detected in a range between 6.79 ± 0.00 U.mg-1 (Trachurus lathami) and 94.35 ± 0.02 U.mg-1 (C. ocellaris). After collagenolytic screening, the selected species was C. ocellaris, being subjected to a preheating, which culminated with an increase of enzymatic activity of 35.07% (up to 127.44 ± 0.09 U.mg-1). The optimal collagenolytic activity recovered from C. ocellaris byproducts was 55 °C (thermostable between 25 and 60 °C) and 7.5 (stable between 6.5 and 11.5) for temperature and pH evaluations, respectively. The kinetic parameters were determined, obtaining Km of 5.92 mM and Vmax of 294.40 U.mg-1. The recovered enzyme was sensitive to the Cu2+, Hg2 and Pb2+ ions, being partially inhibited by phenylmethylsulphonyl fluoride (PMSF), N-p-tosyl-L-lysin chloromethyl ketone (TLCK) and Benzamidine. Furthermore, it was able to cleave native type I collagen, the most important type for industry. Thus, the recovery of biomolecules, besides offering to the industry an alternative source of active molecules, contributes to the reduction of the environmental impact, adding value to the fish product and providing a new source of income.(AU)

As indústrias de peixe e camarão geram uma quantidade significativa de subprodutos. Estes subprodutos podem ser utilizados para a extração de enzimas de interesse biomédico, como fibrinolítica e o colagenolítica. Assim, este trabalho teve como objetivo realizar uma triagem de subprodutos de peixes e camarões como fontes de enzimas com atividade fibrinolítica e colagenolítica e caracterizar as propriedades bioquímicas do extrato bruto com atividade colagenolítica a partir de resíduos de Cichla ocellaris. Enzimas fibrinolíticas foram recuperadas com atividades entre 5,51 ± 0,02 U.mL-1 (Caranx crysos) e 56,16 ± 0,42 U.mL-1 (Litopenaeus vannamei), enquanto enzimas colagenolitícas foram detectadas numa variação entre 6,79 ± 0,00 U.mg-1 (Trachurus lathami) e 94,35 ± 0,02 U.mg-1 (C. ocellaris). Após a triagem colagenolítica, a espécie selecionada foi a C. ocellaris, sendo submetida a um pré-aquecimento, o que culminou com um aumento de atividade enzimática de 35.07% (127.44 ± 0.09 U.mg-1). A atividade colagenolítica ótima recuperada de resíduos de C. ocellaris foi a 55°C (termoestável entre 25 a 60°) e 7,5 (estável entre 6,5 a 11,5) para avaliações de temperatura e de pH, respectivamente. Os parâmetros cinéticos foram determinados, obtendo-se Km de 5,92 mM e Vmax de 294,40 U.mg-1. A enzima recuperada foi sensível aos íons de Cu2+, Hg2+ e Pb2+, sendo parcialmente inibida por Fluoreto de Fenilmetilsulfonil (PMSF), N-α-tosil-L-lisina clorometil cetona (TLCK) e Benzamidina. Ainda, foi capaz de clivar o colágeno nativo tipo I, o tipo mais desejável pela indústria. Assim, a recuperação de biomoléculas, além de oferecer à indústria uma fonte alternativa de moléculas ativas, contribui para a redução do impacto ambiental, agregando valor ao produto pesqueiro e proporcionando nova fonte de renda.(AU)

Recovery of fibrinolytic and collagenolytic enzymes from fish and shrimp byproducts: potential source for biomedical applications / Recuperação de enzimas fibrinolíticas e colagenolitícas de resíduos de peixes e camarões: fonte potencial para aplicações biomédicas

Fish and shrimp industries generate a significant amount of by-products. These by-products can be used for the extraction of enzymes of biomedical interest, such as fibrinolytic and collagenolytic. Thus, this work aimed to perform a screening of fish and shrimp byproducts as sources of enzymes with fibrinolytic and collagenolytic activities and to characterize the biochemical properties of crude extracts with collagenolytic activity from Cichla ocellaris residues. Fibrinolytic enzymes were recovered with activities between 5.51 ± 0.02 U.mL-1 (Caranx crysos) and 56.16 ± 0.42 U.mL-1 (Litopenaeus vannamei), while collagenolytic enzymes were detected in a range between 6.79 ± 0.00 U.mg-1 (Trachurus lathami) and 94.35 ± 0.02 U.mg-1 (C. ocellaris). After collagenolytic screening, the selected species was C. ocellaris, being subjected to a preheating, which culminated with an increase of enzymatic activity of 35.07% (up to 127.44 ± 0.09 U.mg-1). The optimal collagenolytic activity recovered from C. ocellaris byproducts was 55 °C (thermostable between 25 and 60 °C) and 7.5 (stable between 6.5 and 11.5) for temperature and pH evaluations, respectively. The kinetic parameters were determined, obtaining Km of 5.92 mM and Vmax of 294.40 U.mg-1. The recovered enzyme was sensitive to the Cu2+, Hg2 and Pb2+ ions, being partially inhibited by phenylmethylsulphonyl fluoride (PMSF), N-p-tosyl-L-lysin chloromethyl ketone (TLCK) and Benzamidine. Furthermore, it was able to cleave native type I collagen, the most important type for industry. Thus, the recovery of biomolecules, besides offering to the industry an alternative source of active molecules, contributes to the reduction of the environmental impact, adding value to the fish product and providing a new source of income.

As indústrias de peixe e camarão geram uma quantidade significativa de subprodutos. Estes subprodutos podem ser utilizados para a extração de enzimas de interesse biomédico, como fibrinolítica e o colagenolítica. Assim, este trabalho teve como objetivo realizar uma triagem de subprodutos de peixes e camarões como fontes de enzimas com atividade fibrinolítica e colagenolítica e caracterizar as propriedades bioquímicas do extrato bruto com atividade colagenolítica a partir de resíduos de Cichla ocellaris. Enzimas fibrinolíticas foram recuperadas com atividades entre 5,51 ± 0,02 U.mL-1 (Caranx crysos) e 56,16 ± 0,42 U.mL-1 (Litopenaeus vannamei), enquanto enzimas colagenolitícas foram detectadas numa variação entre 6,79 ± 0,00 U.mg-1 (Trachurus lathami) e 94,35 ± 0,02 U.mg-1 (C. ocellaris). Após a triagem colagenolítica, a espécie selecionada foi a C. ocellaris, sendo submetida a um pré-aquecimento, o que culminou com um aumento de atividade enzimática de 35.07% (127.44 ± 0.09 U.mg-1). A atividade colagenolítica ótima recuperada de resíduos de C. ocellaris foi a 55°C (termoestável entre 25 a 60°) e 7,5 (estável entre 6,5 a 11,5) para avaliações de temperatura e de pH, respectivamente. Os parâmetros cinéticos foram determinados, obtendo-se Km de 5,92 mM e Vmax de 294,40 U.mg-1. A enzima recuperada foi sensível aos íons de Cu2+, Hg2+ e Pb2+, sendo parcialmente inibida por Fluoreto de Fenilmetilsulfonil (PMSF), N-α-tosil-L-lisina clorometil cetona (TLCK) e Benzamidina. Ainda, foi capaz de clivar o colágeno nativo tipo I, o tipo mais desejável pela indústria. Assim, a recuperação de biomoléculas, além de oferecer à indústria uma fonte alternativa de moléculas ativas, contribui para a redução do impacto ambiental, agregando valor ao produto pesqueiro e proporcionando nova fonte de renda.

Characterization of brain acetylcholinesterase of bentonic fish Hoplosternum littorale: Perspectives of application in pesticides and metal ions biomonitoring.

Acetylcholinesterase (AChE; EC 3.1.1.7) is a serine hydrolase, whose main function is to modulate neurotransmission at cholinergic synapses. It is, therefore, the primary target of some pesticides and heavy metals. Its inhibition in aquatic organisms has been used as an indicator of the presence of these pollutants in water bodies. The present study aimed to characterize physicochemical and kinetic parameters of brain AChE in the benthic fish Hoplosternum littorale and to analyze the in vitro effects of pesticides (dichlorvos, diazinon, chlorpyrifos, parathion-methyl, temephos, carbaryl, carbofuran, aldicarb, diflubenzuron, novaluron and pyriproxyfen) and metal ions (As3+, Cd2+, Cu2+, Fe2+, Mn2+, Mg2+, K+, Pb2+, Hg2+, Zn2+) investigating the potential of this enzyme as environmental biomarker based on current regulations. Specific substrates and inhibitors have indicated AChE to be the predominant cholinesterase (ChE) in the brain of H. littorale. Peak activity was observed at pH 8.0 and 30 °C. The enzymatic activity is otherwise moderately thermostable (≈ 50% activity at 45 °C). The enzyme can reduce the activation energy of acetylthiocholine hydrolysis reaction to 8.34 kcal mol-1 while reaching a rate enhancement of 106. Among the pesticides under study, dichlorvos presented an IC50 value below the maximum concentrations allowed by legislation. This study presents the first report on the inhibition of brain AChE activity from Siluriformes by the pesticides novaluron and pyriproxyfen. Mercury ion also exerted a strong inhibitory effect on its enzymatic activity. The H. littorale enzyme thus has the potential to function as an in vitro biomarker for the presence of the pesticide dichlorvos as well as mercury in areas of mining and industrial discharge.

Acetylcholinesterase of mangrove oyster Crassostrea rhizophorae: A highly thermostable enzyme with promising features for estuarine biomonitoring.

Enzyme biomarkers from several aquatic organisms have been used for assessing the exposure to contaminants at sublethal levels. Amongst them, the cholinesterases are commonly extracted from several organisms to evaluate/measure organophosphate and carbamate neurotoxic effects. Acetylcholinesterase (AChE; EC 3.1.1.7) is an enzyme of the group of serine esterases that acts on the hydrolysis of the neurotransmitter acetylcholine allowing the intermittence of the nerve impulses responsible for the neuronal communication. This enzyme is the main target for the action of some pesticides and the inhibition of its activity in bivalve mollusks may be used as biomarker due to their filter-feeding habit. In this context, the present study aimed to characterize physicochemical and kinetic parameters of the AChE extracted from gills and viscera of the oyster Crassostrea rhizophorae and investigate the in vitro effect of pesticides (dichlorvos, diazinon, chlorpyrifos, methyl-parathion, temephos, carbaryl, carbofuran, aldicarb, diflubenzuron and novaluron) in search for assessing its potential as biomarker. Specific substrates and inhibitors evidenced the predominance of AChE in both tissues. The optimum pH found for gills and viscera AChE were 8.0 and 8.5, respectively. The maximum peak of activity occurred at 70 °C for gill AChE and 75 °C for viscera AChE. The enzymes of both tissues presented remarkable thermostability. The Michaelis-Menten constant for both enzymes were 1.32 ±â€¯0.20 mM for gills and 0.43 ±â€¯0.12 mM for viscera. The Vmax values for gills and viscera were 53.57 ±â€¯1.72 and 27.71 ±â€¯1.15 mU/mg, respectively. The enzymes were able to reduce the activation energy to 9.75 kcal mol-1 (gills) and 11.87 kcal mol-1 (viscera) obtaining rate enhancements of 3.57 × 105 and 1.01 × 104, respectively, in relation to non-catalyzed reactions. Among the pesticides under study, the carbamates carbaryl and carbofuran exerted the strongest inhibitory effects on the enzyme activity achieving important degrees of inhibition at concentrations below national and international current regulations. The first observation of the effects of benzoylurea pesticides (diflubenzuron and novaluron) on AChE from mollusks is reported here. The gills AChE of C. rhizophorae showed potential to be specific biomarker for the carbamate carbaryl while the viscera AChE showed it for carbofuran. According to their features, these enzymes may be proposed as promising tools for estuarine monitoring as well as biocomponent of biosensor devices.

Collagenase from smooth weakfish: extraction, partial purification, characterization and collagen specificity test for industrial application / Colagenase de pescada branca: extração, purificação parcial, caracterização e teste de especificidade ao colágeno para aplicação industrial

Fish processing residues are rich sources of biomolecules with industrial potential, such as enzymes with collagenolytic properties applied in the pharmaceutical, textile and leather sectors. Here, collagenolytic serine proteases were partially purified from the waste (viscera) of smooth weakfish Cynoscion leiarchus and characterized for the purpose of obtaining a value-added product from fisheries resources. The higher activity of the enzyme (72.5 U mL-1) was verified in optimal temperature and pH of 55oC and 8.0, respectively. The enzyme was stable in wide ranges of temperature (25-60oC) and pH (6.5 to 11.5). The ions Ca2+ and Mg2+ increased the protease activity, whilst Pb2+, Al3+ and Cu2+ had an inhibitory effect, as observed in the presence of Benzamidine and TLCK (inhibitors of serine proteases). Hydrolysis was detected after 48 hours, when the enzyme and bovine collagen type I were incubated together. Thus, digestive viscera of C. leiarchus is suggested as an alternative source of enzymes capable of cleaving type I collagen, with similar biochemical properties to bacterial collagenases commonly employed in industrial processes, reducing costs, adding value to the fisheries product and minimizing the environmental impact caused by its waste.(AU)

Resíduos do processamento do pescado são fontes ricas em biomoléculas com potencial industrial, como as enzimas com propriedades colagenolíticas empregadas nos segmentos farmacêuticos, têxteis e de couro. No presente trabalho, serino-proteases colagenolíticas dos resíduos (vísceras) do processamento de pescada-branca, Cynoscion leiarchus, foram parcialmente purificadas e caracterizadas, visando à obtenção de um produto de valor agregado, maximizando o aproveitamento de recursos pesqueiros. A atividade da melhor etapa de extração foi 72,5 U mL- 1 , com temperatura e pH ótimos de 55oC e 8.0, respectivamente. A enzima manteve-se estável em faixas amplas de temperatura (25-60oC) e pH (6,5-11,5). Os íons Ca2+ e Mg2+ aumentaram a atividade proteolítica, ao passo que Pb2+, Al3+ e Cu2+ inibiram essa atividade assim como os inibidores de serino-proteases (Benzamidina e TLCK). A hidrólise foi detectada após 48h de incubação com colágeno bovino tipo I. Assim, sugere-se vísceras digestivas de C. leiarchus como fonte alternativa para o fornecimento de enzimas com capacidade de clivar o colágeno do tipo I e com propriedades bioquímicas semelhantes às colagenases bacterianas já empregadas nas etapas de processamento industrial, como forma de redução de custo, agregação de valor ao produto pesqueiro e contribuindo para minimizar o impacto ambiental deste tipo de resíduo.(AU)

Collagenase from smooth weakfish: extraction, partial purification, characterization and collagen specificity test for industrial application / Colagenase de pescada branca: extração, purificação parcial, caracterização e teste de especificidade ao colágeno para aplicação industrial

Fish processing residues are rich sources of biomolecules with industrial potential, such as enzymes with collagenolytic properties applied in the pharmaceutical, textile and leather sectors. Here, collagenolytic serine proteases were partially purified from the waste (viscera) of smooth weakfish Cynoscion leiarchus and characterized for the purpose of obtaining a value-added product from fisheries resources. The higher activity of the enzyme (72.5 U mL-1) was verified in optimal temperature and pH of 55oC and 8.0, respectively. The enzyme was stable in wide ranges of temperature (25-60oC) and pH (6.5 to 11.5). The ions Ca2+ and Mg2+ increased the protease activity, whilst Pb2+, Al3+ and Cu2+ had an inhibitory effect, as observed in the presence of Benzamidine and TLCK (inhibitors of serine proteases). Hydrolysis was detected after 48 hours, when the enzyme and bovine collagen type I were incubated together. Thus, digestive viscera of C. leiarchus is suggested as an alternative source of enzymes capable of cleaving type I collagen, with similar biochemical properties to bacterial collagenases commonly employed in industrial processes, reducing costs, adding value to the fisheries product and minimizing the environmental impact caused by its waste.

Resíduos do processamento do pescado são fontes ricas em biomoléculas com potencial industrial, como as enzimas com propriedades colagenolíticas empregadas nos segmentos farmacêuticos, têxteis e de couro. No presente trabalho, serino-proteases colagenolíticas dos resíduos (vísceras) do processamento de pescada-branca, Cynoscion leiarchus, foram parcialmente purificadas e caracterizadas, visando à obtenção de um produto de valor agregado, maximizando o aproveitamento de recursos pesqueiros. A atividade da melhor etapa de extração foi 72,5 U mL- 1 , com temperatura e pH ótimos de 55oC e 8.0, respectivamente. A enzima manteve-se estável em faixas amplas de temperatura (25-60oC) e pH (6,5-11,5). Os íons Ca2+ e Mg2+ aumentaram a atividade proteolítica, ao passo que Pb2+, Al3+ e Cu2+ inibiram essa atividade assim como os inibidores de serino-proteases (Benzamidina e TLCK). A hidrólise foi detectada após 48h de incubação com colágeno bovino tipo I. Assim, sugere-se vísceras digestivas de C. leiarchus como fonte alternativa para o fornecimento de enzimas com capacidade de clivar o colágeno do tipo I e com propriedades bioquímicas semelhantes às colagenases bacterianas já empregadas nas etapas de processamento industrial, como forma de redução de custo, agregação de valor ao produto pesqueiro e contribuindo para minimizar o impacto ambiental deste tipo de resíduo.

Aluminium sulfate exposure: A set of effects on hydrolases from brain, muscle and digestive tract of juvenile Nile tilapia (Oreochromis niloticus).

Aluminium is a major pollutant due to its constant disposal in aquatic environments through anthropogenic activities. The physiological effects of this metal in fish are still scarce in the literature. This study investigated the in vivo and in vitro effects of aluminium sulfate on the activity of enzymes from Nile tilapia (Oreochromis niloticus): brain acetylcholinesterase (AChE), muscle cholinesterases (AChE-like and BChE-like activities), pepsin, trypsin, chymotrypsin and amylase. Fish were in vivo exposed during 14days when the following experimental groups were assayed: control group (CG), exposure to Al2(SO4)3 at 1µg·mL-1 (G1) and 3µg·mL-1 (G3) (concentrations compatible with the use of aluminium sulfate as coagulant in water treatment). In vitro exposure was performed using animals of CG treatment. Both in vivo and in vitro exposure increased cholinesterase activity in relation to controls. The highest cholinesterase activity was observed for muscle BChE-like enzyme in G3. In contrast, the digestive enzymes showed decreased activity in both in vivo and in vitro exposures. The highest inhibitory effect was observed for pepsin activity. The inhibition of serine proteases was also quantitatively analyzed in zymograms using pixel optical densitometry as area under the peaks (AUP) and integrated density (ID). These results suggest that the inhibition of digestive enzymes in combination with activation of cholinesterases in O. niloticus is a set of biochemical effects that evidence the presence of aluminium in the aquatic environment. Moreover, these enzymatic alterations may support further studies on physiological changes in this species with implications for its neurological and digestive metabolisms.

Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste.

A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50°C and 8.5, respectively. The enzyme was thermostable until 55°C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38mM, kcat value was 3.14s(-1), and kcat/km was 8.26s(-1)mM(-1). The catalytic proficiency of the purified enzyme was 2.75×10(12)M(-1) showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93kcalmol(-1) while the resulting rate enhancement of this reaction was found to be approximately in a range from 10(9) to 10(10)-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes.

Brain acetylcholinesterase of jaguar cichlid (Parachromis managuensis): From physicochemical and kinetic properties to its potential as biomarker of pesticides and metal ions.

This contribution aimed to characterize physicochemical and kinetic parameters of the brain cholinesterases (ChEs) from Parachromis managuensis and investigate the in vitro effects of pesticides and metal ions on its activity intending to propose as biomarker. This species is suitable for this investigation because (1) it was recently introduced in Brazil becoming invasive (no restrictions on capture) and (2) occupies the top of the food chain (being subject to bioaccumulation). The enzyme extract was exposed to 10 metal ions (Al(3+), Ba(2+), Cd(2+), Cu(2+), Hg(2+), Mg(2+), Mn(2+), Pb(2+), Fe(2+) and Zn(2+)) and ChEs selective inhibitors (BW284c51, Iso-OMPA, neostigmine and serine). The extract was also incubated with organophosphate (dichlorvos) and carbamate pesticides (carbaryl and carbofuran). Inhibition parameters (IC20, IC50 and ki) were determined. Selective inhibitors and kinetic parameters confirmed acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) -like as responsible for the ChE activities, most AChE. The IC50 values for pesticides were: 1.68µM (dichlorvos); 4.35µM (carbaryl) and 0.28µM (carbofuran). Most of the analyzed ions did not show significant effect at 1mM (p=0.05), whereas the following ions inhibited the enzyme activity in the order: Hg(2+)>Cu(2+)>Cd(2+)>Zn(2+). Mercury ion strongly inhibited the enzyme activity (IC20=0.7µM). The results about allow to conclude that P. managuensis brain AChE is a potential biomarker for heavy metals and pesticides under study, mainly for the carbamate carbofuran once it was capable to detect 6-fold lower levels than the limit concentration internationally recommended.

Digestive enzyme activity in the intestine of Nile tilapia (Oreochromis niloticus L.) under pond and cage farming systems.

The effect of different farming systems (cage, pond) upon digestive enzyme activities of Nile tilapia was evaluated. Juvenile Nile tilapia (87.61 ± 1.52 g) were simultaneously cultured in pond and cage systems during 90 days. Cages used nutritional biphasic plan (35 and 32 % crude protein-CP feeds) and ponds used nutritional triphasic plan (35, 32 and 28 % CP feeds). Biometric measurements were monthly performed for adjustments in feeding regimes and removal of intestine tissues to evaluate the performance of enzyme activities. Total proteolytic, amylase and lipase activities were not statistically different between the treatments throughout the periods analyzed (31, 63 and 94 days of culture). However, trypsin and chymotrypsin activities were higher with 31 and 63 days of culture in fish from pond system, suggesting that natural food may have influenced these activities. A positive correlation was observed between the recommended concentration of essential amino acids for Nile tilapia and specific aminopeptidases activity in fish cage system. Substrate-SDS-PAGE revealed 12 active proteolytic bands in both systems. However, integrated density (ID) values were higher in the bands of ponds. Specimens of either cage or pond exhibited five bands of amylolytic activity. Fish from cage and pond systems showed the highest values of ID within 31 days of cultivation. In this study, the complexity of digestive functions could be verified for animals maintained under commercial conditions. Some of the assessed enzymes may show adaptations of their activities and/or expression that allow the fish to achieve a more efficient nutrient assimilation.

Kinetic and physicochemical properties of brain acetylcholinesterase from the peacock bass (Cichla ocellaris) and in vitro effect of pesticides and metal ions.

Brain acetylcholinesterase (AChE; EC 3.1.1.7) from peacock bass (Cichla ocellaris) was characterized and the effect of organophosphorus and carbamate pesticides as well as ions and heavy metals was evaluated. The kinetic parameters K(m) and V(max) were determined as 0.769 mM and 0.189 U/mg of protein respectively. Optimal pH and temperature were found to be 8.0 and 45°C. The enzyme retained approximately half of the activity after incubation at 50°C for 30 min. Total cholinesterase activity on brain of this species can be ascribed to AChE according to selective inhibitors analysis (neostigmine, eserine and BW284c5 reduced its activity whereas no effect was noticed for Iso-OMPA). Seven pesticides (five organophosphates: dichlorvos, diazinon, chlorpyrifos, temephos, tetraethyl pyrophosphate - TEPP and two carbamates: carbaryl and carbofuran) showed inhibitory effects on C. ocellaris AChE. However, the strongest effect was observed with carbofuran (IC(50)=0.21 µM and K(i)=2.57 × 10(-3) µM). The following ions (1 mM) showed to inhibit its activity (decrescent order): Hg(2+)>As(3+)>Cu(2+)>Zn(2+). EDTA(2-) did not affect enzyme activity. The present study provides assay conditions and data to suggest this enzyme as in vitro biomarker of organophosphorus and carbamate pesticides in routine environmental screening programs.

Comparative effect of pesticides on brain acetylcholinesterase in tropical fish.

Monitoring of pesticides based on acetylcholinesterase (AChE; EC 3.1.1.7) inhibition in vitro avoids interference of detoxification defenses and bioactivation of some of those compounds in non-target tissues. Moreover, environmental temperature, age and stress are able to affect specific enzyme activities when performing in vivo studies. Few comparative studies have investigated the inter-specific differences in AChE activity in fish. Screening studies allow choosing the suitable species as source of AChE to detect pesticides in a given situation. Brain AChE from the tropical fish: pirarucu (Arapaima gigas), cobia (Rachycentron canadum) and Nile tilapia (Oreochromis niloticus) were characterized and their activities were assayed in the presence of pesticides (the organophosphates: dichlorvos, diazinon, chlorpyrifos, temephos, tetraethyl pyrophosphate- TEPP and the carbamates: carbaryl and carbofuran). Inhibition parameters (IC50 and Ki) for each species were found and compared with commercial AChE from electric eel (Electrophorus electricus). Optimal pH and temperature were found to be 8.0 and 35-45 °C, respectively. A. gigas AChE retained 81% of the activity after incubation at 50 °C for 30 min. The electric eel enzyme was more sensitive to the compounds (mainly carbofuran, IC50 of 5 nM), excepting the one from A. gigas (IC50 of 9 nM) under TEPP inhibition. These results show comparable sensitivity between purified and non-purified enzymes suggesting them as biomarkers for organophosphorus and carbamate detection in routine environmental and food monitoring programs for pesticides.

Characterization of acetylcholinesterase from the brain of the Amazonian tambaqui (Colossoma macropomum) and in vitro effect of organophosphorus and carbamate pesticides.

In the present study, acetylcholinesterase (AChE) from the brain of the Amazonian fish tambaqui (Colossoma macropomum) was partially characterized and its activity was assayed in the presence of five organophosphates (dichlorvos, diazinon, chlorpyrifos, and tetraethyl pyrophosphate [TEPP]) and two carbamates (carbaryl and carbofuran) insecticides. Optimal pH and temperature were 7.0 to 8.0 and 45°C, respectively. The enzyme retained approximately 70% of activity after incubation at 50°C for 30 min. The insecticide concentration capable of inhibiting half of the enzyme activity (IC50) for dichlorvos, chlorpyrifos, and TEPP were calculated as 0.04 µmol/L, 7.6 µmol/L, and 3.7 µmol/L, respectively. Diazinon and temephos did not inhibit the enzyme. The IC50 values for carbaryl and carbofuran were estimated as 33.8 µmol/L and 0.92 µmol/L, respectively. These results suggest that AChE from the juvenile C. macropomum brain could be used as an alternative biocomponent of organophosphorus and carbamate biosensors in routine pesticide screening in the environment.

Effect of dichlorvos on the acetylcholinesterase from tambaqui (Colossoma macropomum) brain.

Dichlorvos is an acutely toxic organophosphorous pesticide that is known as a classical acetylcholinesterase (AChE; EC 3.1.1.7) inhibitor. Here, the brain AChE from the important Amazonian fish tambaqui (Colossoma macropomum) was assayed in the presence of this insecticide and also of deltamethrin, a classical sodium and potassium channel inhibitor (negative control). Four tissue homogenates were analyzed in triplicate for AChE activity using acetylthiocholine as the substrate and 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) as the color-developing agent. Each tissue homogenate represented pooled brains from five fish. The inhibitory effect of dichlorvos on AChE activities was determined at concentrations from 0.001 to 10 ppm and compared to controls. This effect followed an exponential decay model (y = 9.420 + 26.192e(-x/5.380); r2 = 0.989), presenting IC50 (the concentration of dichlorvos that is required for 50% of AChE inhibition) of 0.081 ppm (0.368 micromol/L). No effect was observed for the deltamethrin, and the concentration 0.0452 micromol/L of dichlorvos was significantly different from this control. These results suggest that tambaqui brain AChE can be proposed as a biomarker for dichlorvos and can be used as a tool for aquatic environment monitoring.