Extração e purificação de inibidores proteolíticos em sementes de leguminosas arbóreas da Amazônia

A família Fabaceae é importante na indústria madeireira, como fonte de proteínas vegetais e contém uma grande variedade de biomoléculas com potencial para aplicação industrial, incluindo os inibidores proteolíticos, os quais são proteínas que podem atuar como fonte de aminoácidos sulfurados, na regulação de proteinases endógenas e na defesa das plantas contra o ataque de insetos e patógenos. Os objetivos deste trabalho foram a investigação e caracterização parcial dos inibidores proteolíticos bem como suas propriedades bioquímicas em extratos e frações de sementes de leguminosas arbóreas da flora amazônica: Parkia pendula, Parkia discolor, Parkia multijuga, Parkia nitida e Enterolobium schomburgkii. Os extratos obtidos após extração salina, precipitação por acetona e precipitação por sulfato de amônio foram utilizados para a quantificação protéica e nas etapas de detecção da atividade inibitória contra tripsina e quimotripsina bovina. O extrato total (ET), proveniente da extração salina, foi submetido à cromatografia de afinidade em Sepharose tripsina. As espécies P. pendula, P. discolor e P. nitida, mostraram maiores teores de proteínas nos ET, enquanto as espécies E. schomburgkii e P. multijuga mostraram estes resultados nos sobrenadantes submetidos ao fracionamento por sulfato de amônio a 0-30% e 60-80% de saturação. A atividade inibitória sobre tripsina foi maior nos extratos totais para todas as espécies, com possível perda de atividade após precipitação acetônica e precipitação por sulfato de amônio a 30-60% de saturação para E. schomburgkii. Contudo, a inibição da quimotripsina acima de 50%, foi observada apenas no extrato total e no precipitado cetônico de P. nitida. Os valores das constantes de inibição (Ki) foram de 8,92 x 10-8 M; 6,89 x 10-8 M; 6,15 x 10-8 M, para P. pendula, P. discolor e P. multijuga, respectivamente. Os inibidores de tripsina apresentaram massas moleculares aparentes de 17 kDa (PpTI), 21 kDa (PdTI), 19 kDa (PmTI e PnTI) e 18 kDa (EsTI), bem como afinidade diferenciada contra a tripsina e quimotripsina bovina. Os resultados mostram que, provavelmente, os inibidores pertencem à família Kunitz.A família Fabaceae é importante na indústria madeireira, como fonte de proteínas vegetais e contém uma grande variedade de biomoléculas com potencial para aplicação industrial, incluindo os inibidores proteolíticos, os quais são proteínas que podem atuar como fonte de aminoácidos sulfurados, na regulação de proteinases endógenas e na defesa das plantas contra o ataque de insetos e patógenos. Os objetivos deste trabalho foram a investigação e caracterização parcial dos inibidores proteolíticos bem como suas propriedades bioquímicas em extratos e frações de sementes de leguminosas arbóreas da flora amazônica: Parkia pendula, Parkia discolor, Parkia multijuga, Parkia nitida e Enterolobium schomburgkii. Os extratos obtidos após extração salina, precipitação por acetona e precipitação por sulfato de amônio foram utilizados para a quantificação protéica e nas etapas de detecção da atividade inibitória contra tripsina e quimotripsina bovina. O extrato total (ET), proveniente da extração salina, foi submetido à cromatografia de afinidade em Sepharose tripsina. As espécies P. pendula, P. discolor e P. nitida, mostraram maiores teores de proteínas nos ET, enquanto as espécies E. schomburgkii e P. multijuga mostraram estes resultados nos sobrenadantes submetidos ao fracionamento por sulfato de amônio a 0-30% e 60-80% de saturação. A atividade inibitória sobre tripsina foi maior nos extratos totais para todas as espécies, com possível perda de atividade após precipitação acetônica e precipitação por sulfato de amônio a 30-60% de saturação para E. schomburgkii. Contudo, a inibição da quimotripsina acima de 50%, foi observada apenas no extrato total e no precipitado cetônico de P. nitida. Os valores das constantes de inibição (Ki) foram de 8,92 x 10-8 M; 6,89 x 10-8 M; 6,15 x 10-8 M, para P. pendula, P. discolor e P. multijuga, respectivamente. Os inibidores de tripsina apresentaram massas moleculares aparentes de 17 kDa (PpTI), 21 kDa (PdTI), 19 kDa (PmTI e PnTI) e 18 kDa (EsTI), bem como afinidade diferenciada contra a tripsina e quimotripsina bovina. Os resultados mostram que, provavelmente, os inibidores pertencem à família Kunitz

Purificação, caracterização química e atividade de proteases e inibidores de proteases durante a germinação e em eventos pré e pós-germinativos de sementes de Parkia multijuga

During seed germination, storage proteins are accumulated and degraded to support the major metabolic events involved in development new cells and tissues. The mobilization of proteins occurs through the activity of proteases, which can be regulated through the presence of proteases inhibitors that prevent premature hydrolysis of the storage proteins. The objective of this research was to investigate the activity of proteases and protease inhibitors during germination and pre-and postgermination of seeds of Parkia multijuga and purify trypsin inhibitors. Thereby, the seeds after breaking dormancy, they were germinated in plastic pots containing vermiculite and packed in germination chambers at 25ºC. The collection stages were quiescent seeds (SQ), after 24 h of imbibition (EM), radicle protrusion (RA), expansion of cotyledon node (NO) and issue shoot (PA). Proteins were extracted into 0.15 M NaCl. The activities of serine proteinases and trypsin and chymotrypsin inhibitors were performed using BAPNA and azocasein as substrates. The activity of cysteine proteases and papain and bromelain inhibitors using BANA and azocasein, respectively. Trypsin inhibitors were purified by affinity chromatography usin trypsin- Sepharose 4B and ion exchange on HiTrap DEAE FF. The purification and the mobilization of proteins was performed by electrophoresis on SDS-PAGE and 2D gels. During seed germination of P. multijuga observed a significant decrease in the protein content after RA remaining until PA was confirmed by 2D gels, wich show proteins degradation in the range of 35 to 75 kDa in PA, while 9 kDa proteins were synthesized during EM. Regarding the inhibitory activity, serine protease inhibition showed high stability during all stages studied, of which trypsin inhibition was 20 times higher than chymotrypsin. Inhibition of cysteine proteases is more variable, observing for maximum inhibition of papain and bromelain in EM and NO, respectively. The activities of serine and cysteine proteases presented themselves to constant NO, with an increase in PA. The purification of trypsin inhibitors involved two chromatographic steps, which were similar to the profiles obtained in all stages studied, indicating the purification of the same molecule or isoinhibitors in different stages studied. From the purification of trypsin inhibitors (PmTI1 and PmTI2) SQ, these proteins are characterized by presenting against trypsin inhibitory specificity, high stability against variation of temperature and pH and amino acid sequence homology to the Bowman-type inhibitors Birk from other Fabaceae species. Thus, serine and cysteine proteases inhibitors could participate in the regulation of protease activity during germination and events pre and post-germination of seeds of P. multijuga, with the trypsin inhibitors Bowman-Birk type present throughout the process of formation of the seedling.Durante a germinação das sementes, proteínas de reserva são acumuladas e degradadas para dar suporte aos principais eventos metabólicos envolvidos na construção de novas células e tecidos. A mobilização proteica ocorre a partir da atividade de proteases e, pode ser controlada por inibidores de proteases, evitando a hidrólise prematura das proteínas de reserva. O objetivo desta pesquisa foi investigar a atividade de proteases e inibidores de proteases durante a germinação e em períodos pré e pós-germinativos de sementes de Parkia multijuga, bem como purificar inibidores de tripsina. Para tanto, as sementes, após quebra da dormência, foram semeadas em bandejas plásticas contendo vermiculita e acondionadas em câmaras de germinação à 25ºC. Os estádios de coleta foram: sementes quiescentes (SQ), após 24 h de embebição (EM), emissão da radícula (RA), expansão do nó cotiledonar (NO) e emissão da parte aérea (PA). As proteínas foram extraídas em NaCl 0,15 M. As atividades de serinoproteases e de inibidores de tripsina e quimotripsina foram avaliadas utilizando-se como substratos o BAPNA e a azocaseína e, a atividade das cisteínoproteases e de inibidores de papaína e bromelaína, utilizando-se o BANA e azocaseína, respectivamente. Os inibidores de tripsina foram purificados a partir de cromatografias em tripsina-Sepharose 4B e HiTrap DEAE FF. O monitoramento da purificação e da mobilização das proteínas foi realizado por meio eletroforeses em SDS-PAGE e géis 2D. Durante a germinação das sementes de P. multijuga observou-se decréscimo expressivo no conteúdo de proteínas após RA, permanecendo até PA, sendo confirmado por géis 2D, demonstrando a degradação de proteínas na faixa de 35 a 75 kDa durante PA, ao passo que, proteínas de 9 kDa foram sintetizadas durante EM. Quanto às atividades inibitórias, verificou-se estabilidade na inibição das serinoproteases em todos os estádios estudados, sendo a atividade anti-tríptica vinte vezes maior quando comparada à da quimotripsina. A inibição de cisteínoproteases foi mais variável, verificando-se máxima inibição da papaína e da bromelaína em EM e NO, respectivamente. As atividades de serino e cisteínoproteases apresentaram-se constantes até NO, com acréscimo em PA. A purificação dos inibidores de tripsina envolveu duas etapas cromatográficas, onde os perfis obtidos foram similares em todos os estádios estudados, indicando a purificação das mesmas moléculas ou de isoinibidores nos diferentes estádios estudados. A partir da purificação dos inibidores de tripsina (PmTI1 e PmTI2) de SQ, essas proteínas foram caracterizadas por apresentarem especificidade inibitória contra a tripsina, alta estabilidade quanto à variação de temperatura e pH e, homologia à sequência de aminoácidos de inibidores do tipo Bowman-Birk de outras espécies de Fabaceae. Neste sentido, inibidores de serino e cisteínoproteases devem participar da regulação da atividade de proteases durante a germinação e nos eventos pré e pós-germinativos de sementes de P. multijuga, estando os inibidores de tripsina do tipo Bowman-Birk presentes durante todo o processo de formação da plântula

Energetic flux and performance index in copaiba (Copaifera multijuga Hayne) and mahogany (Swietenia macrophylla King) seedlings grown under two irradiance environments

Plant adaptation under conditions of limited irradiance can be investigated by their efficiency while using the available energy with a minimal thermodynamic energy loss. For this reason, we compared the energetic fluxes and the performance index (PIABS) in copaiba (Copaifera multijuga) and mahogany (Swietenia macrophyla) seedlings under both sunlight and shade. The experiment was carried out in Manaus (3°8′S, 59°52′W), Amazonas State, Brazil. The fluorescence transients were obtained in intact and healthy leaves using a Plant Efficiency Analyser (PEA) between 1200 h and 1300 h. Using the O-J-I-P test, the following parameters were calculated: 1) density of reaction centers per cross section (RC/CS), 2) maximum quantum yield for primary photochemistry of photosystem II (φPo), and 3) probability of energy excitation (ψo) or that of an absorbed photon (φEo) to move an electron further than quinone A, and 4) the performance index (PIABS). The highest PIABS was found in seedlings under shade for both species. Mahogany seedlings exhibited values of PIABS about three-fold and 14-fold higher than copaiba seedlings under shade and sunlight, respectively. For RC/CS, reaction centers were inactive under sunlight, 58% for mahogany and 78% for copaiba. The high irradiance induced a decrease of φPo in mahogany (36%) and in copaiba (69%) when compared to shaded seedlings. This likely was a result of need for excess energy dissipation under sunlight. In addition, it was verified that seedlings under sunlight presented a severe decrease in φEo, particularly in copaiba (23-fold), when compared to mahogany (9-fold). Therefore, mahogany seedlings showed the highest performance in energy use under low and high irradiance, thus it may be proposed that mahogany presented lower entropy than copaiba seedlings, as demonstrated by the higher plasticity in the use of energy

Seeds of Amazonian Fabaceae as a source of new lectins

Seeds from fifty native Amazonian Fabaceae species (representing subfamilies Caesalpinioideae, Mimosoideae and Faboideae) were screened for the presence of new lectins. Their crude protein extracts were assayed for hemagglutinating activity (HA). The protein fractions of Anadenanthera peregrina, Dimorphandra caudata, Ormosia lignivalvis and Swartzia laevicarpa exhibited HA, and this activity was inhibited by galactose or lactose but not by glucose or mannose. The crude extract of S. laevicarpa exhibited HA activity only after ion exchange chromatography, and its lectin was further purified by affinity chromatography on immobilized lactose. Despite the large number of lectins that have been reported in leguminous plants, this is the first description of lectins in the genera Anadenanthera, Dimorphandra and Ormosia. The study of lectins from these genera and from Swartzia will contribute to the understanding of the evolutionary relationships of legume lectins in terms of their protein processing properties and structures

Detection of trypsin inhibitors and hemagglutinating activity in tree leguminous seeds of amazonian

Different classes of proteins are common in Leguminosae seeds, including trypsin inhibitors and hemagglutinin proteins, which act on proteolytic enzymes and cell-surface carbohydrates, respectively. The aim of this work was to quantify, to detect and characterize partially these proteins in seeds of Tachigali plumbea, Sesbania exasperata and Ormosia costulata var. trifoliata. Seeds of the three species were powdered and submitted to an extraction with a saline solution (NaCl 0.15M - 10%, p/v). The resulting total extracts were used to quantify proteins content, detect the residual trypsin activity, hemagglutinating activity (AHE) and the proteic profile. Residual trypsin activity was observed only for T. plumbea and S. exasperata, which values were 4 and 19% respectively. AHE was detected in extracts of all three species, total extracts of T. plumbea and S. exasperata hemagglutinated erythrocytes of rats, mice and hamsters, whereas O. costulata had this effect only on hamster erythrocytes. The proteic profile obtained by SDS-PAGE showed that T. plumbea and S. exasperata have a higher content of protein with an apparent molecular mass of 10 - 30 kDa, while O. costulata predominantly contains proteic bands with molecular masses varying between 20 to 25 kDa. It is concluded that total extracts of O. costulata and S. exasperata, species of the subfamily Papilionoideae, present less trypsin inhibitors than T. plumbea (Caesalpinioideae). AHE, both in form of minimum hemagglutinin concentration and the specified interaction with erythrocytes, differed even among species from the same subfamily.Diferentes classes de proteínas são comuns em sementes de leguminosas, incluindo inibidores de tripsina e proteínas hemaglutinantes, as quais atuam sobre enzimas proteolíticas e sobre carboidratos da superfície celular, respectivamente. O objetivo deste trabalho foi quantificar, detectar e caracterizar parcialmente a ocorrência dessas proteínas em sementes de Tachigali plumbea, Sesbania exasperata e Ormosia costulata var. trifoliolata. Sementes das três espécies foram moídas e submetidas à extração salina (NaCl 0,15M - 10 %, p/v). Os extratos totais obtidos foram utilizados para quantificar o conteúdo protéico, detectar a atividade residual da tripsina, a atividade hemaglutinante (AHE) e na obtenção do perfil protéico. A atividade residual da tripsina foi observada somente para T. plumbea e S. exasperata, cujos valores foram 4 e 19 %, respectivamente. A AHE foi detectada nos extratos das três espécies, sendo que os extratos totais de T. plumbea e S. exasperata, hemaglutinaram eritrócitos de rato, camundongo e hamster, enquanto que a espécie O. costulata hemaglutinou somente eritrócitos de hamster. O perfil protéico em SDS-PAGE revelou maior ocorrência de proteínas com massa molecular aparente de 10 a 30 kDa para T. plumbea e S. exasperata, enquanto que para O. costulata prevaleceram bandas protéicas com massa molecular variando entre 20-25 kDa. Conclui-se que os extratos totais de O. costulata e S. exasperata, pertencentes à subfamília Papilionoideae, apresentam menor conteúdo de inibidores de tripsina que T. plumbea (Caesalpinioideae) e, quanto à AHE, os resultados mostraram-se diferenciados, mesmo entre as espécies da mesma subfamília, tanto para a concentração mínima hemaglutinante quanto para a especificidade de interação com os eritrócitos

Production of commercial and Amazonian strains of Pleurotus ostreatus in plant waste / Produção de linhagens comerciais e amazônicas de Pleurotus ostreatus em resíduos de plantas

Pleurotus spp. have the ability to grow on a wide variety of lignocellulosic materials, which opens up a range of options to be explored. The use of locally available residues and native strains can reduce production costs, thus making fungiculture a potential economic activity in developing regions, such as northern Brazil. The aim of this research was to compare the production and nutritional characteristics of different strains of P. ostreatus, cultivated on lignocellulosic residues available in Amazonas state, Brazil. The native (474 and 1467) and commercial (542 and 885) strains were cultivated in marupá (Simarouba amara) and pine (Pinus sp.) sawdust, açaí (Euterpe oleracea) seeds and elephant grass (Pennisetum purpureum Schum.) straw. Productivity was evaluated according to biological efficiency, yield and loss of organic matter. Some physicochemical and nutritional parameters were also evaluated. In general, among the residues evaluated for mushroom cultivation, the elephant grass substrates (EG) showed better physicochemical properties in terms of moisture, ash content (minerals), hemicellulose and lignin. Additionally, the EG-based substrate resulted in the best productive parameters (yield and EB) for all strains studied, with emphasis on strain 1467, which is native to the Amazon. The basidiocarps of the native strains (474 and 1467) presented a nutritional composition that is similar to the commercial strains (885 and 542), and the mushrooms grown in EG-based residue, in general, presented the best nutritional compositions. Thus, strain 1467 of Pleurotus ostreatus, native to the Amazon, shows promise for commercial purposes, when cultivated in elephant grass residue.Pleurotus spp. have the ability to grow on a wide variety of lignocellulosic materials, which opens up a range of options to be explored. The use of locally available residues and native strains can reduce production costs, thus making fungiculture a potential economic activity in developing regions, such as northern Brazil. The aim of this research was to compare the production and nutritional characteristics of different strains of P. ostreatus, cultivated on lignocellulosic residues available in Amazonas state, Brazil. The native (474 and 1467) and commercial (542 and 885) strains were cultivated in marupá (Simarouba amara) and pine (Pinus sp.) sawdust, açaí (Euterpe oleracea) seeds and elephant grass (Pennisetum purpureum Schum.) straw. Productivity was evaluated according to biological efficiency, yield and loss of organic matter. Some physicochemical and nutritional parameters were also evaluated. In general, among the residues evaluated for mushroom cultivation, the elephant grass substrates (EG) showed better physicochemical properties in terms of moisture, ash content (minerals), hemicellulose and lignin. Additionally, the EG-based substrate resulted in the best productive parameters (yield and EB) for all strains studied, with emphasis on strain 1467, which is native to the Amazon. The basidiocarps of the native strains (474 and 1467) presented a nutritional composition that is similar to the commercial strains (885 and 542), and the mushrooms grown in EG-based residue, in general, presented the best nutritional compositions. Thus, strain 1467 of Pleurotus ostreatus, native to the Amazon, shows promise for commercial purposes, when cultivated in elephant grass residue

Use of fruit epicarps to formulate pectin-based bioactive films / Utilização de epicarpos de frutos para formulação de filmes bioativos à base de pectinas

The functional properties of biofilms can vary according to the biopolymer used as the raw material; thus, in the search for alternative sources for preparation of biodegradable films, fruits and vegetables have been used to extract compounds of interest with applications in the food industry. The objective of this work was to obtain and characterize bioactive films based on pectin extracted from the epicarps (skin) of the fruit. The genipap (Genipa americana), red pitaya (Hylocereus polyrhizus) and star fruit (Averrhoa carambola) were collected, washed, pulped and dried at 50 °C for 24 h, and their epicarps were ground and subjected to pectin extraction using the casting method. The films were characterized as to their visual appearance, moisture, pH, water solubility and antioxidant activity. The pectin-based films of G. americana and H. polyrhizus showed a yellowish color, while A. carambola was dark brown. The highest pectin yield (29%) and moisture content (13.9%) were obtained from the H. polyrhizus film, while A. carambola showed the highest solubility in water (98.6%) and had the highest pH (3.9). Additionally, the film based on A. carambola showed greater antioxidant potential against ABTS (30.5%) and DPPH (34%), as well as greater reducing power (0.262 absorbance at 750 nm) and content of total phenolic compounds (553 mg GAE/100 g), whereas H. polyrhizus had a higher percentage of chelating ability (27%). The physicochemical characteristics and bioactive properties exhibited make the film formulation a viable alternative for the food industry