Extração das proteínas de sementes e polpa de Cucurbita maxima: uma análise experimental / Extraction of proteins from Cucurbita maxima seeds and pulp: an experimental analysis

As abóboras (Cucurbitaceae) estão distribuídas em mais de 120 gêneros e 800 espécies. Acredita-se que as abóboras foram cultivadas pela primeira vez no México, em 5.550 a.C. Devido ao melhoramento genético, hoje, as abóboras são cultivadas nas mais variadas condições climáticas em todo o mundo. Por este motivo, estudos acerca do potencial anti-helmíntico, anti-hipertensivo, hipoglicêmico, antimicrobiano e antioxidante de diversas espécies de abóboras são crescentes. Do ponto de vista nutricional, as Cucurbitáceas são fontes de vitaminas A, B, cálcio, ferro, silício e magnésio. Neste estudo descrevemos o processo de extração das proteínas presentes no endosperma das sementes e na polpa de Cucurbita maxima, variedade Duchesne (moranga). A farinha do endosperma das sementes foi utilizada para a extração de proteínas, utilizando tampão fosfato de potássio (KH2PO4) 0,1 M contendo 0,3 M de NaCl, pH7,6. Uma segunda metodologia de extração foi realizada utilizando água destilada. A extração em tampão fosfato apresentou um rendimento de 10,5%, enquanto a extração aquosa apresentou rendimento próximo de 0,6%. A partir da polpa, a recuperação de proteínas foi inversa: 0,2% para o extrato salino e 1,7% para o extrato aquoso. Dado o elevador teor de proteínas presente nas sementes, propomos seu uso como forma de suplementação nutricional e/ou probiótica, sendo necessárias análises complementares para investigar seu valor biológico, bem como as propriedades antimicrobianas e antifúngicas. 

A new Kunitz trypsin inhibitor from Erythrina poeppigiana exhibits antimicrobial and antibiofilm properties against bacteria

Erythrina poeppigiana belongs to Fabaceae family (subfamily Papillionoideae) and is commonly found in tropical and subtropical regions in Brazil. Herein, we described the purification and characterization of a new Kunitz-type inhibitor, obtained from E. poeppigiana seeds (EpTI). EpTI is composed by three isoforms of identical aminoterminal sequences with a molecular weight ranging from 17 to 20 kDa. The physicochemical features showed by EpTI are common to Kunitz inhibitors, including the dissociation constant (13.1 nM), stability against thermal (37–100 ◦C) and pH (2–10) ranging, and the presence of disulfide bonds stabilizing its reactive site. Furthermore, we investigated the antimicrobial, anti-adhesion, and anti-biofilm properties of EpTI against Grampositive and negative bacteria. The inhibitor showed antimicrobial activity with a minimum inhibitory concentration (MIC, 5–10 μM) and minimum bactericidal concentration (MBC) of 10 μM for Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Staphylococcus aureus, and Staphylococcus haemolyticus. The combination of EpTI with ciprofloxacin showed a marked synergistic effect, reducing the antibiotic concentration by 150%. The increase in crystal violet uptake for S. aureus and K. pneumoniae strains was approximately 30% and 50%, respectively, suggesting that the bacteria plasma membrane is targeted by EpTI. Treatment with EpTI at 1x and 10 x MIC significantly reduced the biofilm formation and prompted the disruption of a mature biofilm. At MIC/2, EpTI decreased the bacterial adhesion to polystyrene surface within 2 h. Finally, EpTI showed low toxicity in animal model Galleria mellonella. Given its antimicrobial and anti-biofilm properties, the EpTI sequence might be used to design novel drug prototypes

Dual Insecticidal Effects of Adenanthera pavonina Kunitz-Type Inhibitor on Plodia interpunctella is Mediated by Digestive Enzymes Inhibition and Chitin-Binding Properties

The Indianmeal moth, Plodia interpunctella, is one of the most damaging pests of stored products. We investigated the insecticidal properties of ApKTI, a Kunitz trypsin inhibitor from Adenanthera pavonina seeds, against P. interpunctella larvae through bioassays with artificial diet. ApKTI-fed larvae showed reduction of up to 88% on larval weight and 75% in survival. Trypsin enzymes extracted from P. interpunctella larvae were inhibited by ApKTI, which also demonstrated capacity to bind to chitin. Kinetic studies revealed a non-competitive inhibition mechanism of ApKTI for trypsin, which were further corroborated by molecular docking studies. Furthermore, we have demonstrated that ApKTI exhibits a hydrophobic pocket near the reactive site loop probably involved in chitin interactions. Taken together, these data suggested that the insecticidal activity of ApKTI for P. interpunctella larvae involves a dual and promiscuous mechanisms biding to two completely different targets. Both processes might impair the P. interpunctella larval digestive process, leading to larvae death before reaching the pupal stage. Further studies are encouraged using ApKTI as a biotechnological tool to control insect pests in field conditions

Study of the adaptive mechanism of the fall armyworm (Spodoptera frugiperda) against the plant peptidase inhibitors

Orientador: Maria Lígia Rodrigues MacedoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Inibidores de peptidases vegetais apresentam atividade inseticida para insetos de diferentes ordens. Entretanto, alguns insetos não apresentam os efeitos deletérios esperados ao se alimentarem com estas proteínas. A lagarta-do-cartucho (Spodoptera frugiperda), inseto-praga generalista, é capaz de sintetizar enzimas resistentes à inibição quando alimentada em dieta contendo inibidores enzimáticos vegetais. No intuito de estudar as respostas adaptativas da lagarta-do-cartucho contra inibidores de peptidases realizamos bioensaios utilizando o inibidor de tripsina purificado de sementes de Entada acaciifolia (EATI). Larvas de 6º instar cronicamente alimentadas com dieta contendo 0,5% de inibidor (p/p) apresentaram uma redução de 20% no peso médio. No entanto, nenhum outro parâmetro foi afetado nos demais estágios de desenvolvimento. A presença do inibidor em dieta provocou aumento da atividade enzimática das tripsinas e quimotripsinas digestivas. Estudos de PCR em tempo real quantitativo (qPCR) revelaram que o aumento da atividade enzimática ocorreu devido a transcrição diferencial de genes de tripsinas e quimotripsinas e ensaios bioquímicos revelaram que as tripsinas expressas possuem baixa afinidade por diferentes inibidores vegetais e pelo EATI. Interessantemente, quando as larvas são retiradas da dieta contento inibidor estas enzimas deixam de ser expressas. Estudos de western blot revelaram que EATI está majoritariamente presente no intestino médio e fezes das larvas alimentadas, em comparação aos demais tecidos analisados. Mostramos também que o tempo que o inibidor permanece no intestino de S. frugiperda é ligeiramente maior que o tempo normal de digestão do alimento. Através de ensaios de Imunofluorescência co-localizamos EATI junto a membrana peritrófica do intestino médio e corpo gorduroso. Baseado nestes resultados sugerimos que ao longo do processo evolutivo S. frugiperda adquiriu a capacidade de produzir enzimas resistentes à inibição por inibidores de diferentes famílias, o que explicaria seu hábito alimentar generalista. Verificamos também que as enzimas transcritas na presença do inibidor não tem a função de degradá-loAbstract: Plant peptidase inhibitors show insecticide activity for insects from different orders. However, some insects do not show the negative effects through feeding experiments. The fall armyworm (Spodoptera frugiperda), generalist insect-pest, is able to express resistant enzymes after feeding in diet containing plant inhibitors. In order to evaluate the adaptive responses of the fall armyworm against peptidase inhibitors we carried out bioassays incorporating the trypsin inhibitor from Entada acaciifolia seeds (EATI) into artificial diet. Sixth-instar larvae fed with EATI 0.5% (w/w) showed reduction on larval weight of 20%. Nevertheless, any other evaluated parameter was affected in further development steps. The EATI-dietary triggered the increase of trypsin and chymotrypsin activities. qPCR studies revealed that the increase in enzymatic activity is related to differential transcription of trypsin and chymoytypsin genes and biochemical studies revealed that these trypsins are resistant to inhibition by several plant inhibitors and by EATI. Interestingly, when the EATI is removed from diet the differential transcription is interrupted. Western blot studies showed that EATI is majorly present in midgut and feaces in comparison with other analyzed tissues. The time of contact between the inhibitor and the gut is extended in comparison with normal food. Immunofluorescence assays collocated EATI in the same position of peritrophic membrane and fatty body. Based in these results, we suggested that along the evolution S. frugiperda evolved the capability to synthetize wide-range resistant enzymes, explicating it polyphagia. The enzymes transcribed during adaptive response are no involved with inhibitor degradationDoutoradoBioquimicaDoutor em Biologia Funcional e MolecularCNPQFAPES

Functional and structural aspects of trypsin inhibitor from Entada acaciifolia

Orientadores: Maria Lígia Rodrigues Macedo, Ricardo AparícioDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Diversas pesquisas buscam candidatos para o controle alternativo de pragas agrícolas, uma vez conhecidos os malefícios oriundos do emprego de inseticidas químicos nas lavouras. Neste trabalho, um inibidor de tripsina isolado de sementes de Entada acaciifolia (Mimosoideae) - (EATI) foi purificado e caracterizado sob o ponto de vista funcional e estrutural. O inibidor mostrou-se estável a variações térmicas e de pH, alem de apresentar sua atividade inibitória inalterada tanto na forma oxidada quanto na forma reduzida com 100 mM de DTT. Quando submetido a cromatografia liquida de alta eficiência em coluna C-18, EATI foi resolvido em 4 picos, indicando a presença de 4 isoformas, sendo que a isoforma majoritária apresentou uma massa acurada de 19.725 Daltons, revelada através de espectrometria de massa MALDI-TOF. Outras características como a estequiometria de inibição, a constante de inibição (Ki), a analise da composição global de aminoácidos e o sequenciamento N-terminal permitiram classificar EATI como membro da família Kunitz de inibidores de serinoprotease. Estudos de dicroísmo circular revelaram um alto conteúdo de fohas-? e estruturas não ordenadas alem da aparente ausência de ?-helices, padrão comum a esta classe de proteínas. A incubação em 1 mM de DTT resultou em perturbações em sua cadeia polipeptídica, corroborando os ensaios in vitro. EATI foi resistente a desnaturação por uréia, característica relatada para outros inibidores vegetais. O inibidor apresentou uma interessante atividade inibitória in vitro contra as proteases de diversos insetos-praga. De acordo com estes resultados, apontamos EATI como uma ferramenta promissora no combate a pragas agrícolasAbstract: Several studies seeking alternative candidates for the control of agricultural pests, once known harm from the use of chemical insecticides in crops. In this work, a trypsin inhibitor isolated from Entada acaciifolia seeds (Mimosoideae) - (EATI) was purified and characterized from the point of view unctional and structural. The inhibitor was stable to thermal heat and pH range, in addition to its inhibitory activity unchanged in both the oxidized and in reduced form with 100 mM DTT. When subjected to high performance liquid chromatography on C-18 column, EATI was resolved in four peaks, indicating the presence of four isoforms, with the major isoform showing an accurate mass of 19,725 Daltons, as revealed by MALDI-TOF mass spectrometry. Other characteristic such as stoichiometry of inhibition, the dissociation constant (Ki), the analysis of global amino acid composition and N-terminal sequencing allowed to classify EATI as member of the Kunitz family of serine protease inhibitors. Circular dichroism studies revealed a high content of ?- sheets and unordered structures beyond the apparent absence of ?-helix, typical of this class of proteins. Incubation in 1 mM DTT resulted in small perturbations in their polypeptide chain, corroborate the in vitro assays. EATI was resistant to denaturation by urea, a characteristic reported for other plant inhibitors. The inhibitor presented an interesting inhibitory activity in vitro against the proteases of several insect pests. According to these results, we focused EATI as a promising tool in the fight against agricultural pestsMestradoBioquimicaMestre em Biologia Funcional e Molecula

Purification and characterization of a Kunitz inhibitor from Poincianella pyramidalis with insecticide activity against the Mediterranean flour moth

CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFINEP - FINANCIADORA DE ESTUDOS E PROJETOSFUNDECT - FUNDAÇÃO DE APOIO AO DESENVOLVIMENTO DO ENSINO, CIÊNCIA E TECNOLOGIA DO ESTADO DO MATO GROSSO DO SULThis paper describes the characterization of a trypsin inhibitor from Poincianella pyramidalis seeds (PpyTI). The partial sequencing of PpyTI revealed homology to Kunitz inhibitors, clustered as a member of Family 103 in MEROPS database. PpyTI has a single polypeptide chain of 19,042 Da and presents stability at high temperatures (up to 70 degrees C) and a wide range of pH. In vitro assays showed that disulfide bridges have an important stabilization role of reactive site in PpyTI, a characteristic shared among several Kunitz inhibitors. Bioassays carried out with the Mediterranean flour moth (Anagasta kuehniella) revealed a significant decrease in both larval weight and survival of PpyTI-fed larvae, besides a larval stage extension. Through biochemical analysis, we demonstrated that the PpyTI insecticide effects were triggered by digestion process commitment, through the inhibition of trypsin and chymotrypsin activities, the major digestive enzymes in this species. The insecticide effects and biochemical characterization of PpyTI encourage further studies using this inhibitor for insect pest control. (C) 2014 Elsevier Inc. All rights reserved.This paper describes the characterization of a trypsin inhibitor from Poincianella pyramidalis seeds (PpyTI). The partial sequencing of PpyTI revealed homology to Kunitz inhibitors, clustered as a member of Family 103 in MEROPS database. PpyTI has a single polypeptide chain of 19,042 Da and presents stability at high temperatures (up to 70 degrees C) and a wide range of pH. In vitro assays showed that disulfide bridges have an important stabilization role of reactive site in PpyTI, a characteristic shared among several Kunitz inhibitors. Bioassays carried out with the Mediterranean flour moth (Anagasta kuehniella) revealed a significant decrease in both larval weight and survival of PpyTI-fed larvae, besides a larval stage extension. Through biochemical analysis, we demonstrated that the PpyTI insecticide effects were triggered by digestion process commitment, through the inhibition of trypsin and chymotrypsin activities, the major digestive enzymes in this species. The insecticide effects and biochemical characterization of PpyTI encourage further studies using this inhibitor for insect pest control11819CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFINEP - FINANCIADORA DE ESTUDOS E PROJETOSFUNDECT - FUNDAÇÃO DE APOIO AO DESENVOLVIMENTO DO ENSINO, CIÊNCIA E TECNOLOGIA DO ESTADO DO MATO GROSSO DO SULCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFINEP - FINANCIADORA DE ESTUDOS E PROJETOSFUNDECT - FUNDAÇÃO DE APOIO AO DESENVOLVIMENTO DO ENSINO, CIÊNCIA E TECNOLOGIA DO ESTADO DO MATO GROSSO DO SULsem informaçãosem informaçãosem informaçã

Purification and biochemical properties of a Kunitz-type trypsin inhibitor from Entada acaciifolia (Benth.) seeds

A new trypsin inhibitor (EATI) was isolated from Entada acaciifolia (Benth.) seeds. EATI is a competitive inhibitor with a molecular mass of 20 kDa and an inhibition stoichiometry of 1:1 for bovine trypsin. The dissociation constant (K-i) calculated was 1.75 nmol/L, displaying a high affinity between enzyme and inhibitor. Both Native PAGE and RP-HPLC revealed that EATI is composed of four isoinhibitors that share the amino acid composition and the amino-terminal sequence homolog to Kunitz-type inhibitors. EATI is stable to denaturation by heat (up to 70 degrees C), pH (2-10), urea (8 mol/L) and its inhibitory activity was unaltered in different concentrations of DTT (up to 100 mmol/L). CD analysis revealed that EATI in reduced form underwent structural modifications associated with a decrease in thermal and pH stabilities, suggesting that their disulfide bonds are not involved in the structuring of its reactive site, but are important for maintenance of its conformational stability. This behavior makes EATI one of the few inhibitors described in the literature with high DTT resistance476929935FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPFUNDAÇÃO DE APOIO AO DESENVOLVIMENTO DO ENSINO, CIÊNCIA E TECNOLOGIA DO ESTADO DE MATO GROSSO DO SUL - FUNDECT2011/09361-0sem informaçã

Evaluation of the Synthetic Multifunctional Peptide Hp-MAP3 Derivative of Temporin-PTa

In recent years, antimicrobial peptides isolated from amphibian toxins have gained attention as new multifunctional drugs interacting with different molecular targets. We aimed to rationally design a new peptide from temporin-PTa. Hp-MAP3 (NH2-LLKKVLALLKKVL-COOH), net charge (+4), hydrophobicity (0.69), the content of hydrophobic residues (69%), and hydrophobic moment (0.73). For the construction of the analog peptide, the physicochemical characteristics were reorganized into hydrophilic and hydrophobic residues with the addition of lysines and leucines. The minimum inhibitory concentration was 2.7 to 43 μM against the growth of Gram-negative and positive bacteria, and the potential for biofilm eradication was 173.2 μM. Within 20 min, the peptide Hp-MAP3 (10.8 μM) prompted 100% of the damage to E. coli cells. At 43.3 μM, eliminated 100% of S. aureus within 5 min. The effects against yeast species of the Candida genus ranged from 5.4 to 86.6 μM. Hp-MAP3 presents cytotoxic activity against tumor HeLa at a concentration of 21.6 μM with an IC50 of 10.4 µM. Furthermore, the peptide showed hemolytic activity against murine erythrocytes. Structural studies carried out by circular dichroism showed that Hp-MAP3, while in the presence of 50% trifluoroethanol or SDS, an α-helix secondary structure. Finally, Amphipathic Hp-MAP3 building an important model for the design of new multifunctional molecules

Purification and biochemical properties of a Kunitz-type trypsin inhibitor from Entada acaciifolia (Benth.) seeds

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)A new trypsin inhibitor (EATI) was isolated from Entada acaciifolia (Benth.) seeds. EATI is a competitive inhibitor with a molecular mass of 20 kDa and an inhibition stoichiometry of 1:1 for bovine trypsin. The dissociation constant (K-i) calculated was 1.75 nmol/L, displaying a high affinity between enzyme and inhibitor. Both Native PAGE and RP-HPLC revealed that EATI is composed of four isoinhibitors that share the amino acid composition and the amino-terminal sequence homolog to Kunitz-type inhibitors. EATI is stable to denaturation by heat (up to 70 degrees C), pH (2-10), urea (8 mol/L) and its inhibitory activity was unaltered in different concentrations of DTT (up to 100 mmol/L). CD analysis revealed that EATI in reduced form underwent structural modifications associated with a decrease in thermal and pH stabilities, suggesting that their disulfide bonds are not involved in the structuring of its reactive site, but are important for maintenance of its conformational stability. This behavior makes EATI one of the few inhibitors described in the literature with high DTT resistance. (C) 2012 Elsevier Ltd. All rights reserved.476929935Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Foundation to Support the Development of Education, Science and Technology of State of Mato Grosso do Sul (FUNDECT - Fundacao de Apoio ao Desenvolvimento do Ensino, Ciencia e Tecnologia do Estado de Mato Grosso do Sul)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Novel Peptidase Kunitz Inhibitor from <i>Platypodium elegans</i> Seeds Is Active against <i>Spodoptera frugiperda</i> Larvae

A novel Kunitz-type inhibitor from <i>Platypodium elegans</i> seeds (PeTI) was purified and characterized. The mass spectrometry analyses of PeTI indicated an intact mass of 19 701 Da and a partial sequence homologous to Kunitz inhibitors. PeTI was purified by ion exchange and affinity chromatographies. A complex with a 1:1 ratio was obtained only for bovine trypsin, showing a <i>K</i>_i = 0.16 nM. Stability studies showed that PeTI was stable over a wide range of temperature (37–80 °C) and pH (2–10). The inhibitory activity of PeTI was affected by dithiothreitol (DTT). Bioassays of PeTI on <i>Spodoptera frugiperda</i> showed negative effects on larval development and weight gain, besides extending the insect life cycle. The activities of digestive enzymes, trypsin and chymotrypsin, were reduced by feeding larvae with 0.2% PeTI in an artificial diet. In summary, we describe a novel Kunitz inhibitor with promising biotechnological potential for pest control