RESUMO
New approaches are needed to reduce risks to the environment and natural enemies and to avoid or delay the onset of insecticide resistance. The use of insecticides based on proteinase inhibitors of hemolymph is an alternative for the control of Lepidoptera pests primarily by having low toxicity and short persistence in the environment. Thus, in this study, we describe the purification process and identification of protease inhibitors from hemolymph Anticarsia gemmatalis and their activities against trypsin enzymes. Furthermore, the three-dimensional (3D) structure of the inhibitor and binding mode to trypsin enzymes was determined, and the stability of the inhibitory activity in several pHs and temperature values was evaluated. The inhibitor was characterized as a serpin family inhibitor and named A. gemmatalis hemolymph serpin inhibitor (AHSI), with an approximate mass of 38 ± 2 kDa, highly stable to temperature and pH variations, and with inhibitory capacity on bovine trypsin and gut trypsin of A. gemmatalis demonstrated by calculated Ki values and affinity energy through molecular docking, being a reversible competitive inhibitor that binds to the active site of trypsin-like enzymes. We conclude that the AHSI inhibitor identified from the hemolymph of the soybean pest A. gemmatalis preserves the original structure of the serpin family with a good overall stereochemical quality confirmed from molecular modeling. The docking analysis showed that the reactive site of the inhibitor is in contact with the catalytic cavity of the trypsin with high-affinity energy.
Assuntos
Lepidópteros , Mariposas , Animais , Bovinos , Hemolinfa , Larva , Simulação de Acoplamento Molecular , Inibidores de Proteases , Glycine max , TripsinaRESUMO
Drought is one of the major constraints for soybean production in Brazil. In this study we investigated the physiological traits of two soybean parental genotypes under progressive soil drying and rewetting. The plants were evaluated under full irrigation (control) conditions and under water deficit imposed by suspending irrigation until the plants reached predawn leaf water potentials (Ψam) of -1.0 MPa (moderate) and -1.5 MPa (severe). Physiological analyses showed that these genotypes exhibit different responses to water deficit. The Embrapa 48 genotype reached moderate and severe water potential two days after the BR16 genotype and was able to maintain higher levels of A, ETR and ΦPSII even under deficit conditions. This result was not related to changes in gs, 13C isotopic composition and presence of a more efficient antioxidant system. In addition, Fv/Fm values did not decrease in Embrapa 48 genotype in relation to irrigated condition showing that stress was not causing photochemical inhibition of photosynthesis. The greater reduction in the relative growth of the shoots, with concomitant greater growth of the root system under drought, indicates that the tolerant genotype is able to preferentially allocated carbon to the roots, presenting less damage to photosynthesis. Therefore, the physiological responses revealed that the tolerant genotype postponed leaf dehydration by a mechanism involving a more efficient use and translocation of water from root to shoot to maintain cell homeostasis and photosynthetic metabolism under stress.
Assuntos
Secas , Glycine max/fisiologia , Estresse Fisiológico , Brasil , Genótipo , Fotossíntese , Folhas de Planta/fisiologia , Raízes de Plantas/fisiologia , Água/fisiologiaRESUMO
Conidia of entomopathogenic fungi can penetrate the insect exoskeleton both by mechanic action of the germinative tube and by production of multiple proteases, chitinases, and lipases in response to the composition of the insect cuticle. Therefore the purpose of this work was to produce, to purify, and to characterize the structure of the proteases produced by the fungus Beauveria bassiana CG432, previously activated in adults of coffee berry borer (Hypothenemus hampei) grown under submerged culture conditions. A suspension containing 106 activated conidia/mL was inoculated to a culture medium at 28ºC and 150 rpm for 3 days. Protease extracts, were obtained by centrifugation at 8000g for 20 minutes, were fractionated and were concentrated by ultrafiltration using controlled porosity membranes of 100 kDa and 3 kDa, respectively. Gel filtration chromatography on Sephadex G-100 isolated one protein peak (peak II), which contained 56% of residues of the aminoacid aspartic acid, characterized by HPLC using an ODS-C18 reverse phase column. The peak protein showed specific activity 43 times superior when compared to the free-cell extract in bovine serum albumin, subtilisin-like protease activity, and a single protein band reveled by Brilliant Coomassie Blue on a gelatin zimogram PAGE electrophoresis, by native conditions. The homogeneity of peak II was confirmed by revelation of a sin
Conídios de fungos entomopatogênicos atravessam o exoesqueleto do inseto pela ação mecânica do tubo germinativo e produção de múltiplas isoformas de proteases, quitinases e lipases em resposta à composição da cutícula do inseto. Desta forma o objetivo deste trabalho foi extrair, purificar e caracterizar a estrutura de proteases produzidas em cultivo submerso por Beauveria bassiana CG432 previamente ativada em adultos vivos de broca-do-café (Hypothenemus hampei). Uma suspensão contendo 106 conídios ativados/mL foi inoculada em meio de cultura líquido a 28ºC, 150 rpm por 3 dias. O extrato de proteases (EP) foi obtido da centrifugação a 8000 g por 20 minutos, fracionado e concentrado por ultrafiltração em membrana de porosidade controlada 100 kDa e 3 kDa, respectivamente. A cromatografia de gel filtração em Sephadex G-100 separou um pico proteico (Pico II) que apresentou 56% de resíduos do aminoácido ácido aspártico quando analisado por HPLC em coluna de fase reversa ODS-C18; atividade específica 43 vezes superior ao EP sobre soro albumina bovina; atividade de protease tipo-subtilisina e uma única banda proteica revelada por nitrato de prata e Coomassie Brilhant Blue em zimograma sobre gelatina por eletroforese PAGE em condições nativas. A homogeneidade do Pico II foi confirmada pela revelação de uma única banda durante a determinação do pH isoelétrico igual a 4,5, porém a determi
RESUMO
Conidia of entomopathogenic fungi can penetrate the insect exoskeleton both by mechanic action of the germinative tube and by production of multiple proteases, chitinases, and lipases in response to the composition of the insect cuticle. Therefore the purpose of this work was to produce, to purify, and to characterize the structure of the proteases produced by the fungus Beauveria bassiana CG432, previously activated in adults of coffee berry borer (Hypothenemus hampei) grown under submerged culture conditions. A suspension containing 106 activated conidia/mL was inoculated to a culture medium at 28ºC and 150 rpm for 3 days. Protease extracts, were obtained by centrifugation at 8000g for 20 minutes, were fractionated and were concentrated by ultrafiltration using controlled porosity membranes of 100 kDa and 3 kDa, respectively. Gel filtration chromatography on Sephadex G-100 isolated one protein peak (peak II), which contained 56% of residues of the aminoacid aspartic acid, characterized by HPLC using an ODS-C18 reverse phase column. The peak protein showed specific activity 43 times superior when compared to the free-cell extract in bovine serum albumin, subtilisin-like protease activity, and a single protein band reveled by Brilliant Coomassie Blue on a gelatin zimogram PAGE electrophoresis, by native conditions. The homogeneity of peak II was confirmed by revelation of a sin
Conídios de fungos entomopatogênicos atravessam o exoesqueleto do inseto pela ação mecânica do tubo germinativo e produção de múltiplas isoformas de proteases, quitinases e lipases em resposta à composição da cutícula do inseto. Desta forma o objetivo deste trabalho foi extrair, purificar e caracterizar a estrutura de proteases produzidas em cultivo submerso por Beauveria bassiana CG432 previamente ativada em adultos vivos de broca-do-café (Hypothenemus hampei). Uma suspensão contendo 106 conídios ativados/mL foi inoculada em meio de cultura líquido a 28ºC, 150 rpm por 3 dias. O extrato de proteases (EP) foi obtido da centrifugação a 8000 g por 20 minutos, fracionado e concentrado por ultrafiltração em membrana de porosidade controlada 100 kDa e 3 kDa, respectivamente. A cromatografia de gel filtração em Sephadex G-100 separou um pico proteico (Pico II) que apresentou 56% de resíduos do aminoácido ácido aspártico quando analisado por HPLC em coluna de fase reversa ODS-C18; atividade específica 43 vezes superior ao EP sobre soro albumina bovina; atividade de protease tipo-subtilisina e uma única banda proteica revelada por nitrato de prata e Coomassie Brilhant Blue em zimograma sobre gelatina por eletroforese PAGE em condições nativas. A homogeneidade do Pico II foi confirmada pela revelação de uma única banda durante a determinação do pH isoelétrico igual a 4,5, porém a determi