RESUMEN
Functional screening of metagenomic libraries is an important tool for the discovery of new molecules. The metabolic diversity of microorganisms enables survival in harsh environments and is related to the production of enzymes. In this study, we identified a protease-producing clone from a metagenomic library derived from mangrove sediment. The protease was purified by ammonium sulphate precipitation and gel filtration chromatography, with a yield of 77.27% and a specific activity of 8.57 U µg-1 . It had a molecular weight of approximately 70 kDa. MS/MS in ESI-Q-TOF revealed nine peptides similar to a peptidase of Bacillus safensis. The aligned partial sequence showed 47.48% identity and 82.74% similarity to the conserved domains of a glutamyl aminopeptidase from the human gut metagenome and 32.12% total coverage. The protease had an optimal pH of 8.5 and optimal activity at 60°C. At pH 9-12, its activity was greater than 80%. It had moderate thermotolerance and thermostability at temperatures of 40 and 50 °C. The KM and Vmax values were estimated to be 0.92 mg ml-1 , and 13.15 mmol min-1 for azocasein. Substrate specificity analysis showed that PR4A3 was active on gelatin, blood, egg yolk, and milk. These results support the potential use of PR4A3 in biotechnological applications.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Endopeptidasas/química , Endopeptidasas/metabolismo , Sedimentos Geológicos/microbiología , Metagenómica , Humedales , Secuencia de Aminoácidos , Bacillus/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Biotecnología , Brasil , Cromatografía en Gel , Endopeptidasas/genética , Endopeptidasas/aislamiento & purificación , Activación Enzimática , Pruebas de Enzimas , Estabilidad de Enzimas , Calor , Concentración de Iones de Hidrógeno , Cinética , Metagenoma , Peso Molecular , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Péptido Hidrolasas/aislamiento & purificación , Sales (Química) , Alineación de Secuencia , Especificidad por Sustrato , Espectrometría de Masas en Tándem , TemperaturaRESUMEN
Proteins are abundant biomolecules found in human cells, as well as pathogenic bacteria and viruses. Some of them become pollutants when released into water. Adsorption is an advantageous method for separating proteins in aqueous media since proteins are already immobilized on solid surfaces. Adsorbents with surfaces rich in tannins are efficient due to their affinity for strong interactions with the various amino acids that make up proteins. This work aimed to develop an adsorbent for protein adsorption in aqueous medium using lignocellulosic materials modified from eucalyptus bark and vegetable tannins. A more efficient resin was prepared containing 10% eucalyptus bark fibers and 90% tannin mimosa by condensation with formaldehyde, and it was characterized by UV-Vis, FTIR-ATR spectroscopy and determinations of degree of swelling, bulk and bulk density and specific mass. For UV-Vis spectroscopy the percentage of condensed and hydrolysable tannins in the extracts of fibers of the dry husks of Eucalyptus Citriodora was estimated and it was also determined your soluble solids. The study of bovine serum albumin (BSA) adsorption was carried out in batch with quantification by UV-Vis spectroscopy. The most efficient prepared resin obtained 71.6 ± 2.78% removal in a solution of 260 mg L-1 of BSA working in a better pH range of the aqueous solution of BSA in its isoelectric point, ~ 5, 32 ± 0.02, under these conditions, the synthesized resin can reach a maximum BSA adsorption capacity of ~ 26.7 ± 0.29 mg g-1 in 7 min. The new synthesized resin presents good prospects for adsorption of proteins or species that in their structure have higher percentages of amino functional groups or amino acids with aliphatic, acidic and/or basic hydrophilic characteristics.
RESUMEN
This article addresses the interactions of the synthetic antimicrobial peptide dermaseptin 01 (GLWSTIKQKGKEAAIAAA- KAAGQAALGAL-NH(2) , DS 01) with phospholipid (PL) monolayers comprising (i) a lipid-rich extract of Leishmania amazonensis (LRE-La), (ii) zwitterionic PL (dipalmitoylphosphatidylcholine, DPPC), and (iii) negatively charged PL (dipalmitoylphosphatidylglycerol, DPPG). The degree of interaction of DS 01 with the different biomembrane models was quantified from equilibrium and dynamic liquid-air interface parameters. At low peptide concentrations, interactions between DS 01 and zwitterionic PL, as well as with the LRE-La monolayers were very weak, whereas with negatively charged PLs the interactions were stronger. For peptide concentrations above 1 µg/ml, a considerable expansion of negatively charged monolayers occurred. In the case of DPPC, it was possible to return to the original lipid area in the condensed phase, suggesting that the peptide was expelled from the monolayer. However, in the case of DPPG, the average area per lipid molecule in the presence of DS 01 was higher than pure PLs even at high surface pressures, suggesting that at least part of DS 01 remained incorporated in the monolayer. For the LRE-La monolayers, DS 01 also remained in the monolayer. This is the first report on the antiparasitic activity of AMPs using Langmuir monolayers of a natural lipid extract from L. amazonensis.
Asunto(s)
Proteínas Anfibias/química , Péptidos Catiónicos Antimicrobianos/química , Antiprotozoarios/química , Leishmania/efectos de los fármacos , Fosfolípidos/metabolismo , Secuencia de Aminoácidos , Proteínas Anfibias/farmacocinética , Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/farmacocinética , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Leishmania/metabolismo , Membranas Artificiales , Modelos BiológicosRESUMEN
Beta-amyloid peptide (Abeta) is a primary protein component of senile plaques in Alzheimer's disease (AD) and plays an important, but not fully understood role in neurotoxicity. Model peptides with the demonstrated ability to mimic the structural and toxicity behavior of Abeta could provide a means to evaluate the contributions to toxicity that are common to self-associating peptides from many disease states. In this work, we have studied the peptide-membrane interactions of a model beta-sheet peptide, P(11-2) (CH(3)CO-Gln-Gln-Arg-Phe-Gln-Trp-Gln-Phe-Glu-Gln-Gln-NH(2)), by fluorescence, infrared spectroscopy, and hydrogen-deuterium exchange. Like Abeta(1-40), the peptide is toxic, and conditions which produce intermediate oligomers show higher toxicity against cells than either monomeric forms or higher aggregates of the peptide. Further, P(11-2) also binds to both zwitterionic (POPC) and negatively charged (POPC:POPG) liposomes, acquires a partial beta-sheet conformation in presence of lipid, and is protected against deuterium exchange in the presence of lipids. The results show that a simple rationally designed model beta-sheet peptide recapitulates many important features of Abeta peptide structure and function, reinforcing the idea that toxicity arises, at least in part, from a common mode of action on membranes that is independent of specific aspects of the amino acid sequence. Further studies of such well-behaved model peptide systems will facilitate the investigation of the general principles that govern the molecular interactions of aggregation-prone disease-associated peptides with cell and/or membrane surfaces.
Asunto(s)
Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/toxicidad , Liposomas/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/toxicidad , Péptidos/química , Péptidos/toxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Medición de Intercambio de Deuterio , Humanos , Espectrometría de Masas , Fosfatidilgliceroles/química , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
A strategy that has been gaining increased application for the study of the conformation, dynamics, orientation, and physicochemical properties of peptides is labeling with the paramagnetic amino acid TOAC. This approach was used to gain a deeper understanding on the mechanism of action of the antimicrobial peptide tritrpticin (TRP3). TRP3 was labeled with TOAC at the N-terminus (prior to V1, TOAC0-TRP3) or internally (replacing P5, TOAC5-TRP3). Functional studies showed that labeling led to peptides with higher activity against Gram-positive bacteria and lower hemolytic activity with respect to TRP3. Peptide-induced model membranes permeabilization and ion channel-like activity studies corroborated the functional assays qualitatively, showing higher activity of the peptides against negatively charged membranes, which had the purpose of mimicking bacterial membranes. TOAC presented a greater freedom of motion at the N-terminus than at the internal position, as evinced by EPR spectra. EPR and fluorescence spectra reported on the peptides conformational properties, showing acquisition of a more packed conformation in the presence of the secondary structure-inducing solvent, TFE. CD studies showed that TOAC0-TRP3 acquires a conformation similar to that of TRP3, both in aqueous solution and in TFE, while TOAC5-TRP3 presents a different conformation in all environments. While the mechanism of action of TRP3 was impacted to some extent by TOAC labeling at the N-terminus, it did change upon replacement of P5 by TOAC. The results demonstrated that TOAC-labeling could be used to modulate TRP3 activity and mechanism of action and, more importantly, the critical role of P5 for TRP3 pore formation.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Óxidos N-Cíclicos/química , Oligopéptidos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/toxicidad , Membrana Celular/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Humanos , Micrococcus luteus/efectos de los fármacos , Oligopéptidos/farmacología , Oligopéptidos/toxicidadRESUMEN
This work combines experimental and theoretical approaches to investigate terahertz absorption spectra of the DNA formed by the sequence oligomer 5'-CCGGCGCCGG-3'. The three-dimensional structure of this self-complimentary DNA decamer has been well-studied, permitting us to perform direct identification of the low-frequency phonon modes associated with specific conformation and to conduct comprehensive computer simulations. Two modeling techniques, normal-mode analysis and nanosecond molecular dynamics with explicit solvent molecules, were employed to extract the low-frequency vibrational modes based on which the absorption spectra were calculated. The absorption spectra of the DNA decamer in aqueous solution were measured in the frequency range 10-25 cm(-1) using the terahertz Fourier transform infrared spectroscopy. Multiple well-resolved and reproducible resonance modes were observed. When calculated and experimental spectra were compared, the spectrum based on molecular dynamics simulations showed a better correlation with the experimental spectra than the one based on normal-mode analysis. These results demonstrate that there exist a considerable number of active low-frequency phonon modes in this short DNA duplex.
Asunto(s)
ADN/química , Oligodesoxirribonucleótidos/química , Absorción , Secuencia de Bases , Dicroismo Circular , Simulación por Computador , ADN/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/genéticaRESUMEN
Investigating interactions of designed peptide-based biomaterials with lipid membranes is important for applications in nanobiotechnology. Here the interaction of an ornithine-rich pH-responsive peptide called P11-5 with a model membrane was investigated employing Fourier transform infrared spectroscopy (FTIR). The results showed that in the range of pH 6.0-8.0 the peptide P11-5 shows spectral features which are evidence of the presence of peptides with antiparallel beta-sheet conformation (bands in the range 1625-1615 cm-1), as also spectral features which indicate the existence of random coil conformation (bands in the range 1640-1648 cm-1). Two types of membranes were used, 1,2- dipalmitoyl-sn-3-glycero-phosphocholine (DPPC) membranes with zwitterionic head groups and 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DPPG) membranes with anionic head groups. It was showed that there was a distinct peptide interaction under different pH values, pH 6.0, 7.0 and 8.0 for each type of lipid membrane with DPPC membrane preventing the peptide self-association even at basic pH, while for DPPG membrane there was a more evident peptide-lipid interaction. FTIR measurements indicate that in the presence of DPPC membrane the peptide was prevented to form beta-sheet aggregates at basic pH, while in the presence of DPPG membranes the self-association behavior of the peptide was more similar to its behavior when in aqueous solution in the absence of lipid membranes. Such results are important for the potential development of novel biomolecular nanostructured materials by the physico-chemical understanding of the peptide-lipid interactions.
Asunto(s)
Membrana Celular/metabolismo , Membrana Dobles de Lípidos/química , Ornitina/química , Péptidos/metabolismo , Concentración de Iones de Hidrógeno , Modelos Biológicos , Modelos Moleculares , Conformación Molecular , Nanotecnología , Péptidos/química , Unión Proteica , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Developing functional biointerfaces is important for technological applications. We investigated the interaction and adsorption of the antimicrobial peptide tritrpticin (VRRFPWWWPFLRR, TRP3) onto solid and liquid surfaces and the influence of ions on these processes by several techniques. Surface tension measurements showed that salt addition to TRP3 solution causes a high decrease of surface tension due to the adsorption of TRP3 at air-liquid surface. Ellipsometry studies show the TRP3 adsorption on silicon surfaces forming nanometric films that are able to further interact with liposomes. Contact angle measurements gave insight on the nature of thin film and its roughness. AFM shows the topology of the film on the solid substrates. In addition, those techniques also showed that anions can act as modulators on adsorption phenomena and are correlated with the Hofmeister series. The findings of the current work are relevant for the development of functional interfaces such as biocidal surfaces.
Asunto(s)
Antiinfecciosos/química , Oligopéptidos/química , Adsorción , Secuencia de Aminoácidos , Antiinfecciosos/metabolismo , Iones/química , Microscopía de Fuerza Atómica , Oligopéptidos/metabolismo , Electricidad Estática , Propiedades de SuperficieRESUMEN
The cationic peptide tritrpticin (VRRFPWWWPFLRR, Trp3) has a broad action spectrum, acting against Gram-positive and Gram-negative bacteria, as well as some fungi, while also displaying hemolytic activity. We have studied the behavior of Trp3 in planar lipid bilayers (or black lipid membrane - BLM) and were able to demonstrate its ion channel-like activity. Channel-like activity was observed in negatively charged azolectin BLM as a sudden appearance of discrete current fluctuations upon application of a constant voltage across the membrane. Trp3 formed large conductance channels (500-2000 pS) both at positive and negative potentials. In azolectin bilayers, the predominant ion-channel activity was characterized by very regular and discrete current steps (corresponding to openings) of uniform amplitude, which exhibited relatively long residence times (of the order of seconds). Occasionally, multiple conductance steps were observed, indicating the simultaneous presence of more than one open pore. In bilayers of zwitterionic diphytanoylphosphatidyl choline (DPhPC) Trp3 also showed ion-channel activity, but in a much less frequent and less prominent way. Studies of ion selectivity indicated that Trp3 forms a cation-selective channel. These results should contribute to the understanding of the molecular interactions and mechanism of action of Trp3 in lipid bilayers and biological membranes.
Asunto(s)
Antibacterianos/farmacología , Canales Iónicos/metabolismo , Membrana Dobles de Lípidos/química , Oligopéptidos/farmacología , Canales de Calcio/química , Cationes , Membrana Celular/metabolismo , Electrofisiología , Iones , Membrana Dobles de Lípidos/metabolismo , Lípidos/química , Modelos Químicos , Péptidos/química , Fosfatidilcolinas/química , Fosfolípidos/química , Canales Catiónicos TRPCRESUMEN
We examined the interaction of the cationic antimicrobial peptide (AMP) tritrpticin (VRRFPWWWPFLRR, TRP3) with Langmuir monolayers of zwitterionic (dipalmitoyl phosphatidylcholine, DPPC, and dipalmitoyl phosphatidylethanolamine, DPPE) and negatively charged phospholipids (dipalmitoyl phosphatidic acid, DPPA, and dipalmitoyl phosphatidylglycerol, DPPG). Both surface pressure and surface potential isotherms became more expanded upon addition of TRP3 (DPPE~DPPC<Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química
, Péptidos Catiónicos Antimicrobianos/química
, Oligopéptidos/química
, Ácidos Fosfatidicos/química
, Fosfatidiletanolaminas/química
, Fosfatidilgliceroles/química
, Antibacterianos/química
, Bacterias/química
, Membrana Celular/química
, Células Eucariotas/química
, Membranas Artificiales
, Modelos Moleculares
, Electricidad Estática
, Propiedades de Superficie
, Termodinámica