RESUMEN

The angiotensin-converting enzyme (ACE) plays a key role in blood pressure regulation process, and its inhibition is one of the main drug targets for the treatment of hypertension. Though various peptides from milk proteins are well-known for their ACE-inhibitory capacity, research devoted to understand the molecular bases of such property remain scarce, specifically for such peptides. Therefore, in this work, computational molecular docking and molecular dynamics calculations were performed to enlighten the intermolecular interactions involved in ACE inhibition by six different casein-derived peptides (FFVAPFPEVFGK, FALPQYLK, ALNEINQFYQK, YLGYLEQLLR, HQGLPQEVLNENLLR and NAVPITPTLNR). Two top ranked docking poses for each peptide (one with N- and the other C-terminal peptide extremity oriented towards the ACE active site) were selected for dynamic simulations (50 ns; GROMOS53A6 force field), and the results were correlated to in vitro ACE inhibition capacity. Two molecular features appeared to be essential for peptides to present high ACE inhibition capacity in vitro: i) to interact with the S1 active site residues (Ala354, Glu384, and Tyr523) by hydrogen bonds; ii) to interact with Zn2+ coordinated residues (His383, His387, and Glu411) by short-lenght hydrogen bonds, as observed in the cases of ALNEINQFYQK (IACE = 80.7%), NAVPITPTLNR (IACE = 80.7%), and FALPQYLK (IACE = 79.0%). Regardless of the temporal stability of these strong interactions, they promoted some disruption of Zn2+ tetrahedral coordination during the molecular dynamics trajectories, and were pointed as the main reason for the greatest ACE inhibition by these peptides. On the other hand, peptides with intermediate inhibition capacity (50% < IACE < 45%) interacted mainly by weaker interactions (e.g.: electrostatic and hydrophobic) with the Zn2+ coordinated residues, and were not able to change significantly its tetrahedral coordination structure. These findings may: i) assist the discrimination in silico of "good" and "bad" ACE-inhibitory peptides from other food sources, and/or ii) aid in designing de novo new molecules with ACE-inhibitory capacity. Communicated by Ramaswamy Sarma.

Asunto(s)

Inhibidores de la Enzima Convertidora de Angiotensina , Caseínas , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Angiotensinas , Animales , Bovinos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Péptidos , Peptidil-Dipeptidasa A/metabolismo

RESUMEN

BACKGROUND: Endo-1,4-ß-xylanases have marked hydrolytic activity towards arabinoxylans. Xylanases (xynA) produced by the anaerobic fungus Orpinomyces sp. strain PC-2 have been shown to be superior in specific activity, which strongly suggests their applicability in the bakery industry for the processing of whole-wheat flour containing xylans. In the present study, two xylanases from this source, the small wild-type xylanase SWT and the small mutant xylanase SM2 (V108A, A199T), were expressed in Escherichia coli, purified, characterized, tested for their ability to hydrolyze whole-wheat flour and applied in dough processing. RESULTS: Both purified SM2 and SWT showed high specific activity against oat spelt xylan and wheat arabinoxylan, exhibiting maximum activity at pH 3-7 and 60 °C. SM2 was more thermostable than SWT, which suggests that the mutations enhanced its stability. Both SWT and SM2 were able to hydrolyze whole-wheat flour, and evaluation of their applicability in dough processing by the sponge method indicated that use of these enzymes increased dough volume by 60% and reduced texture hardness by more than 50%, while gumminess and chewiness were reduced by 40%. CONCLUSION: The recombinant xylanases showed potential for application in bakery processing and can improve techno-functional properties in sponges. © 2018 Society of Chemical Industry.

Asunto(s)

Endo-1,4-beta Xilanasas/química , Proteínas Fúngicas/química , Neocallimastigales/enzimología , Triticum/química , Biocatálisis , Pan/análisis , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/metabolismo , Harina/análisis , Manipulación de Alimentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Neocallimastigales/genética , Ingeniería de Proteínas , Xilanos/química

RESUMEN

This review is focused on the state-of-art of peptides with inhibitory activity towards angiotensin I-converting enzyme (ACE) - thus, with anti-hypertensive potential - derived from enzymatic hydrolysis of caseins. Firstly, molecular characteristics of caseins relevant to a better understanding of this subject were concisely commented. Next, a brief description of the pathophysiology of hypertension was explained, focusing on the ACE role in regulation of blood pressure in human body. Then, casein-derived peptides with ACE inhibitory capacity were specifically addressed. The main in vitro and in vivo bioassays often reported in literature to assess the anti-hypertensive potential of peptides were presented, illustrated with recently published studies, and discussed in terms of advantages and limitations of both approaches. Characteristics related to amino acid composition and sequence of peptides with high ACE-inhibitory potential were also commented. Process parameters of enzymatic hydrolysis (types and origins of casein substrates, types of enzymes, pH, temperature, and times of reactions) were discussed. Patents dealing with casein-derived anti-hypertensive peptides were examined not only in terms of amino acid sequences, but also regarding their novelty claims in hydrolysis process parameters. Finally, some trends, challenges, and opportunities inferred from this literature analysis were commented, emphasizing the importance of this research topic in food products development.

Asunto(s)

Inhibidores de la Enzima Convertidora de Angiotensina/química , Caseínas/química , Manipulación de Alimentos , Peptidil-Dipeptidasa A , Animales , Bovinos , Humanos , Hidrólisis

RESUMEN

Combination of ß-lactoglobulin (ß-Lg) and lactoferrin (Lf), biomacromolecules derived from bovine whey, was used in the formation of supramolecular structures by thermal gelation technique to adjust the pH. Furthermore, the influence of the molar ratio, temperature, pH, and heating time in the formation of supramolecular structures were also studied. The characterization of the protein supramolecular structures was performed using dynamic light scattering, zeta potential measurements, molecular spectrofluorimetry, and circular dichroism spectroscopy. The thermal behavior of the pure proteins was investigated by differential scanning calorimetry. The protein denaturation temperatures were of around 85°C for the ß-Lg and around 52°C and 85°C (a small portion) for the Lf. The protein molar ratio of 2:1 Lf/ß-Lg was used to form the structures, whose characterization showed that the best conditions of supramolecular structure formation occurred at pH6.5 and at temperatures of 62.5°C. In those conditions, more stable systems with reduced hydrophobic surface and average sizes between 30 and 100nm were generated. The correlation between pH and temperature suggests that the method of preparation of the supramolecular structure affects its size during storage.

Asunto(s)

Lactoferrina/química , Lactoferrina/metabolismo , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Animales , Rastreo Diferencial de Calorimetría , Bovinos , Calor , Interacciones Hidrofóbicas e Hidrofílicas , Lactoferrina/análisis , Lactoglobulinas/análisis , Complejos Multiproteicos/análisis , Estabilidad Proteica , Análisis Espectral

RESUMEN

The products formed by glycosylation of food proteins with carbohydrates via the Maillard reaction, also known as conjugates, are agents capable of changing and improving techno-functional characteristics of proteins. The Maillard reaction uses the covalent bond between a group of a reducing carbohydrates and an amino group of a protein. This reaction does not require additional chemicals as it occurs naturally under controlled conditions of temperature, time, pH, and moisture. Moreover, there is growing interest in modifying proteins for industrial food applications. This review analyses the current state of art of the Maillard reaction on food protein functionalities. It also discusses the influence of the Maillard reaction on the conditions and formulation of reagents that improve desirable techno-functional characteristics of food protein.

Asunto(s)

Proteínas en la Dieta/química , Alimentos , Reacción de Maillard , Polisacáridos/química , Fenómenos Químicos , Carbohidratos de la Dieta , Glicosilación , Calor

RESUMEN

Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.

Asunto(s)

Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Niacinamida/análogos & derivados , Piperidinas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Antineoplásicos/química , Antineoplásicos/metabolismo , Sitios de Unión , Western Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regulación Leucémica de la Expresión Génica , Células HL-60 , Células HeLa , Humanos , Células Jurkat , Células K562 , Leucemia/genética , Leucemia/metabolismo , Leucemia/patología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Niacinamida/química , Niacinamida/metabolismo , Niacinamida/farmacología , Piperidinas/química , Piperidinas/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Fluorescencia

RESUMEN

Peptides inhibiting the activity of angiotensin converting enzyme (ACE) were obtained by trypsin-catalyzed hydrolysis of bovine milk casein, performed at 37°C, during 1, 2, 5, 8 and 24h. Results of in vitro inhibitory activity ranged between 13.4% and 78.5%. The highest ACE inhibitory activity was evidenced for hydrolysates obtained after 2h of reaction. Aqueous two-phase systems (ATPS) formed by polyethylene glycol of 1500gmol-1 (PEG 1500)+sodium phosphate or potassium phosphates were produced and evaluated, in terms of partition coefficients (K) and extraction yields (y), to recovery the casein hydrolysates at room temperature. In ATPS containing sodium phosphate, the peptides showed a slightly greater affinity toward the bottom salt-rich phase (0.1≤K≤0.9; 5.7%≤y≤47%). In the case of ATPS containing potassium phosphates, these molecules showed substantially greater affinity toward the top polymer-rich phase (137≤K≤266; y≥99%). These results point out extraction using PEG 1500/potassium phosphate ATPS is an efficient technique to recover casein hydrolysates containing ACE inhibitors peptides. Outlined data will be helpful in integrating such unit operation to larger scale processes.