Fitoproteasas de aplicación en la industria alimentaria: purificación y caracterización de proteinasas de Asclepias fruticosa L.

Objetivos de la investigación: - Aislar, purificar y caracterizar nuevas peptidasas presentes en plantas que crecen en nuestro país. - Aplicar estas nuevas fitopeptidasas para hidrolizar parcialmente proteínas alimenticias con el objeto de mejorar sus propiedades funcionales. - Contar con nuevas fuentes de enzimas proteolíticas que eventualmente reemplacen a las preparaciones comerciales importadas que actualmente se usan en biotecnología.Centro de Investigación de Proteínas Vegetale

Biochemical analysis of a papain-like protease isolated from the latex of Asclepias curassavica L.

Most of the species belonging to Asclepiadaceae family usually secrete an endogenous milk-like fluid in a network of laticifer cells in which sub-cellular organelles intensively synthesize proteins and secondary metabolites. A new papain-like endopeptidase (asclepain cII) has been isolated and characterized from the latex extracted from petioles of Asclepias curassavica L. (Asclepiadaceae). Asclepain cII was the minor proteolytic component in the latex, but showed higher specific activity than asclepain cI, the main active fraction previously studied. Both enzymes displayed quite distinct biochemical characteristics, confirming that they are different enzymes. Crude extract was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry, were isolated. Asclepain cII displayed a molecular mass of 23,590 Da, a pI higher than 9.3, maximum proteolytic activity at pH 9.4-10.2, and showed poor thermostability. The activity of asclepain cII is inhibited by cysteine proteases inhibitors like E-64, but not by any other protease inhibitors such as 1,10-phenantroline, phenylmethanesulfonyl fluoride, and pepstatine. The N-terminal sequence (LPSFVDWRQKGVVFPIRNQGQCGSCWTFSA) showed a high similarity with those of other plant cysteine proteinases. When assayed on N-α-CBZ-amino acid-p-nitrophenyl esters, the enzyme exhibited higher preference for the glutamine derivative. Determinations of kinetic parameters were performed with N-α-CBZ-l-Gln-p-nitrophenyl ester as substrate: Km = 0.1634 mM, kcat = 121.48 s-1, and kcat/Km = 7.4 × 105 s-1/mM.Centro de Investigación de Proteínas VegetalesFacultad de Ciencias Exacta

Papain purification insights: monitoring by electrophoretic approaches and MALDI-TOF peptide mass fingerprint analyses

Papain was purified from dried Carica papaya latex by fractioned salt precipitation in presence of sodium tetrathionate to preserve enzymatic activity. Purification was followed by different electrophoretic methods. Identification of the purified product was afforded by submitting the peptides obtained by tryptic digestion of papain to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS) analysis. Comparison of the peptide masses analyzed by peptide mass fingerprinting (PMF) MALDI-TOF and those obtained by theoretical tryptic digestion, revealed the presence of some peptides belonging the other three endopeptidases contained in papaya latex (very similar to papain in molecular weight and pI) in the purified fraction of papain. The PMF by MALDI-TOF could be applied as a method to follow papain purification.Centro de Investigación de Proteínas Vegetale

Papain purification insights: monitoring by electrophoretic approaches and MALDI-TOF peptide mass fingerprint analyses

Papain was purified from dried Carica papaya latex by fractioned salt precipitation in presence of sodium tetrathionate to preserve enzymatic activity. Purification was followed by different electrophoretic methods. Identification of the purified product was afforded by submitting the peptides obtained by tryptic digestion of papain to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS) analysis. Comparison of the peptide masses analyzed by peptide mass fingerprinting (PMF) MALDI-TOF and those obtained by theoretical tryptic digestion, revealed the presence of some peptides belonging the other three endopeptidases contained in papaya latex (very similar to papain in molecular weight and pI) in the purified fraction of papain. The PMF by MALDI-TOF could be applied as a method to follow papain purification.Centro de Investigación de Proteínas Vegetale

Papain purification insights: monitoring by electrophoretic approaches and MALDI-TOF peptide mass fingerprint analyses

Papain was purified from dried Carica papaya latex by fractioned salt precipitation in presence of sodium tetrathionate to preserve enzymatic activity. Purification was followed by different electrophoretic methods. Identification of the purified product was afforded by submitting the peptides obtained by tryptic digestion of papain to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS) analysis. Comparison of the peptide masses analyzed by peptide mass fingerprinting (PMF) MALDI-TOF and those obtained by theoretical tryptic digestion, revealed the presence of some peptides belonging the other three endopeptidases contained in papaya latex (very similar to papain in molecular weight and pI) in the purified fraction of papain. The PMF by MALDI-TOF could be applied as a method to follow papain purification.Centro de Investigación de Proteínas Vegetale

Plasticity in the Oxidative Folding Pathway of the High Affinity Nerita Versicolor Carboxypeptidase Inhibitor (NvCI)

Nerita Versicolor carboxypeptidase inhibitor (NvCI) is the strongest inhibitor reported so far for the M14A subfamily of carboxypeptidases. It comprises 53 residues and a protein fold composed of a two-stranded antiparallel β sheet connected by three loops and stabilized by three disulfide bridges. Here we report the oxidative folding and reductive unfolding pathways of NvCI. Much debate has gone on whether protein conformational folding guides disulfide bond formation or instead they are disulfide bonds that favour the arrangement of local or global structural elements. We show here that for NvCI both possibilities apply. Under physiological conditions, this protein folds trough a funnelled pathway involving a network of kinetically connected native-like intermediates, all sharing the disulfide bond connecting the two β-strands. In contrast, under denaturing conditions, the folding of NvCI is under thermodynamic control and follows a "trial and error" mechanism, in which an initial quasi-stochastic population of intermediates rearrange their disulfide bonds to attain the stable native topology. Despite their striking mechanistic differences, the efficiency of both folding routes is similar. The present study illustrates thus a surprising plasticity in the folding of this extremely stable small disulfide-rich inhibitor and provides the basis for its redesign for biomedical applications.Instituto Multidisciplinario de Biología Celula

Propiedades funcionales de proteínas de soja parcialmente hidrolizadas empleando la peptidasa de Asclepias fruticosa

La harina de soja es el subproducto más relevante en el procesamiento de esta leguminosa y contiene un alto contenido de proteínas. La hidrólisis controlada de dichas proteínas puede generar ingredientes de alto valor añadido, con mejores características nutricionales y propiedades funcionales. La preparación proteolítica denominada Af, obtenida por centrifugación del látex de Asclepias fruticosa (Asclepiadaceae) recolectado en buffer cítrico-citrato, posee una única peptidasa cisteínica de PM 23,7 kDa y pI>9,3 denominada asclepaína f, la cual ha sido clonada y expresada en Pichia pastoris. La preparación Af posee una actividad proteolítica de 1,1 Ucas/ml, alta estabilidad térmica a 37° y 45°C, un rango de pH óptimo de actividad entre 7,5 y 10,5 y ha demostrado capacidad para hidrolizar las proteínas de soja, degradando fuertemente la fracción 7S y en menor medida las globulinas 11S. El objetivo del trabajo fue evaluar las propiedades funcionales (solubilidad, capacidad de retención de agua y aceite, y espumado) de un hidrolizado de proteínas de soja obtenido con la preparación Af. Se utilizó una dispersión de concentrado de soja (conteniendo 62,1 % de proteínas determinadas por Kjeldhal) al 8% p/v y una cantidad de extracto enzimático equivalente a 8,9 Ucas por gramo de proteína, con el agregado de cisteína 20 mM. La hidrólisis se llevó a cabo a diferentes valores de pH (8, 9 y 10) durante 180 minutos a 45ºC. La actividad enzimática se detuvo por calentamiento en microondas, las suspensiones fueron centrifugadas y los pellets secados en estufa. Se realizó un blanco de reacción en las mismas condiciones, reemplazando la preparación enzimática por igual volumen de agua. Posteriormente, se determinó la concentración de proteínas solubles mediante el método de Bradford, la capacidad de retención de agua a diferentes temperaturas (5, 20 y 45°C), la retención de aceite de soja y girasol, y la formación de espuma. La capacidad de retención de agua se ensayó mezclando 1 gr de muestra con 10 ml de agua destilada a diferentes temperaturas, centrifugando las suspensiones y determinando el peso de las muestras húmedas; del mismo modo se determinó la capacidad de retención de aceite. Para ensayar la capacidad espumante se prepararon suspensiones al 1% p/v de los hidrolizados, se agitaron durante 1 minuto a temperatura ambiente y posteriormente se midió el volumen de espuma y de suspensión. El mayor grado de hidrólisis de las proteínas de soja con la preparación Af se obtuvo a pH 10 y fue del 7%. A través de dicho proceso de hidrólisis se logró incrementar la concentración de proteínas solubles un 60%, la capacidad de retención de agua a 20°C se incrementó un 71% y a 45°C un 134%, mientras que la capacidad de retención de aceite de soja a 20°C aumentó un 92% y la de girasol 73,5%. En tanto que la capacidad espumante casi no fue afectada, se detectó solo un ligero aumento (3%). Empleando la preparación Af se logró obtener un hidrolizado de proteínas de soja con propiedades funcionales particulares que podrá emplearse en el desarrollo de alimentosInstituto Multidisciplinario de Biología Celula

Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of <i>Funastrum clausum</i>

A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70°C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0–10.0 for casein and 6.2–6.8 for PFLNA). Kinetic parameters were determined for N-α-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s–1) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s–1). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.Centro de Investigación de Proteínas Vegetale

Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of <i>Funastrum clausum</i>

A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70°C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0–10.0 for casein and 6.2–6.8 for PFLNA). Kinetic parameters were determined for N-α-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s–1) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s–1). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.Centro de Investigación de Proteínas Vegetale

Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of <i>Funastrum clausum</i>

A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70°C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0–10.0 for casein and 6.2–6.8 for PFLNA). Kinetic parameters were determined for N-α-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s–1) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s–1). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.Centro de Investigación de Proteínas Vegetale