RESUMO
Proteolytic enzymes are widely distributed in nature, playing essential roles in important biological functions. Recently, the use of plant proteases at the industrial level has mainly increased in the food industry (e.g., cheesemaking, meat tenderizing, and protein hydrolysate production). Current technological and scientific advances in the detection and characterization of proteolytic enzymes have encouraged the search for new natural sources. Thus, this work aimed to explore the milk-clotting and proteolytic properties of different tissues of Vallesia glabra. Aqueous extracts from the leaves, fruits, and seeds of V. glabra presented different protein profiles, proteolytic activity, and milk-clotting activity. The milk-clotting activity increased with temperature (30-65 °C), but this activity was higher in leaf (0.20 MCU/mL) compared with that in fruit and seed extracts (0.12 and 0.11 MCU/mL, respectively) at 50 °C. Proteolytic activity in the extracts assayed at different pH (2.5-12.0) suggested the presence of different types of active proteases, with maximum activity at acidic conditions (4.0-4.5). Inhibitory studies indicated that major activity in V. glabra extracts is related to cysteine proteases; however, the presence of serine, aspartic, and metalloproteases was also evident. The hydrolytic profile of caseins indicated that V. glabra leaves could be used as a rennet substitute in cheesemaking, representing a new and promising source of proteolytic enzymes.
Assuntos
Apocynaceae/enzimologia , Leite/química , Peptídeo Hidrolases/química , Folhas de Planta/enzimologia , Proteínas de Plantas/química , Proteólise , Sementes/enzimologia , Animais , Concentração de Íons de HidrogênioRESUMO
The objective of this study was to produce vinegar from mangaba pulp using semi-solid alcoholic fermentation combined with the enzymatic activity of pectinase and to investigate the chemical composition and sensory characteristics of the final product. was evaluated for volatile acidity and the reduced dry extract was evaluated for ashes, alcohol content, sulfates, pH, total phenolic compounds, total carotenoids, color parameters, yield, productivity, and sensory analysis. Average and standard deviation was used for descriptive statistics. Principal component analysis (PCA) was applied to all variables except total carotenoid content. Physicochemical characterization of the raw and alcoholically fermented pulp was also carried out. The main results showed that, in the vinegar, the reduced dry extract, volatile acidity, pH, and ashes were 44.3±1.5 (g/L), 4.4±0.1 (% w/v), 3.1±0.0, and 3.0±0.41 (g/L), respectively. The total phenolic compound content and total carotenoid content for the mangaba vinegar were 19.2±8.20. mg/100 g and 2.6±0.6. mg/100 g, respectively. The conversion yield from ethanol to acetic acid was 90%. PCA showed that pH and volatile acidity had a strong influence on the product, and there was a strong positive correlation between color and aroma. The final product met all legal requirements, showing that it is possible to produce mangaba vinegar with antioxidant potential for consumers. In the sensory evaluation, it was favored by the tasters, demonstrating potential economic value in the Cerrado fruit.(AU)
Objetivou-se produzir vinagre, a partir da polpa de mangaba por fermentação alcoólica semi-sólida com ação enzimática através da pectinase, investigar a composição química e avaliação sensorial do produto final. O vinagre foi avaliado através da acidez volátil, extrato seco reduzido, cinzas, teor alcoólico, sulfatos, pH, compostos fenólicos totais, carotenoides totais, parâmetros de cor, rendimento, produtividade e análise sensorial. Os dados foram submetidos a estatística descritiva com média e desvio padrão. Foi aplicado a análise de componentes principais (ACP) para todas as variáveis, exceto para análise de carotenoides totais. Também foi realizada a caracterização físico-química da polpa e fermentado alcoólico. Os principais resultados mostraram que, no vinagre, extrato seco reduzido, acidez volátil, pH e cinzas foram, respectivamente, 44,3±1,5 (g/L), 4,4±0,1 (% m/v), 3,1±0,0, 3,0±0,41 (g/L). Os compostos fenólicos totais e carotenoides totais apresentaram valores de 19,2±8,20 (mg/100 g) e 2,6±0,6 (mg/100 g), respectivamente. O rendimento de conversão de etanol a ácido acético foi de 90%. ACP foi aplicada nas variáveis físico-químicas do vinagre no qual os parâmetros de cor, pH e acidez volátil apresentaram forte influência no produto e, para os atributos da análise sensorial, cor e aroma apresentaram uma forte correlação positiva entre si. O produto final atendeu a todos os quesitos legais, demonstrando ser possível a produção de vinagre de mangaba com potencial antioxidante. Na avaliação sensorial teve boa aceitação pelos provadores, valorizando o uso deste fruto do Cerrado.(AU)
Assuntos
Apocynaceae/química , Apocynaceae/enzimologia , Sucos de Frutas e Vegetais/análiseRESUMO
The objective of this study was to produce vinegar from mangaba pulp using semi-solid alcoholic fermentation combined with the enzymatic activity of pectinase and to investigate the chemical composition and sensory characteristics of the final product. was evaluated for volatile acidity and the reduced dry extract was evaluated for ashes, alcohol content, sulfates, pH, total phenolic compounds, total carotenoids, color parameters, yield, productivity, and sensory analysis. Average and standard deviation was used for descriptive statistics. Principal component analysis (PCA) was applied to all variables except total carotenoid content. Physicochemical characterization of the raw and alcoholically fermented pulp was also carried out. The main results showed that, in the vinegar, the reduced dry extract, volatile acidity, pH, and ashes were 44.3±1.5 (g/L), 4.4±0.1 (% w/v), 3.1±0.0, and 3.0±0.41 (g/L), respectively. The total phenolic compound content and total carotenoid content for the mangaba vinegar were 19.2±8.20. mg/100 g and 2.6±0.6. mg/100 g, respectively. The conversion yield from ethanol to acetic acid was 90%. PCA showed that pH and volatile acidity had a strong influence on the product, and there was a strong positive correlation between color and aroma. The final product met all legal requirements, showing that it is possible to produce mangaba vinegar with antioxidant potential for consumers. In the sensory evaluation, it was favored by the tasters, demonstrating potential economic value in the Cerrado fruit.
Objetivou-se produzir vinagre, a partir da polpa de mangaba por fermentação alcoólica semi-sólida com ação enzimática através da pectinase, investigar a composição química e avaliação sensorial do produto final. O vinagre foi avaliado através da acidez volátil, extrato seco reduzido, cinzas, teor alcoólico, sulfatos, pH, compostos fenólicos totais, carotenoides totais, parâmetros de cor, rendimento, produtividade e análise sensorial. Os dados foram submetidos a estatística descritiva com média e desvio padrão. Foi aplicado a análise de componentes principais (ACP) para todas as variáveis, exceto para análise de carotenoides totais. Também foi realizada a caracterização físico-química da polpa e fermentado alcoólico. Os principais resultados mostraram que, no vinagre, extrato seco reduzido, acidez volátil, pH e cinzas foram, respectivamente, 44,3±1,5 (g/L), 4,4±0,1 (% m/v), 3,1±0,0, 3,0±0,41 (g/L). Os compostos fenólicos totais e carotenoides totais apresentaram valores de 19,2±8,20 (mg/100 g) e 2,6±0,6 (mg/100 g), respectivamente. O rendimento de conversão de etanol a ácido acético foi de 90%. ACP foi aplicada nas variáveis físico-químicas do vinagre no qual os parâmetros de cor, pH e acidez volátil apresentaram forte influência no produto e, para os atributos da análise sensorial, cor e aroma apresentaram uma forte correlação positiva entre si. O produto final atendeu a todos os quesitos legais, demonstrando ser possível a produção de vinagre de mangaba com potencial antioxidante. Na avaliação sensorial teve boa aceitação pelos provadores, valorizando o uso deste fruto do Cerrado.
Assuntos
Apocynaceae/enzimologia , Apocynaceae/química , Sucos de Frutas e Vegetais/análiseRESUMO
OBJECTIVE: To determine the enzymatic properties of asclepain f, a plant cysteine protease isolated and purified from the latex of Asclepias fruticosa, and to investigate its potential application to hydrolyze soybean proteins. RESULTS: Kinetic parameters were determined by hydrolysis of p-Glu-Phe-Leu-p-nitroanilide (PFLNA). The Km value for asclepain f was 6 to 8 times higher than those achieved for papain, bromelain and ficin, the main plant cysteine proteases. Asclepain f showed 12 cut-off points toward the oxidized B chain insulin, revealing that the enzyme possesses broad substrate specificity. The cut specificity was governed by the presence of hydrophobic residues (F, L, V) in the P2 position. Asclepain f was able to selectively hydrolyze soybean proteins at pH 10, employing an enzyme/substrate ratio of 0.2% (w/w). The enzymatic hydrolysis allowed a strong increase in the solubility, water and oil holding capacity. CONCLUSIONS: Asclepain f was revealed as a successful enzyme for biocatalysis of protein hydrolysis processes at alkaline pH. This new plant protease has a broad substrate specificity and is capable of selectively degrading the fractions of soy proteins and improving its functional properties.
Assuntos
Apocynaceae/enzimologia , Cisteína Proteases/metabolismo , Proteínas de Soja/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Proteólise , Especificidade por SubstratoRESUMO
The present study evaluated four laticifer fluids as a novel source of peptidases capable of hydrolyzing proteins in cow's milk. The latex peptidases from Calotropis procera (CpLP), Cryptostegia grandiflora (CgLP), and Carica papaya (CapLP) were able to perform total hydrolysis of caseins after 30â¯min at pH 6.5, as confirmed by a significant reduction in the residual antigenicity. Casein hydrolysis by Plumeria rubra latex peptidases (PrLP) was negligible. Moreover, whey proteins were more resistant to proteolysis by latex peptidases; however, heat pretreatment of the whey proteins enhanced the degree of hydrolysis and reduced the residual antigenicity of the hydrolysates. The in vivo assays show that the cow's milk proteins hydrolysed by CgLP and CapLP exhibited no immune reactions in mice allergic to cow's milk, similar to a commercial partially hydrolysed formula. Thus, these peptidases are promising enzymes for the development of novel hypoallergenic formulas for children with a milk allergy.
Assuntos
Caseínas/metabolismo , Hipersensibilidade a Leite/patologia , Peptídeo Hidrolases/metabolismo , Animais , Apocynaceae/enzimologia , Calotropis/enzimologia , Carica/enzimologia , Caseínas/imunologia , Bovinos , Humanos , Hidrólise , Látex/metabolismo , Masculino , Camundongos , Leite/metabolismo , Hipersensibilidade a Leite/imunologia , Hipersensibilidade a Leite/veterinária , Proteínas do Soro do Leite/imunologia , Proteínas do Soro do Leite/metabolismoRESUMO
A espécie Hancornia speciosa Gomes é uma frutífera nativa do Brasil sobre a qual tem crescido o interesse por parte da indústria de polpas, sucos e sorvetes. A exploração do fruto da mangabeira é dificultada pela sua reduzida vida útil pós-colheita. A perda rápida das características apreciáveis desse fruto está associada à atividade de enzimas oxidativas como a peroxidase (POD) e a polifenoloxidase (PPO). Neste trabalho foi quantificada a atividade dessas duas enzimas na polpa de frutos de mangaba submetida a três temperaturas de refrigeração (6, 10 e 18°C) em função de diferentes tempos de armazenamento (0, 3, 6, 9, 12 e 15 dias). Os níveis de PDO na polpa dos frutos de mangaba foram sempre superiores aos de PPO para as três temperaturas testadas, para todos os tempos de armazenamento analisados. Analisando-se cada enzima em particular, não foi detectada diferença na atividade de POD ou de PPO entre as temperaturas testadas e ao desdobrar-se o tempo dentro de cada temperatura testada, foi encontrada uma correlação de 0,035, para a atividade de PPO.(AU)
The species Hancornia speciosa is a native fruit of Brazil over which it has been growing interest by industry of the pulps, juices and ice cream. The exploration of the fruit of mangabeira is hampered by their limited shelf-life. The rapid loss of the appreciable features of this fruit is associated with the activity of oxidative enzymes such as peroxidase and polyphenol oxidase. In this study we quantified activity of these two enzymes in the fruits pulp of the mangaba subjected to three refrigeration temperatures (6, 10 and 18 º C) for different storage times (0 ,3, 6, 9, 12 and 15 days). Levels of PDO in the pulp mangaba were always higher than the PPO for the three temperatures tested, for ali storage periods analyzed. Analyzing each particular enzyme, no difference was detected in the activity of POD and PPO between the temperatures tested and unfold the time within each temperature tested, found a correlation of 0.035 for the PPO activity.(AU)
Assuntos
Armazenamento de Alimentos , Conservação de Alimentos/métodos , Temperatura , Apocynaceae/enzimologia , Polifenóis/metabolismo , Sucos de Frutas e Vegetais , Ativação Enzimática , OxidaçãoRESUMO
Araujiain aII, the protease with highest specific activity purified from latex of Araujia angustifolia (Apocynaceae), shows optimum proteolytic activity at alkaline pH, and it is completely inhibited by the irreversible inhibitor of cysteine proteases trans-epoxysucciny-L: -leucyl-amido(4-guanidino) butane. It exhibits esterolytic activity on several N-α-Cbz-amino acid p-nitrophenyl esters with a preference for Gln, Ala, and Gly derivatives. Kinetic enzymatic assays were performed with the thiol proteinase substrate p-Glu-Phe-Leu-p-nitroanilide (K (m) = 0.18 ± 0.03 mM, k (cat) = 1.078 ± 0.055 s(-1), k (cat)/K (m) = 5.99 ± 0.57 s(-1) mM(-l)). The enzyme has a pI value above 9.3 and a molecular mass of 23.528 kDa determined by mass spectrometry. cDNA of the peptidase was obtained by reverse transcription-PCR starting from total RNA isolated from latex. The deduced amino acid sequence was confirmed by peptide mass fingerprinting analysis. The N-terminus of the mature protein was determined by automated sequencing using Edman's degradation and compared with the sequence deduced from cDNA. The full araujiain aII sequence was thus obtained with a total of 213 amino acid residues. The peptidase, as well as other Apocynaceae latex peptidases, is a member of the subfamily C1A of cysteine proteases. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was suggested by molecular modeling.
Assuntos
Apocynaceae/metabolismo , Cisteína Proteases/química , Cisteína Proteases/metabolismo , Látex/química , Sequência de Aminoácidos , Apocynaceae/enzimologia , Apocynaceae/genética , Sequência de Bases , Clonagem Molecular , Cisteína Proteases/genética , Cisteína Proteases/isolamento & purificação , DNA Complementar/genética , Frutas/enzimologia , Frutas/genética , Frutas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Ponto Isoelétrico , Cinética , Modelos Químicos , Dados de Sequência Molecular , Peso Molecular , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
Two cysteine endopeptidases from latex of Araujia angustifolia (araujiain aI and araujiain aIII) were purified and characterized by means of conventional and proteomics techniques (MALDI-TOF). N-terminal sequences showed a high percentage of identity with cysteine proteinases belonging to the papain family. The peptide mass fingerprint analysis demonstrated a close homology among both proteinases.
Assuntos
Apocynaceae/enzimologia , Cisteína Proteases/química , Papaína/química , Mapeamento de Peptídeos/métodos , Proteínas de Plantas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Biologia Computacional/métodos , Cisteína Proteases/isolamento & purificação , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/química , Ésteres/metabolismo , Frutas/enzimologia , Concentração de Íons de Hidrogênio , Látex/química , Papaína/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismoRESUMO
A crude extract rich in plant cysteine peptidases was obtained from the latex of the fruits of Araujia hortorum, a South American climbing plant. The highly concentrated extract was immobilized onto titanium dioxide to produce biocatalysts through a simple adsorption procedure. Absorbance measurement at 280 nm and Bradford's method for protein quantification revealed that the protein content of the crude extract was selectively adsorbed onto the titanium dioxide surface at a very high rate. In 5 min of contact with the support all protein present in the crude extract was selectively withdrawn from the solution, leading to an immobilized biocatalyst with a high protein concentration. Caseinolytic assays indicated that, except for the catalyst obtained with the highest crude amount contacted with the support, all the proteolytic activity present in the crude extract was adsorbed onto TiO(2). The amidasic activity of the immobilized catalysts (Ah/TiO(2)) was tested in the hydrolysis of a synthetic chromogenic substrate (PFLNA) showing partial deactivation with respect to the native enzyme. In amidasic activity assays the ionic strength of the buffer medium showed to be a key feature to consider in order to avoid protease desorption from the support, indicating the importance of electrostatic interactions between the enzymes and TiO(2). Reuse of the produced biocatalysts with PFLNA as substrate revealed that after five successive uses Ah/TiO(2) retained more than 20% of its initial activity.
Assuntos
Apocynaceae/enzimologia , Cisteína Endopeptidases/metabolismo , Titânio/metabolismo , Adsorção , Amidoidrolases/metabolismo , Biocatálise , Caseínas/metabolismo , Enzimas Imobilizadas/metabolismo , Peso Molecular , Extratos Vegetais/metabolismo , Soluções , Espectrofotometria Ultravioleta , Fatores de TempoRESUMO
In this paper we studied the effect of different organic solvents (1-octanol, trichloroethylene, ethanol, ethyl acetate, tetrahydrofuran, cyclohexane, propanone, acetonitrile, dichloromethane, chlorobenzene, N,N-dimethylformamide, acetophenone, diethyl ether, methanol, ethylene glycol and toluene) with low and constant water content on substrate preferences, thermostability and stability (caseinolytic activity retention after 4 h) of proteases of Araujia hortorum Fourn. (Asclepiadaceae). The stability of araujiain was high in N,N-dimethylformamide and ethanol at 40 ºC, but decreased at higher temperature. Araujiain substrates preferences in buffer Tris-HCl (pH 8), ethylene glycol and N,N-dimethylformamide exhibited different patterns, but the enzyme showed a high preference by glutamine derivative in all cases. According to FTIR spectroscopy studies, araujiain changed its secondary structure and as a consequence, it also changed its substrate preferences. This enzyme showed lower beta-helical character and greater beta-sheet folding in buffer than in organic media. A larger amount of antiparallel beta-sheet residues indicates the formation of tighter intermolecular hydrogen bonds and enzymatic aggregates. These facts could explain the higher esterolytic activities, the greater stability and good hydrolytic potential of araujiain in some organic media such as N,N-dimethylformamide.
Assuntos
Apocynaceae/enzimologia , Cisteína Endopeptidases/metabolismo , Frutas/enzimologia , Solventes , Catálise , Caseínas/metabolismo , Estabilidade Enzimática , Espectroscopia de Infravermelho com Transformada de Fourier , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/metabolismo , Especificidade por Substrato , Temperatura , ÁguaRESUMO
A new papain-like cysteine peptidase isolated from latex of Philibertia gilliesii Hook. et Arn., Apocynaceae (formerly Asclepiadaceae) has been purified and characterized. The enzyme, named philibertain g I, is the most basic component present in latex extracts and was purified by acetone fractionation followed by cation exchange chromatography (SP-Sepharose HR) using FPLC system. Homogeneity was confirmed by SDS-PAGE and mass spectroscopy (MS). Molecular mass of the enzyme was 23,530 Da (MALDI-TOF MS), its isoelectric point was >10.25, and maximum proteolytic activity (casein) was achieved at pH 7-8. The new protease was inhibited by E-64 a cysteine peptidases inhibitor. Km was 0.15 mM, using PFLNA as substrate. The N-terminal sequence of philibertain g I (LPASVDWRKEGAVLPIRHQGQCG) was compared with those of twenty plant proteases. Philibertain g I showed the higher degree of identity (73%) with caricain, one of the Carica papaya endopepetidases.
Assuntos
Apocynaceae , Cisteína Endopeptidases/isolamento & purificação , Látex/química , Proteínas de Plantas/isolamento & purificação , Sequência de Aminoácidos , Animais , Apocynaceae/química , Apocynaceae/enzimologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Alinhamento de SequênciaRESUMO
As part of a screening of latex endopeptidases from plants growing in Argentina, the presence of proteolytic activity in the latex of Funastrum clausum stems is reported. The proteases present in the crude extract showed the main characteristics of the cysteine proteolytic class, i.e. optimum pH at alkaline range, isoelectric point (pI) higher than 9.0, and inhibition of proteolytic activity by thiol blocking reagents. A remarkable thermal stability was also evident in the crude extract. Endosterolytic preference tried on p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids was higher for the alanine, asparagine and tyrosine derivatives. Preliminary peptidase purification by two-step ionic exchange showed the presence of two proteolytic fractions with molecular masses of approximately 24.0 kDa according to SDS-PAGE.
Assuntos
Apocynaceae/enzimologia , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/farmacologia , Látex/química , Fitoterapia , Argentina , Cisteína Endopeptidases/isolamento & purificação , Humanos , Medicina Tradicional , Caules de PlantaRESUMO
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 degrees 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-alpha-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.
Assuntos
Apocynaceae/enzimologia , Cisteína Endopeptidases/metabolismo , Látex/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Cinética , Látex/química , Espectrometria de Massas , Fatores de TempoRESUMO
A new cysteine endopeptidase (morrenain b I) has been purified and characterized from the latex of stems and petiols of Morrenia brachystephana Griseb. (Asclepiadaceae). Morrenain b I was the minor proteolytic component in the latex but showed higher specific activity than morrenain b II, which was the main active fraction. Both enzymes showed similar pH profiles and molecular masses, but kinetic parameters and N-terminal sequences were quite distinct, demonstrating that they are different enzymes instead of different forms of the same enzyme.
Assuntos
Apocynaceae/enzimologia , Cisteína Endopeptidases/química , Papaína/química , Sequência de Aminoácidos , Cisteína Endopeptidases/isolamento & purificação , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Látex/química , Dados de Sequência Molecular , Peso Molecular , Especificidade por Substrato , TemperaturaRESUMO
Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 microg of protein/mL and the proteolytic activity reached 1.2 caseinolytic U/mL. This enzyme preparation was very stable even after 2 hours at 45 degrees C, but was quickly inactivated after 5 minutes at 80 degrees C. Chromatographic purification was achieved by FPLC using a cation exchanger (SP-Sepharose FF). Thus, a unique proteolitically active fraction could be isolated, being homogeneous by bidimensional electrophoresis and mass spectrometry (Mr = 23,652). The optimum pH range was achieved at 8.5-10.5. The enzyme activity was completely inhibited by specific cysteine peptidases inhibitors. Isoelectric focusing followed by zymogram showed the enzyme had a pI greater than 9.3. The N-terminus sequence (LPDSVDWREKGVVFPIRNQGK) shows a great deal of similarity to those of the other cysteine endopeptidases isolated from latices of Asclepiadaceae even when a high degree of homology could be observed with other plant cysteine endopeptidases.