G-site residue S67 is involved in the fungicide-degrading activity of a tau class glutathione S-transferase from Carica papaya.

Thiram is a toxic fungicide extensively used for the management of pathogens in fruits. Although it is known that thiram degrades in plant tissues, the key enzymes involved in this process remain unexplored. In this study, we report that a tau class glutathione S-transferase (GST) from Carica papaya can degrade thiram. This enzyme was easily obtained by heterologous expression in Escherichia coli, showed low promiscuity toward other thiuram disulfides, and catalyzed thiram degradation under physiological reaction conditions. Site-directed mutagenesis indicated that G-site residue S67 shows a key influence for the enzymatic activity toward thiram, while mutation of residue S13, which reduced the GSH oxidase activity, did not significantly affect the thiram-degrading activity. The formation of dimethyl dithiocarbamate, which was subsequently converted into carbon disulfide, and dimethyl dithiocarbamoylsulfenic acid as the thiram degradation products suggested that thiram undergoes an alkaline hydrolysis that involves the rupture of the disulfide bond. Application of the GST selective inhibitor 4-chloro-7-nitro-2,1,3-benzoxadiazole reduced papaya peel thiram-degrading activity by 95%, indicating that this is the main degradation route of thiram in papaya. GST from Carica papaya also catalyzed the degradation of the fungicides chlorothalonil and thiabendazole, with residue S67 showing again a key influence for the enzymatic activity. These results fill an important knowledge gap in understanding the catalytic promiscuity of plant GSTs and reveal new insights into the fate and degradation products of thiram in fruits.

Evaluation of supplemented protein-L-isoaspartate-O-methyltransferase (PIMT) gene of Carica papaya and Ricinus communis in stress survival of Escherichia coli BL21(DE3) cells.

In growing plant population, effect of stress is a perturb issue affecting its physiological, biochemical, yield loss and developmental growth. Protein-L-isoaspartate-O-methyltransferase (PIMT) is a broadly distributed protein repair enzyme which actuate under stressful environment or aging. Stress can mediate damage converting protein bound aspartate (Asp) residues to isoaspartate (iso-Asp). This spontaneous and deleterious conversion occurs at an elevated state of stress and aging. Iso-Asp formation is associated with protein inactivation and compromised cellular survival. PIMT can convert iso-Asp back to Asp, thus repairing and contributing to cellular survival. The present work describes the isolation, cloning, sequencing and expression of PIMT genes of Carica papaya (Cp pimt) and Ricinus communis (Rc pimt) Using gene specific primers, both the pimts were amplified from their respective cDNAs and subsequently cloned in prokaryotic expression vector pProEXHTa. BL21(DE3) strain of E. coli cells were used as expression host. The expression kinetics of both the PIMTs were studied with various concentrations of IPTG and at different time points. Finally, the PIMT supplemented BL21(DE3) cells were evaluated against different stresses in comparison to their counterparts with the empty vector control.

Biochemical Properties and Anti-Biofilm Activity of Chitosan-Immobilized Papain.

Chitosan, the product of chitin deacetylation, is an excellent candidate for enzyme immobilization purposes. Here we demonstrate that papain, an endolytic cysteine protease (EC: 3.4.22.2) from Carica papaya latex immobilized on the matrixes of medium molecular (200 kDa) and high molecular (350 kDa) weight chitosans exhibits anti-biofilm activity and increases the antimicrobials efficiency against biofilm-embedded bacteria. Immobilization in glycine buffer (pH 9.0) allowed adsorption up to 30% of the total protein (mg g chitosan-1) and specific activity (U mg protein-1), leading to the preservation of more than 90% of the initial total activity (U mL-1). While optimal pH and temperature of the immobilized papain did not change, the immobilized enzyme exhibited elevated thermal stability and 6-7-fold longer half-life time in comparison with the soluble papain. While one-half of the total enzyme dissociates from both carriers in 24 h, this property could be used for wound-dressing materials design with dosed release of the enzyme to overcome the relatively high cytotoxicity of soluble papain. Our results indicate that both soluble and immobilized papain efficiently destroy biofilms formed by Staphylococcus aureus and Staphylococcus epidermidis. As a consequence, papain, both soluble and immobilized on medium molecular weight chitosan, is capable of potentiating the efficacy of antimicrobials against biofilm-embedded Staphylococci. Thus, papain immobilized on medium molecular weight chitosan appears a presumably beneficial agent for outer wound treatment for biofilms destruction, increasing antimicrobial treatment effectiveness.

Papain-like cysteine proteases in Carica papaya: lineage-specific gene duplication and expansion.

BACKGROUND: Papain-like cysteine proteases (PLCPs), a large group of cysteine proteases structurally related to papain, play important roles in plant development, senescence, and defense responses. Papain, the first cysteine protease whose structure was determined by X-ray crystallography, plays a crucial role in protecting papaya from herbivorous insects. Except the four major PLCPs purified and characterized in papaya latex, the rest of the PLCPs in papaya genome are largely unknown. RESULTS: We identified 33 PLCP genes in papaya genome. Phylogenetic analysis clearly separated plant PLCP genes into nine subfamilies. PLCP genes are not equally distributed among the nine subfamilies and the number of PLCPs in each subfamily does not increase or decrease proportionally among the seven selected plant species. Papaya showed clear lineage-specific gene expansion in the subfamily III. Interestingly, all four major PLCPs purified from papaya latex, including papain, chymopapain, glycyl endopeptidase and caricain, were grouped into the lineage-specific expansion branch in the subfamily III. Mapping PLCP genes on chromosomes of five plant species revealed that lineage-specific expansions of PLCP genes were mostly derived from tandem duplications. We estimated divergence time of papaya PLCP genes of subfamily III. The major duplication events leading to lineage-specific expansion of papaya PLCP genes in subfamily III were estimated at 48 MYA, 34 MYA, and 16 MYA. The gene expression patterns of the papaya PLCP genes in different tissues were assessed by transcriptome sequencing and qRT-PCR. Most of the papaya PLCP genes of subfamily III expressed at high levels in leaf and green fruit tissues. CONCLUSIONS: Tandem duplications played the dominant role in affecting copy number of PLCPs in plants. Significant variations in size of the PLCP subfamilies among species may reflect genetic adaptation of plant species to different environments. The lineage-specific expansion of papaya PLCPs of subfamily III might have been promoted by the continuous reciprocal selective effects of herbivore attack and plant defense.

Soluble expression of biologically active methionine sulfoxide reductase B1 (PaMsrB1) from Carica papaya in Escherichia coli and isolation of its protein targets.

Plant methionine sulfoxide reductase B1 (MsrB1) protects the photosynthetic apparatus from oxidative damage by scavenging reactive oxygen species to repair Met-oxidized proteins in response to abiotic stresses and biotic attack. Papaya MsrB1 (PaMsrB1) was identified previously to interact with papaya ringspot virus NIa-Pro, and this interaction inhibits the import of PaMsrB1 into the chloroplast. Further functional characterization of PaMsrB1 requires the production of a biologically active purified recombinant protein. In this report, PaMsrB1 as a fusion protein containing an N-terminal maltose-binding protein (MBP) was expressed in Escherichia coli Rosetta (DE3) cells and purified. Production of soluble fusion protein was greater when the cells were cultured at 16 °C than at 37 °C. The Factor Xa protease digested MBP-PaMsrB1 fusion protein and subsequently purified recombinant PaMsrB1 specifically reduced the R-diastereomer of methionine sulfoxide (MetSO) and Dabsyl-MetSO to Met in the presence of dithiothreitol. Eight chloroplast-localized and five non-chloroplast-localized candidate proteins that interact with PaMsrB1 were isolated by affinity chromatography and liquid chromatography coupled to tandem mass spectrometry. The results provide a platform to further understand the anti-oxidative defense mechanism of PaMsrB1.

Chemoenzymatic synthesis of new derivatives of glycyrrhetinic acid with antiviral activity. Molecular docking study.

We present an efficient approach to the synthesis of a series of glycyrrhetinic acid derivatives. Six derivatives, five of them new compounds, were obtained through chemoenzymatic reactions in very good to excellent yield. In order to find the optimal reaction conditions, the influence of various parameters such as enzyme source, nucleophile:substrate ratio, enzyme:substrate ratio, solvent and temperature was studied. The excellent results obtained by lipase catalysis made the procedure very efficient considering their advantages such as mild reaction conditions and low environmental impact. Moreover, in order to explain the reactivity of glycyrrhetinic acid and the acetylated derivative to different nucleophiles in the enzymatic reactions, molecular docking studies were carried out. In addition, one of the synthesized compounds exhibited remarkable antiviral activity against TK + and TK- strains of Herpes simplex virus type 1 (HSV-1), sensitive and resistant to acyclovir (ACV) treatment.

A wild origin of the loss-of-function lycopene beta cyclase (CYC-b) allele in cultivated, red-fleshed papaya (Carica papaya).

PREMISE OF THE STUDY: The red flesh of some papaya cultivars is caused by a recessive loss-of-function mutation in the coding region of the chromoplast-specific lycopene beta cyclase gene (CYC-b). We performed an evolutionary genetic analysis of the CYC-b locus in wild and cultivated papaya to uncover the origin of this loss-of-function allele in cultivated papaya. METHODS: We analyzed the levels and patterns of genetic diversity at the CYC-b locus and six loci in a 100-kb region flanking CYC-b and compared these to genetic diversity levels at neutral autosomal loci. The evolutionary relationships of CYC-b haplotypes were assessed using haplotype network analysis of the CYC-b locus and the 100-kb CYC-b region. KEY RESULTS: Genetic diversity at the recessive CYC-b allele (y) was much lower relative to the dominant Y allele found in yellow-fleshed wild and cultivated papaya due to a strong selective sweep. Haplotype network analyses suggest the y allele most likely arose in the wild and was introduced into domesticated varieties after the first papaya domestication event. The shared haplotype structure between some wild, feral, and cultivated haplotypes around the y allele supports subsequent escape of this allele from red cultivars back into wild populations through feral intermediates. CONCLUSIONS: Our study supports a protracted domestication process of papaya through the introgression of wild-derived traits and gene flow from cultivars to wild populations. Evidence of gene flow from cultivars to wild populations through feral intermediates has implications for the introduction of transgenic papaya into Central American countries.

A novel assay for the detection of anthelmintic activity mediated by cuticular damage to nematodes: validation on Caenorhabditis elegans exposed to cysteine proteinases.

Plant cysteine proteinases (CPs) from Carica papaya kill parasitic and free-living nematodes in vitro by hydrolysis of the worm cuticle, a mechanism that is different to all commercially available synthetic anthelmintics. We have developed a cheap and effective, rapid-throughput Caenorhabditis elegans-based assay for screening plant CP extracts for anthelmintic activity targeting cuticular integrity. The assay exploits colorimetric methodology for assessment of cuticular damage, and is based on the ability of viable cells to incorporate and bind Neutral red dye within lysosomes and to release the dye when damaged. Living worms are pre-stained with the dye, exposed to CPs and then leakage of the dye through the damaged cuticle is quantified by spectrophotometry. In contrast to motility assays and semi-subjective interpretation of microscopical images, this colorimetric assay is independent of observer bias. Our assay was applied to a series of C. elegans bus mutant strains with leaky cuticles and to cystatin knockout mutants. At ambient temperature and over 0.5-24 h, both bus mutants and the cystatin knockouts were highly susceptible to CPs, whereas wild-type Bristol N2 worms were essentially unstained by Neutral red and unaffected by CPs, providing validation for the utility of this assay.

Reverse micellar extraction of papain with cationic detergent based system: An optimization approach.

In this study, reverse micellar extraction of papain model system was performed using cetyltrimethylammonium bromide (CTAB)/iso-octane/hexanol/butanol system to optimize the forward and back extraction efficiency (BEE). A maximum forward extraction efficiency of 55.0, 61.0, and 54% was achieved with an aqueous phase pH of 11.0, 150 mM CTAB/iso-octane and 0.1 M NaCl, respectively. Taguchi's orthogonal array was applied to optimize the pH of stripping phase, concentration of isopropyl alcohol (IPA) and potassium chloride (KCl) for maximizing BEE. The optimal levels of stripping phase pH, concentration of IPA and KCl were found to be 6, 20% (v/v), and 0.8 M, respectively. Under these optimal levels, the BEE was found to be 88% after which enzyme activity was recovered with 2.5-fold purification. Further optimization was performed using artificial neural network-linked genetic algorithm, where the BEE was improved to 90.52% with pH 6, IPA (%) = 19.938, and KCl (M) = 0.729.

Biosynthesis of (S)-naproxen starch ester by Carica papaya lipase in intermittent opening reaction mode.

Inorder to brought S-naproxen into small intestine, an optically pure (S)-naproxen starch ester was produced by lipase through enantio-selective trans-esterification of racemic naproxen methyl ester with pretreatment starch in solvent system. With carefully selection of the reaction medium (isooctane), lipase (Carica Papaya Lipase, CPL) and the reaction mode (intermittent opening), a high conversion rate (48.6%) and enantiomeric excess of product (99.6%) was obtained. The slow release macromolecular (S)-Naproxen had been synthesized to improve the efficacy of racemic naproxen and overcome its side effects. The enanitomeric ratio of CPL (E=52.5) was higher than CRL (E=22) and greatly influenced by the byproduct methyl alcohol. The intermittent opening reaction mode was the effective way to remove the inhibition of methyl alcohol and to improve the enantio-selectivity of CPL. S-naproxen starch was confirmed by HPLC and 1H NMR. This method may also apply to preparation the other optically pure 2-phenylpropionic acid derivatives. S-naproxen starch was a new optically pure derivatives possessing emulsifying and slow release properties would be widely applied to the food, pharmaceutical and biomedical industries.

The anthelmintic efficacy of natural plant cysteine proteinases against the equine tapeworm, Anoplocephala perfoliata in vitro.

Papaya latex has been demonstrated to be an efficacious anthelmintic against murine, porcine, ovine and canine nematode parasites, and even those infecting poultry, and it has some efficacy against rodent cestodes. The active ingredients of papaya latex are known to be cysteine proteinases (CPs). The experiments described in this paper indicate that CPs in papaya latex, and also those in pineapples, are highly efficacious against the equine cestode Anoplocephala perfoliata in vitro, by causing a significant reduction in motility leading to death of the worms. The susceptibility of A. perfoliata to damage by CPs was considerably greater than that of the rodent cestodes Hymenolepis diminuta and H. microstoma. Our results are the first to report anthelmintic efficacy of CPs against an economically important equine helminth. Moreover, they provide further evidence that the spectrum of activity of CPs is not restricted to nematodes and support the idea that these plant-derived enzymes can be developed into useful broad-spectrum anthelmintics.

Comparison of proteases in DNA extraction via quantitative polymerase chain reaction.

We compared four proteases in the QIAamp DNA Investigator Kit (Qiagen) to extract DNA for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate alternate proteases for improved DNA recovery as compared with proteinase K for forensic, biochemical research, genetic paternity and immigration, and molecular diagnostic purposes. The Quantifiler Kit TaqMan quantitative PCR assay was used to measure the recovery of DNA from human blood, semen, buccal cells, breastmilk, and earwax in addition to low-template samples, including diluted samples, computer keyboard swabs, chewing gum, and cigarette butts. All methods yielded amplifiable DNA from all samples.

Host genetic influences on the anthelmintic efficacy of papaya-derived cysteine proteinases in mice.

Eight strains of mice, of contrasting genotypes, infected with Heligmosomoides bakeri were studied to determine whether the anthelmintic efficacy of papaya latex varied between inbred mouse strains and therefore whether there is an underlying genetic influence on the effectiveness of removing the intestinal nematode. Infected mice were treated with 330 nmol of crude papaya latex or with 240 nmol of papaya latex supernatant (PLS). Wide variation of response between different mouse strains was detected. Treatment was most effective in C3H (90·5-99·3% reduction in worm counts) and least effective in CD1 and BALB/c strains (36·0 and 40·5%, respectively). Cimetidine treatment did not improve anthelmintic efficacy of PLS in a poor drug responder mouse strain. Trypsin activity, pH and PLS activity did not differ significantly along the length of the gastro-intestinal (GI) tract between poor (BALB/c) and high (C3H) drug responder mouse strains. Our data indicate that there is a genetic component explaining between-mouse variation in the efficacy of a standard dose of PLS in removing worms, and therefore warrant some caution in developing this therapy for wider scale use in the livestock industry, and even in human medicine.

Antiangiogenesis, loss of cell adhesion and apoptosis are involved in the antitumoral activity of Proteases from V. cundinamarcensis (C. candamarcensis) in murine melanoma B16F1.

The proteolytic enzymes from V. cundinamarcensis latex, (P1G10), display healing activity in animal models following various types of lesions. P1G10 or the purified isoforms act as mitogens on fibroblast and epithelial cells by stimulating angiogenesis and wound healing in gastric and cutaneous ulcers models. Based on evidence that plant proteinases act as antitumorals, we verified this effect on a murine melanoma model. The antitumoral effect analyzed mice survival and tumor development after subcutaneous administration of P1G10 into C57BL/6J mice bearing B16F1 low metastatic melanoma. Possible factors involved in the antitumoral action were assessed, i.e., cytotoxicity, cell adhesion and apoptosis in vitro, haemoglobin (Hb), vascular endothelial growth factor (VEGF), tumor growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α) content and N-acetyl-glucosaminidase (NAG) activity. We observed that P1G10 inhibited angiogenesis measured by the decline of Hb and VEGF within the tumor, and TGF-ß displayed a non-significant increase and TNF-α showed a minor non-significant reduction. On the other hand, there was an increase in NAG activity. In treated B16F1 cells, apoptosis was induced along with decreased cell binding to extracellular matrix components (ECM) and anchorage, without impairing viability.

Amino derivatives of glycyrrhetinic acid as potential inhibitors of cholinesterases.

The development of remedies against the Alzheimer's disease (AD) is one of the biggest challenges in medicinal chemistry nowadays. Although not completely understood, there are several strategies fighting this disease or at least bringing some relief. During the progress of AD, the level of acetylcholine (ACh) decreases; hence, a therapy using inhibitors should be of some benefit to the patients. Drugs presently used for the treatment of AD inhibit the two ACh controlling enzymes, acetylcholinesterase as well as butyrylcholinesterase; hence, the design of selective inhibitors is called for. Glycyrrhetinic acid seems to be an interesting starting point for the development of selective inhibitors. Although its glycon, glycyrrhetinic acid is known for being an AChE activator, several derivatives, altered in position C-3 and C-30, exhibited remarkable inhibition constants in micro-molar range. Furthermore, five representative compounds were subjected to three more enzyme assays (on carbonic anhydrase II, papain and the lipase from Candida antarctica) to gain information about the selectivity of the compounds in comparison to other enzymes. In addition, photometric sulforhodamine B assays using murine embryonic fibroblasts (NiH 3T3) were performed to study the cytotoxicity of these compounds. Two derivatives, bearing either a 1,3-diaminopropyl or a 1H-benzotriazolyl residue, showed a BChE selective inhibition in the single-digit micro-molar range without being cytotoxic up to 30µM. In silico molecular docking studies on the active sites of AChE and BChE were performed to gain a molecular insight into the mode of action of these compounds and to explain the pronounced selectivity for BChE.

Developing a rapid throughput screen for detection of nematicidal activity of plant cysteine proteinases: the role of Caenorhabditis elegans cystatins.

Plant cysteine proteinases (CPs) from papaya (Carica papaya) are capable of killing parasitic nematode worms in vitro and have been shown to possess anthelmintic effects in vivo. The acute damage reported in gastrointestinal parasites has not been found in free-living nematodes such as Caenorhabditis elegans nor among the free-living stages of parasitic nematodes. This apparent difference in susceptibility might be the result of active production of cysteine proteinase inhibitors (such as cystatins) by the free-living stages or species. To test this possibility, a supernatant extract of refined papaya latex (PLS) with known active enzyme content was used. The effect on wild-type (Bristol N2) and cystatin null mutant (cpi-1(-/-) and cpi-2(-/-)) C. elegans was concentration-, temperature- and time-dependent. Cysteine proteinases digested the worm cuticle leading to release of internal structures and consequent death. Both cystatin null mutant strains were highly susceptible to PLS attack irrespective of the temperature and concentration of exposure, whereas wild-type N2 worms were generally resistant but far more susceptible to attack at low temperatures. PLS was able to induce elevated cpi-1 and cpi-2 cystatin expression. We conclude that wild-type C. elegans deploy cystatins CPI-1 and CPI-2 to resist CP attack. The results suggest that the cpi-1 or cpi-2 null mutants (or a double mutant combination of the two) could provide a cheap and effective rapid throughput C. elegans-based assay for screening plant CP extracts for anthelmintic activity.

Inhibitory effect of post-micellar SDS concentration on thermal aggregation and activity of papain.

Papain, a cysteine protease isolated from the latex of Carica papaya, is known to undergo irreversible thermal unfolding. In this study, we found that thermal unfolding of papain is accompanied by a simultaneous self-assembly process where this protein is observed to aggregate above 50°C. The extent of aggregation increased with increasing protein concentration from 3-40 µM. The aggregation was confirmed by enhanced turbidity, light scattering intensity, 1-anilino-8-naphthalene sulfonate (ANS) fluorescence intensity and by transmission electron microscopy. Furthermore, we noted that post-micellar concentration of sodium dodecyl sulfate (SDS) remarkably suppresses the thermal aggregation of papain. Far-UV circular dichroism studies revealed that SDS significantly enhances α-helical content of the protein and also tends to prevent its unfolding, and thus inhibits aggregation. Additionally, papain showed maximal activity at 65°C in neutral buffer. However, in the presence of 6 mM SDS (above its critical micellar concentration), the enzyme lost activity by about 10-fold. Thus, promoting the helical propensity of the protein does not appear to be a suitable strategy to overcome the aggregation related problems of industrially important proteins such as papain, which are not only required to be protected against aggregation but also need to remain functionally active in the presence of aggregation inhibitors.

Peptidases from latex of Carica candamarcensis upregulate COX-2 and IL-1 mRNA transcripts against Salmonella enterica ser. Typhimurium-mediated inflammation.

The immunomodulatory properties of a mixture of cysteine peptidases (P1G10) obtained from the fruit lattice of Carica candamarcensis were investigated. P1G10 was obtained from fresh latex samples by chromatography in a Sephadex column and initially administered to Swiss mice (n = 5; 1 or 10 mg/kg) via i.p. After 30 min, the mice were injected with carrageenan (0.5 mg/mouse) or heat-killed S. Typhimurium (10(7) CFU/mL; 100°C/30 min) into the peritoneal cavity. Afterwards, two animal groups were i.p. administered with P1G10 (n = 6; 1, 5, or 10 mg/Kg) or PBS 24 hours prior to challenge with live S. Typhimurium (10(7) CFU/mL). P1G10 stimulated the proliferation of circulating neutrophils and lymphocytes, 6 h after injection of carrageenan or heat-killed bacteria, respectively. Furthermore, survival after infection was dose-dependent and reached 60% of the animal group. On the other hand, control mice died 1-3 days after infection. The examination of mRNA transcripts in liver cells 24 h after infection confirmed fold variation increases of 5.8 and 4.8 times on average for IL-1 and COX-2, respectively, in P1G10 pretreated mice but not for TNF-α, IL-10, γ-IFN and iNOS, for which the results were comparable to untreated animals. These data are discussed in light of previous reports.

Effects of nitric oxide treatment on the cell wall softening related enzymes and several hormones of papaya fruit during storage.

Papaya fruits (Carica papaya L. cv 'Sui you 2') harvested with < 5% yellow surface at the blossom end were fumigated with 60 microL/L of nitric oxide for 3 h and then stored at 20 degrees C with 85% relative humility for 20 days. The effects of nitric oxide treatment on ethylene production rate, the activities of cell wall softening related enzymes including polygalacturonase, pectin methyl esterase, pectate lyase and cellulase and the levels of hormones including indole acetic acid, abscisic acid, gibberellin and zeatin riboside were examined. The results showed that papaya fruits treated with nitric oxide had a significantly lower rate of ethylene production and a lesser loss of firmness during storage. A decrease in polygalacturonase, pectin methyl esterase, pectate lyase and cellulase activities was observed in nitric oxide treated fruit. In addition, the contents of indole acetic acid, abscisic acid and zeatin riboside were reduced in nitric oxide treated fruit, but no significant reduction in the level of gibberellin was found. These results indicate that nitric oxide treatment can effectively delay the softening and ripening of papaya fruit, likely via the regulation of cell wall softening related enzymes and certain hormones.

Biochemical characterization of VQ-VII, a cysteine peptidase with broad specificity, isolated from Vasconcellea quercifolia latex.

The latex from Vasconcellea quercifolia ("oak leaved papaya"), a member of the Caricaceae family, contains at least seven cysteine endopeptidases with high proteolytic activity, which helps to protect these plants against injury. In this study, we isolated and characterized the most basic of these cysteine endopeptidases, named VQ-VII. This new purified enzyme was homogeneous by bidimensional electrophoresis and MALDI-TOF mass spectrometry, and exhibited a molecular mass of 23,984 Da and an isoelectric point >11. The enzymatic activity of VQ-VII was completely inhibited by E-64 and iodoacetic acid, confirming that it belongs to the catalytic group of cysteine endopeptidases. By investigating the cleavage of the oxidized insulin B-chain to establish the hydrolytic specificity of VQ-VII, we found 13 cleavage sites on the substrate, revealing that it is a broad-specificity peptidase. The pH profiles toward p-Glu-Phe-Leu-p-nitroanilide (PFLNA) and casein showed that the optimum pH is about 6.8 for both substrates, and that in casein, it is active over a wide pH range (activity higher than 80 % between pH 6 and 9.5). Kinetic enzymatic assays were performed with the thiol peptidase substrate PFLNA (K m = 0.454 ± 0.046 mM, k cat = 1.57 ± 0.07 s(-1), k cat/K m = 3.46 × 10(3) ± 14 s(-1) M(-1)). The N-terminal sequence (21 amino acids) of VQ-VII showed an identity >70 % with 11 plant cysteine peptidases and the presence of highly conserved residues and motifs shared with the "papain-like" family of peptidases. VQ-VII proved to be a new latex enzyme of broad specificity, which can degrade extensively proteins of different nature in a wide pH range.