Structural Analysis and Construction of a Thermostable Antifungal Chitinase.

Chitin is a biopolymer of N-acetyl-d-glucosamine with ß-1,4-bond and is the main component of arthropod exoskeletons and the cell walls of many fungi. Chitinase (EC 3.2.1.14) is an enzyme that hydrolyzes the ß-1,4-bond in chitin and degrades chitin into oligomers. It has been found in a wide range of organisms. Chitinase from Gazyumaru (Ficus microcarpa) latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields. However, the enzyme's thermostability is an important factor; chitinase is not thermostable enough to maintain its activity under the actual application conditions. In addition to the fact that thermostable chitinases exhibiting antifungal activity can be used under various conditions, they have some advantages for the production process and long-term preservation, which are highly demanded in industrial use. We solved the crystal structure of chitinase to explore the target sites to improve its thermostability. We rationally introduced proline residues, a disulfide bond, and salt bridges in the chitinase using protein-engineering methods based on the crystal structure and sequence alignment among other chitinases. As a result, we successfully constructed the thermostable mutant chitinases rationally with high antifungal and specific activities. The results provide a useful strategy to enhance the thermostability of this enzyme family. IMPORTANCE We solved the crystal structure of the chitinase from Gazyumaru (Ficus microcarpa) latex exhibiting antifungal activity. Furthermore, we demonstrated that the thermostable mutant enzyme with a melting temperature (Tm) 6.9°C higher than wild type (WT) and a half-life at 60°C that is 15 times longer than WT was constructed through 10 amino acid substitutions, including 5 proline residues substitutions, making disulfide bonding, and building a salt bridge network in the enzyme. These mutations do not affect its high antifungal activity and chitinase activity, and the principle for the construction of the thermostable chitinase was well explained by its crystal structure. Our results provide a useful strategy to enhance the thermostability of this enzyme family and to use the thermostable mutant as a seed for antifungal agents for practical use.

Drupin, a thrombin-like protease prompts platelet activation and aggregation through protease-activated receptors.

Hemostasis is a proteolytically regulated process that requires activation of platelets and the blood coagulation cascade upon vascular injury. Activated platelets create a thrombogenic environment and amplify the coagulation process. Plant latex proteases (PLPs) have been used as therapeutic components to treat various ailments by folk healers. One of the main applications of plant latices is to stop bleeding from minor injuries and to enhance wound healing activity. Although many studies have reported the pro-coagulant activities of PLPs, an in-depth investigation is required to understand the mechanism of action of PLPs on platelets. Here, the effect of PLPs on platelet aggregation was studied systematically to validate the observed pharmacological effect by folk healers. Among 29 latices from the Ficus genus tested, Ficus drupacea exhibited potent pro-coagulant and thrombin-like activity. Drupin, a thrombin-like cysteine protease responsible for platelet aggregation was purified from F. drupacea latex. Drupin exhibits pro-coagulant activity and reduces the bleeding time in mice tail. It induces platelet aggregation by activating mitogen-activated protein kinases and the nuclear factor-κB and PI3K/Akt signalling cascade, which, in turn, phosphorylats, cytosolic phospholipase A2  leading to the release of thromboxane A2 from the granules to activate the nearby platelets to aggregate. Furthermore, we investigated the involvement of protease-activated receptors in drupin-induced platelet aggregation using specific protease activated receptor 1 (PAR1) and PAR4 receptor antagonists. The results confirmed that the drupin-induced platelet aggregation was mediated by both PAR1 and PAR4, synergistically. Overall, drupin reduces the bleeding time by exerting pro-coagulant activity and induces platelet aggregation by activating the intracellular signalling cascade.

Novel biotechnological formulations of cysteine proteases, immobilized on chitosan. Structure, stability and activity.

Bromelain, papain, and ficin are studied the most for meat tenderization, but have limited application due to their short lifetime. The aim of this work is to identify the adsorption mechanisms of these cysteine proteases on chitosan to improve the enzymes' stability. It is known that immobilization can lead to a significant loss of enzyme activity, which we observed during the sorption of bromelain (protease activity compared to soluble enzyme is 49% for medium and 64% for high molecular weight chitosan), papain (34 and 28% respectively) and ficin (69 and 70% respectively). Immobilization on the chitosan matrix leads to a partial destruction of protein helical structure (from 5 to 19%). Using computer modelling, we have shown that the sorption of cysteine proteases on chitosan is carried out by molecule regions located on the border of domains L and R, including active cites of the enzymes, which explains the decrease in their catalytic activity upon immobilization. The immobilization on chitosan does not shift the optimal range of pH (7.5) and temperature values (60 °C for bromelain and papain, 37-60 °C for ficin), but significantly increases the stability of biocatalysts (from 5.8 times for bromelain to 7.6 times for papain).

Insight into the collagen-degrading activity of a serine protease in the latex of Ficus carica cultivar Masui Dauphine.

Ficus carica produces, in addition to the cysteine protease ficin, a serine protease. Earlier study on a serine protease from F. carica cultivar Brown Turkey showed that it specifically degraded collagen. In this study, we characterized the collagenolytic activity of a serine protease in the latex of F. carica cultivar Masui Dauphine. The serine protease degraded denatured, but not undenatured, acid-solubilized type I collagen. It also degraded bovine serum albumin, while the collagenase from Clostridium histolyticum did not. These results indicated that the serine protease in Masui Dauphine is not collagen-specific. The protease was purified to homogeneity by two-dimensional gel electrophoresis, and its partial amino acid sequence was determined by liquid chromatography-tandem mass spectrometry. BLAST searches against the Viridiplantae (green plants) genome database revealed that the serine protease was a subtilisin-like protease. Our results contrast with the results of the earlier study stating that the serine protease from F. carica is collagen-specific.

Drupin, a cysteine protease from Ficus drupacea latex accelerates excision wound healing in mice.

Wound healing is a tightly regulated physiological process that restores tissue integrity after injury. Plant latex proteases (PLPs) are considered an integral part in herbal wound care as it interferes at different phases of the wound healing process. Although many studies have reported the involvement of PLPs in healing process, an in-depth investigation is required to understand the molecular mechanism. Hence, the effect of PLPs with fibrinolytic activity on wound healing was investigated systematically using mouse excision wound model. Among 29 latices from Ficus genus tested, Ficus drupacea exhibited potent fibrinolytic activity. Cysteine protease responsible for fibrinolysis was purified from the F. drupacea latex named it as drupin, tested for its wound healing efficacy. The accelerated wound healing was mediated by downregulation of matrix metalloprotease (MMP)-9 without altering MMP-8 expression. Besides, drupin enhanced the rate of collagen synthesis at the wound site by increasing arginase 1 activity. And also, drupin increased the expression of arginase 1 in macrophages and involved in cell proliferation, and migration via MAP kinase and PI3K/Akt pathways. Overall, the present study highlights the interference of drupin in wound healing by increased arginase 1 activity and collagen synthesis, and cell proliferation and migration.

Broad range of substrate specificities in papain and fig latex enzymes preparations improve enumeration of Listeria monocytogenes.

Numerous applications of proteolytic enzymes include dissociation of fermented meat products for the enumeration of `foodborne pathogenic bacteria. The use of trypsin for this cause is abandoned due to the high concentration of the enzyme affecting released bacteria. Papain, as a suggested replacement, and fig latex preparation with high extent of papain-like enzymes have the potential to be applied for bacteria enumeration. Both enzymatic preparations, originating from papaya and fig, showed a broader range of substrate specificities including gelatinolytic activity, especially prominent in the case of ficin and attributed to both, cysteine protease ficin and serine protease by the analysis of 2D zymography with specific inhibitors. The activity towards native collagen, mild in the case of papain, and extensive in the case of fig latex was proved by structural analysis of digested collagen by infrared spectroscopy. Further exploration of their potential for dissociation of fermented meat products showed that both papain and fig latex enzymes are stable in the presence of detergents Tween 20 and Triton X-100 and effective in the enumeration of Listeria monocytogenes. Gelatenolytic activity, and at least partial collagenolytic activity and stability in procedure conditions make papaya and fig latex proteases potent for this application in significantly lower concentrations than previously used enzymes. As a mixture of proteolytic enzymes with divergent characteristics, fig latex preparation shows higher efficiency in Listeria monocytogenes release than papain, conserved even in the presence of stronger non-ionic detergent Triton X-100.

Characterization of proteases activities in Ficus carica cultivars.

In this study, we characterized protease activities of 23 Ficus carica cultivars. Extracts of fruit, branch, and leaf of Masui Dauphine, one of the most representative F. carica cultivars in Japan, exhibited gelatin-hydrolyzing activity, both in the absence and presence of a cysteine protease-specific inhibitor, E-64, suggesting that not only ficin (classified as cysteine protease) but also collagenase (classified as serine protease) were involved in the digestion of gelatin. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-l-Lys-l-Pro-l-Leu-Gly-l-Leu-[N3 -(2,4-dinitrophenyl)-l-2,3-diaminopropionyl]-l-Ala-l-Arg-NH2 , all branch extracts of 23 F. carica cultivars exhibited the activity both in the absence and presence of cysteine protease-specific inhibitor E-64, indicating that they contain ficin and collagenase. During digestion of acid-solubilized type I collagen by the branch extract of Masui Dauphine at 40-55 °C, collagen was completely digested in the absence of E-64, while it was partially digested in the presence of the inhibitor, indicating that the manner of digestion differed between ficin and collagenase contained in the extract. These results suggest that F. carica is attractive for industrial use to digest collagen. PRACTICAL APPLICATION: The industrial use of F. carica might be enhanced by efficiently utilizing these proteases and/or selecting the appropriate F. carica cultivar. Collagen is one of the targets to which our results might be applied. It is widely accepted today that collagen and its digestion products could be useful as functional food. F. carica is a potential candidate for use in not only complete but also partial digestion of collagen.

Analysis of apocarotenoid volatiles during the development of Ficus carica fruits and characterization of carotenoid cleavage dioxygenase genes.

In plants the oxidative cleavage of carotenoid substrates produces volatile apocarotenoids, including ß-ionone, 6-methyl-5-hepten-2-ol, and α-ionone; these compounds are important in herbivore-plant communication. Combined chemical, biochemical, and molecular studies were conducted to evaluate the differential accumulation of carotenoids and volatile apocarotenoids during the development of pollinated and parthenocarpic fig fruits. Pollinated fig fruits showed less emission of apocarotenoid volatiles than the parthenocarpic figs, while in the case of carotenoid pigments, pollinated figs manifested higher accumulation. The apocarotenoids, 6-methyl-5-hepten-2-ol and ß-cyclogeraniol, showed a marked increase after the two weeks of hand-pollination in pollinated and parthenocarpic figs; but afterwards these volatile levels decreased during further fruit development. In addition, we report a transcriptome-based identification and functional characterization of the carotenoid cleavage dioxygenase (FcCCD) genes. These genes were overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant FcCCD1A enzyme showed specificity for the 9,10 (9',10') double bond position of cyclic carotenoids to generate α-ionone and ß-ionone, while FcCCD1B cleaved lycopene and an acyclic moiety of δ-carotene, producing 6-methyl-5-hepten-2-one. The qRT-PCR analysis of FcCCD genes revealed differential gene expression during fig fruit development. Our results suggest a role for the FcCCD1genes in apocarotenoid biosynthesis in fig fruits.

Complex Tannins Isolated from Jelly Fig Achenes Affect Pectin Gelation through Non-Specific Inhibitory Effect on Pectin Methylesterase.

Jelly fig (Ficus awkeotsang Makino) is used to prepare drinks and desserts in Asia, owing to the gelling capability of its pectin via endogenous pectin methylesterase (PE) catalyzation. Meanwhile, substances with PE inhibitory activity (SPEI) in jelly fig achenes (JFA) residue were noticed to be able to impede the gelation. In this study, we characterized and isolated SPEI from JFA by a series of PE inhibition-guided isolations. Crude aqueous extract of JFA residue was mixed with acetone, and 90% acetone-soluble matter was further fractionated by Diaion HP-20 chromatography. The retained fraction with dominant PE inhibitory activity was collected from 100% methanol eluate. Results from high-performance liquid chromatography mass spectrometry (HPLC/MS) and hydrolysis-induced chromogenic transition revealed the SPEI as complex tannins. Total tannins content was determined in each isolated fraction, and was closely related to PE inhibitory activity. In addition, SPEI in this study could inhibit activities of digestive enzymes in vitro and may, therefore, be assumed to act as non-specific protein binding agent.

A novel milk-clotting cysteine protease from Ficus johannis: Purification and characterization.

Due to the need for calf rennet alternatives, many attempts have been made to find new proteases. A novel cysteine protease with milk-clotting activity was purified from Ficus johannis by cation exchange chromatography. The protease was stable in various pH (3.0-10.5) with the optimum at 6.5 and showed its maximum activity at 60 °C. The Km and Vmax values of the enzyme were obtained to be 0.604 mg/ml and 0.0273 µmol Tyr/min, respectively. The purified protease exhibited considerable activity towards κ-casein in comparison to α-casein and ß-casein. The enzyme was almost completely active in the presence of high salt concentrations. Besides, it had high stability against autodigestion. The content of free amino acids was determined by HPLC, where leucine, lysine, valine, γ-aminobutyric acid and tyrosine were the most abundant amino acids. The cheese manufactured by using the purified protease showed similar textural properties and physico-chemical compositions to cheese produced using commercial rennet. Considering the special characteristics, including high milk-clotting activity, considerable stability over wide ranges of pH and temperature, resistance towards solvents, salts, and surfactants, the new protease might be the promising candidate for the dairy industry as well as other food and biotechnological industries.

Crystallization and preliminary X-ray analysis of four cysteine proteases from Ficus carica latex.

The latex of the common fig (Ficus carica) contains a mixture of at least five cysteine proteases commonly known as ficins (EC 3.4.22.3). Four of these proteases were purified to homogeneity and crystals were obtained in a variety of conditions. The four ficin (iso)forms appear in ten different crystal forms. All diffracted to better than 2.10 Šresolution and for each form at least one crystal form diffracted to 1.60 Šresolution or higher. Ficin (iso)forms B and C share a common crystal form, suggesting close sequence and structural similarity. The latter diffracted to a resolution of 1.20 Šand belonged to space group P3121 or P3221, with unit-cell parameters a = b = 88.9, c = 55.9 Å.

A cysteine protease isolated from the latex of Ficus microcarpa: purification and biochemical characterization.

A plant protease named microcarpain was purified from the latex of Ficus microcarpa by acetonic (20-40 % saturation) precipitation, Sephadex G-75 filtration, and Mono Q-Sefinose FF chromatography. The protease was purified with a yield of 9.25 % and a purification factor of 8. The molecular weight of the microcarpain was estimated to be 20 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme showed maximum activity at pH 8.0 and at a temperature of 70 °C. Proteolytic activity was strongly inhibited by dithio-bis-nitrobenzoic acid (DTNB), Hg(2+), and Cu(2+). The N-terminal amino acid sequence of the purified microcarpain "VPETVDWRSKGAV" showed high homology with a protease from Arabidopsis thaliana. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine peptidases family.

Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees.

MAIN CONCLUSION: Targeting a gene in apple or fig with ZFN, introduced by transient or stable transformation, should allow genome editing with high precision to advance basic science and breeding programs. Genome editing is a powerful tool for precise gene manipulation in any organism; it has recently been shown to be of great value for annual plants. Classical breeding strategies using conventional cross-breeding and induced mutations have played an important role in the development of new cultivars in fruit trees. However, fruit-tree breeding is a lengthy process with many limitations. Efficient and widely applied methods for targeted modification of fruit-tree genomes are not yet available. In this study, transgenic apple and fig lines carrying a zinc-finger nuclease (ZFNs) under the control of a heat-shock promoter were developed. Editing of a mutated uidA gene, following expression of the ZFN genes by heat shock, was confirmed by GUS staining and PCR product sequencing. Finally, whole plants with a repaired uidA gene due to deletion of a stop codon were regenerated. The ZFN-mediated gene modifications were stable and passed onto regenerants from ZFN-treated tissue cultures. This is the first demonstration of efficient and precise genome editing, using ZFN at a specific genomic locus, in two different perennial fruit trees-apple and fig. We conclude that targeting a gene in apple or fig with a ZFN introduced by transient or stable transformation should allow knockout of a gene of interest. Using this technology for genome editing allows for marker gene-independent and antibiotic selection-free genome engineering with high precision in fruit trees to advance basic science as well as nontransgenic breeding programs.

Post-translational modification and extended glycosylation pattern of a plant latex peroxidase of native source characterized by X-ray crystallography.

UNLABELLED: The crystal structure of banyan peroxidase purified from the latex of Ficus benghalensis has been solved at 1.67 Å resolution by single-wavelength anomalous diffraction phasing. The refined structure includes 306 amino acid residues, a heme and two calcium ions. The protein belongs to class III peroxidases and is the first one from plant latex. Extensive glycosylation was observed with N-linked glycans attached to seven asparagine residues. The enzyme is stable with respect to a wide pH range, temperature, chemical denaturants and organic solvents, probably as a result of its high glycosylation. An unexpected post-translational modification of Asp290 was identified as succinimide moiety. Kinetic parameters of banyan peroxidase have been determined using various hydrogen donor substrates and hydrogen peroxide. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank under accession number 4CUO.

Identification, purification and characterization of a novel collagenolytic serine protease from fig (Ficus carica var. Brown Turkey) latex.

A novel collagenolytic serine protease was identified and then purified (along with ficin) to apparent homogeneity from the latex of fig (Ficus carica, var. Brown Turkey) by two step chromatographic procedure using gel and covalent chromatography. The enzyme is a monomeric protein of molecular mass of 41 ± 9 kDa as estimated by analytical gel filtration chromatography. It is an acidic protein with a pI value of approximately 5 and optimal activity at pH 8.0-8.5 and temperature 60°C. The enzymatic activity was strongly inhibited by PMSF and Pefabloc SC, indicating that the enzyme is a serine protease. The enzyme showed specificity towards gelatin and collagen (215 GDU/mg and 24.8 CDU/mg, respectively) and non-specific protease activity (0.18 U/mg against casein). The enzyme was stable and retained full activity over a broad range of pH and temperature. The fig latex collagenolytic protease is potentially useful as a non-microbial enzyme with collagenolytic activity for various applications in the fields of biochemistry, biotechnology and medicine.

Purification, crystallization and preliminary crystallographic analysis of banyan peroxidase.

Plant peroxidases are extensively used in a wide range of biotechnological applications owing to their high environmental and thermal stability. A new peroxidase, named banyan peroxidase, was purified from the latex of Ficus benghalensis and crystallized. X-ray diffraction data were collected from native crystals and from bromide and xenon derivatives to resolutions of up to 1.66 Šin the trigonal space group P3(2)21, with unit-cell parameters a = b = 73.1, c = 164.6 Å. The anomalous signal of the intrinsic iron and calcium ions was sufficient for structure solution by SAD, although the sequence is not yet known.

Construction of new Pichia pastoris X-33 strains for production of lycopene and ß-carotene.

In this study, we used the non-carotenogenic yeast Pichia pastoris X33 as a receptor for ß-carotene-encoding genes, in order to obtain new recombinant strains capable of producing different carotenoidic compounds. We designed and constructed two plasmids, pGAPZA-EBI* and pGAPZA-EBI*L*, containing the genes encoding lycopene and ß-carotene, respectively. Plasmid pGAPZA-EBI*, expresses three genes, crtE, crtB, and crtI*, that encode three carotenogenic enzymes, geranylgeranyl diphosphate synthase, phytoene synthase, and phytoene desaturase, respectively. The other plasmid, pGAPZA-EBI*L*, carried not only the three genes above mentioned, but also the crtL* gene, that encodes lycopene ß-cyclase. The genes crtE, crtB, and crtI were obtained from Erwinia uredovora, whereas crtL* was cloned from Ficus carica (JF279547). The plasmids were integrated into P. pastoris genomic DNA, and the resulting clones Pp-EBI and Pp-EBIL were selected for either lycopene or ß-carotene production and purification, respectively. Cells of these strains were investigated for their carotenoid contents in YPD media. These carotenoids produced by the recombinant P. pastoris clones were qualitatively and quantitatively analyzed by high-resolution liquid chromatography, coupled to photodiode array detector. These analyses confirmed that the recombinant P. pastoris clones indeed produced either lycopene or ß-carotene, according to the integrated vector, and productions of 1.141 µg of lycopene and 339 µg of ß-carotene per gram of cells (dry weight) were achieved. To the best of our knowledge, this is the first time that P. pastoris has been genetically manipulated to produce ß-carotene, thus providing an alternative source for large-scale biosynthesis of carotenoids.

Partial characterization of a novel amylase activity isolated from Tunisian Ficus carica latex.

CONTEXT: A large number of plants still need to be investigated through screening of amylases suitable for industry. In the present study, and for the first time, we describe the amylolytic activity of Saint Pedro Ficus carica L. (Moraceae) crude latex of Kahli and Bidhi varieties. OBJECTIVE: Effects of temperature, pH, metal ions, and inhibitors and compatibility with some commercial detergents were investigated for amylase activity. MATERIALS AND METHODS: Amylase activity was screened in crude latex using the DNS method and potato starch as a substrate. Analyses of amylolytic reaction products by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were performed. RESULTS: Bidhi and Kahli amylases were active in optimal pH of 6.5 and 7 at 45°C, respectively, displaying a half life of 85 and 60 min, respectively, at 80°C, and they were very stable in a wide range of pH (4-12). Bidhi amylase activity increased to 260% by addition of 10(-3) mM Fe(2+) or 10(-2) mM Cu(2+), and was strongly inhibited by Mg(2+) and EDTA. In the presence of Ca(2+) and Mg(2+), Kahli amylase activity was dramatically enhanced by 220 and 260%, respectively. The compatibility of both amylases with certain commercial detergents was also shown to be good as enzymes retained up to 98% of their activities after 30 min of incubation at 80°C. DISCUSSION AND CONCLUSION: Analysis of amylolytic reaction products by TLC and HPLC suggested that Kahli amylase was an amyloglucosidase and Bidhi amylase was ß-fructose, α(1-4) glucose. Bidhi amylase is a good choice for application in starch, food, detergents and medical industries.

cDNA cloning of a novel gene codifying for the enzyme lycopene ß-cyclase from Ficus carica and its expression in Escherichia coli.

Lycopene beta-cyclase (ß-LCY) is the key enzyme that modifies the linear lycopene molecule into cyclic ß-carotene, an indispensable carotenoid of the photosynthetic apparatus and an important source of vitamin A in human and animal nutrition. Owing to its antioxidant activity, it is commercially used in the cosmetic and pharmaceutical industries, as well as an additive in foodstuffs. Therefore, ß-carotene has a large share of the carotenoidic market. In this study, we used reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE)-PCR to obtain and clone a cDNA copy of the gene Lyc-ß from Ficus carica (Lyc-ß Fc), which codes for the enzyme lycopene ß-cyclase (ß-LCY). Expression of this gene in Escherichia coli produced a single polypeptide of 56 kDa of weight, containing 496 amino acids, that was able to cycle both ends of the lycopene chain. Amino acid analysis revealed that the protein contained several conserved plant cyclase motifs. ß-LCY activity was revealed by heterologous complementation analysis, with lycopene being converted to ß-carotene as a result of the enzyme's action. The ß-LCY activity of the expressed protein was confirmed by high-performance liquid chromatography (HPLC) identification of the ß-carotene. The lycopene to ß-carotene conversion rate was 90%. The experiments carried out in this work showed that ß-LYC is the enzyme responsible for converting lycopene, an acyclic carotene, to ß-carotene, a bicyclic carotene in F. carica. Therefore, by cloning and expressing ß-LCY in E. coli, we have obtained a new gene for ß-carotene production or as part of the biosynthetic pathway of astaxanthin. So far, this is the first and only gene of the carotenoid pathway identified in F. carica.

Selective and reversible thiol-pegylation, an effective approach for purification and characterization of five fully active ficin (iso)forms from Ficus carica latex.

The latex of Ficus carica constitutes an important source of many proteolytic components known under the general term of ficin (EC 3.4.22.3) which belongs to the cysteine proteases of the papain family. So far, no data on the purification and characterization of individual forms of these proteases are available. An effective strategy was used to fractionate and purify to homogeneity five ficin forms, designated A, B, C, D1 and D2 according to their sequence of elution from a cation-exchange chromatographic support. Following rapid fractionation on a SP-Sepharose Fast Flow column, the different ficin forms were chemically modified by a specific and reversible monomethoxypolyethylene glycol (mPEG) reagent. In comparison with their un-derivatized counterparts, the mPEG-protein derivatives behaved differently on the ion-exchanger, allowing us for the first time to obtain five highly purified ficin molecular species titrating 1mol of thiol group per mole of enzyme. The purified ficins were characterized by de novo peptide sequencing and peptide mass fingerprinting analyzes, using mass spectrometry. Circular dichroism measurements indicated that all five ficins were highly structured, both in term of secondary and tertiary structure. Furthermore, analysis of far-UV CD spectra allowed calculation of their secondary structural content. Both these data and the molecular masses determined by MS reinforce the view that the enzymes belong to the family of papain-like proteases. The five ficin forms also displayed different specific amidase activities against small synthetic substrates like dl-BAPNA and Boc-Ala-Ala-Gly-pNA, suggesting some differences in their active site organization. Enzymatic activity of the five ficin forms was completely inhibited by specific cysteine and cysteine/serine proteases inhibitors but was unaffected by specific serine, aspartic and metallo proteases inhibitors.