Insights into the hydrolytic activity of Asclepias fruticosa L. protease.

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.

Structural Properties of Macrodontain I, a Cysteine Protease from Pseudananas macrodontes (Morr.) Harms (Bromeliaceae).

The primary structure of macrodontain I, a peptidase from Pseudananas macrodontes fruits, was determined using Edman's degradation. The enzyme is a non-glycosylated peptidase composed by 213 amino acids with a calculated molecular weight of 23,486.18 Da, pI value 6.99, and a molar extinction coefficient at 280 nm of 61,685 M-1 cm-1. The alignment of the sequence of macrodontain I with those cysteine peptidases from species belonging to the family Bromeliaceae showed the highest identity degree (87.74%) against fruit bromelain. A remarkable fact is that all these peptidase sequences show two Met contiguous residues (Met121 and 122) and the nonapeptide VPQSIDWRD located in the mature N-terminal region. Residues Cys26 and His159, which constitute the catalytic dyad in all cysteine peptidases, as well as active site residues Gln20 and Asn176, characteristic of Clan C1A, are conserved in macrodontain I. The 3-D model suggests that the enzyme belongs to the α + ß class of proteins, with two disulfide bridges (Cys23-Cys63 and Cys57-Cys96) in the α domain, while the ß domain is stabilized by another disulfide bridge (Cys153-Cys201). Further, we were able to establish that the cysteine peptidases from P. macrodontes are involved in the anti-inflammatory activity.

Latex peptidases of Calotropis procera for dehairing of leather as an alternative to environmentally toxic sodium sulfide treatment.

Dehairing of crude leather is a critical stage performed at the beginning of its processing to obtain industrially useful pieces. Tanneries traditionally apply a chemical process based on sodium sulfide. Since this chemical reactive is environmentally toxic and inefficiently recycled, innovative protocols for reducing or eliminating its use in leather depilation are welcomed. Therefore, latex peptidases from Calotropis procera (CpLP) and Cryptostegia grandiflora (CgLP) were assayed for this purpose. Enzyme activity on substrates representative of skin such as hide powder azure (UHPA), elastin (UE), azocollagen (UAZOCOL), keratin (UK), and epidermis (UEP) was determined, while depilation activity was assayed on cow hide. Only CpLP was active against keratin (13.4 UK) and only CgLP was active against elastin (0.12 UE). CpLP (93.0 UHPA, 403.6 UAZOCOL, 36.3 UEP) showed higher activity against the other substrates than CgLP (47.6 UHPA, 261.5 UAZOCOL, 8.5 UEP). In pilot assays, CpLP (0.05% w/v with sodium sulfite 0.6% w/v as activator) released hairs from cow hide pieces. Macroscopic and microscopic analyses of the hide revealed that the dehairing process was complete and the leather structure was preserved. The proteolytic system of C. procera is a suitable bioresources to be exploited by tanneries.

A Bowman-Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid.

MAIN CONCLUSION: A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family. In this work, we report a Bowman-Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3'RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M-1 cm-1. MpBBI inhibits strongly trypsin with K i in the 10-10 M range and was stable in a wide array of pH and extreme temperatures. MpBBI comparative modeling was applied to gain insight into its 3D structure and highlighted some distinguishing features: (1) two non-identical loops, (2) loop 1 (CEEESRC) is completely different from any known BBI, and (3) the amount of disulphide bonds is also different from any reported BBI from dicot plants.

The Proteolytic Activity of Philibertia gilliesii Latex. Purification of Philibertain g II.

The latex from the patagonic plant Philibertia gilliesii Hook. et Arn. (Apocynaceae) is a milky-white suspension containing a proteolytic system constituted by several cysteine endopeptidases. A proteolytic preparation (philibertain g) from the latex of P. gilliesii fruits was obtained and characterized to evaluate its potential use in bioprocesses. Philibertain g contained 1.2 g/L protein and a specific (caseinolytic) activity of 7.0 Ucas/mg protein. It reached 80 % of its maximum caseinolytic activity in the pH 7-10 range, retained 80 % of the original activity after 2 h of incubation at temperatures ranging from 25 to 45 °C and could be fully inactivated after 5 min at 75 °C. Philibertain g retained 60 % of the initial activity even at 1 M NaCl and was able to hydrolyze proteins from stickwater one, of the main waste effluents generated during fishmeal production. Furthermore, as a contribution to the knowledge of the proteolytic system of P. gilliesii, we are reporting the purification of a new peptidase, named philibertain g II (pI 9.4, molecular mass 23,977 Da, N-terminus LPESVDWREKGVVFPXRNQ) isolated from philibertain g through a purification scheme including acetone fractionation, cation exchange, molecular exclusion chromatography, and ultrafiltration.

Production, characterization, and identification using proteomic tools of a polygalacturonase from Fusarium graminearum.

Since enzymatic degradation is a mechanism or component of the aggressiveness of a pathogen, enzymatic activities from a Fusarium graminearum isolate obtained from infected wheat spikes of Argentina Pampa region were studied in order to understand the disease progression, tending to help disease control. In particular, the significance of the study of polygalacturonase activity is based on that such activity is produced in the early stages of infection on the host, suggesting a crucial role in the establishment of disease. In this sense, polygalacturonase activity produced by this microorganism has been purified 375 times from 2-day-old culture filtrates by gel filtration and ion-exchange chromatography successively. The purified sample showed two protein bands in sodium dodecyl sulfate-polyacrylamide gels, with a molecular mass of 40 and 55 kDa. The protein bands were identified as an endopolygalacturonase and as a serine carboxypeptidase of F. graminearum, respectively, by peptide mass fingerprinting (matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF/TOF) fragment ion analysis). The pattern of substrate degradation analyzed by thin layer chromatography confirmed the mode of action of the enzyme as an endopolygalacturonase. High activity of the polygalacturonase against polygalacturonic acid was observed between 4 and 6 of pH, and between 30 and 50 °C, being 5 and 50 °C the optimum pH and temperature, respectively. The enzyme was fully stable at pH 5 for 120 min and 30 °C and sensible to the presence of some metal ions. This information would contribute to understand the most favorable environmental conditions for establishment of the disease.

Purification and characterization of a cysteine endopeptidase from Vasconcellea quercifolia A. St.-Hil. latex displaying high substrate specificity.

A new proteolytic preparation from Vasconcellea quercifolia ("oak leaved papaya") latex containing several cysteine endopeptidases with high proteolytic activity has been obtained. The specific activity of the new enzymatic preparation (VQ) was higher than that of Carica papaya latex. VQ was able to coagulate milk and to hydrolyze caseins and then could be used to produce cheeses and/or casein hydrolysates. Ion exchange chromatography of VQ allowed the isolation of a new protease, named quercifoliain I, homogeneous when analyzed by SDS-PAGE, IEF and MALDI-TOF-MS. Molecular mass was 24195 Da, and its isoelectric point was >9.3. The N-terminal sequence was determined (YPESVDWRQ). Insulin B-chain cleavage showed higher specificity than that of papain and was restricted to glycyl and alanyl residues at P1' position. The tryptic peptide mass fingerprint of quercifoliain I analyzed with the MASCOT search tool did not find a match with papain or any other plant cysteine proteases.

Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from Asclepias fruticosa latex.

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. 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 determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZalpha vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.

Purification and characterization of hieronymain III. Comparison with other proteases previously isolated from Bromelia hieronymi Mez.

A new proteolytic enzyme, named hieronymain III, has been purified by ion-exchange chromatography from unripe fruits of Bromelia hieronymi Mez. The new peptidase belongs to the cysteine catalytic type, as well as hieronymain I and II, the other two peptidases previously isolated from this species. Hieronymain III showed optimum alkaline pH range (8.6-9.3) and the molecular mass (MALDI-TOF) was 23713 Da. The N-terminal sequence (AVPQSIDWRRYGAVTTSRNQG) exhibited a higher percentage identity with hieronymain II (93%) than with hieronymain I (71%). The three peptidases showed notable differences on synthetic substrates degradation: whereas hieronymain III was the only one able to hidrolyze Z-Arg-Arg-p-nitroanilide, hieronymain I and II could degrade Z-Phe-Arg-p-nitroanilide; on the other hand, PFLNA was only split by hieronymain I. Finally, the three proteases showed different preferences on N-alpha-CBZ-p-nitrophenyl aminoacid ester substrates. From a biotechnological point of view, cleavage specificity differences are significant enough to use these enzymes as potential tools in that area.

Granulosain I, a cysteine protease isolated from ripe fruits of Solanum granuloso-leprosum (Solanaceae).

A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.

Isolation and characterization of hieronymain II, another peptidase isolated from fruits of Bromelia hieronymi Mez (Bromeliaceae).

From unripe fruits of Bromelia hieronymi Mez (Bromeliaceae), a partially purified protease preparation was obtained by acetone fractionation of the crude extract. Purification was achieved by anionic exchange chromatography (FPLC) on Q-Sepharose HP followed by cationic exchange chromatography (SP-Sepharose HP). Homogeneity of the new enzyme, named hieronymain II, was confirmed by SDS-PAGE and mass spectroscopy (MALDI-TOF-TOF). The molecular mass of was 23,411 Da, and maximum proteolytic activity (more than 90% of maximum activity) was achieved at pH 7.5-9.0 on casein and at pH 7.30-8.3 on Z-Phe-Arg-p-nitroanilide. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine. The N-terminal sequence of hieronymain II (AVPQSIDWRVYGAV) was compared with those of 12 plant cysteine proteases which showed more than 70% of identity. Kinetic enzymatic assays were made on Z-Phe-Arg-p-nitroanilide (Km = 0.72mM, kcat = 1.82 seg(-1) , kcat/ Km = 2.54seg(-1) mM(-l)). No detectable activity could be found on PFLNA or Z-Arg-Arg-p-nitroanilide.

Proteolytic activity in some Patagonian plants from Argentina.

Six Patagonian plants were screened for proteolytic activity: Colliguaja integerrima, Euphorbia collina, E. peplus and Stillingia patagonica (Euphorbiaceae), Philibertia gilliesii (Asclepiadaceae) and Grindelia chiloensis (Asteraceae). P. gilliesii extracts showed the highest specific activity, followed by S. patagonica and E. collina. Proteolytic activity was unnoticeable in the other three species studied. Inhibition assays revealed that P. gilliesii and S. patagonica extracts contain cysteine-type peptidases and that in E. collina serine-type peptidases are present.

Hieronymain I, a new cysteine peptidase isolated from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae).

A new peptidase, named hieronymain I, was purified to homogeneity from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae) by acetone fractionation followed by cation exchange chromatography (FPLC) on CM-Sepharose FF. Homogeneity of the enzyme was confirmed by mass spectroscopy (MALDI-TOF), isoelectric focusing, and SDS-PAGE. Hieronymain is a basic peptidase (pI > 9.3) and its molecular mass was 24,066 Da. Maximum proteolytic activity on casein (>90% of maximum activity) was achieved at pH 8.5-9.5. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine; these results strongly suggest that the isolated protease should be included within the cysteine group. The N-terminal sequence of hieronymain (ALPESIDWRAKGAVTEVKRQDG) was compared with 25 plant cysteine proteases that showed more than 50% of identity.