Constanza Liggieri

Purification and biochemical characterization of asclepain c I from the latex of Asclepias curassavica L.

In this work we report the isolation, purification and characterization of a new protease from latex of Asclepias curassavica L. Crude extract (CE) was obtained by gathering latex on 0.1M citric-phosphate buffer with EDTA and cysteine with subsequent ultracentrifugation. Proteolytic assays were made on casein or azocasein as substrates. Caseinolytic activity was completely inhibited by E-64. Stability at different temperatures, optimum pH and ionic strength were evaluated by measuring the residual caseinolytic activity at different times after the incubation. CE showed the highest caseinolytic activity at pH 8.5 in the presence of 12 mM cysteine. CE was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by SDS-PAGE, were isolated. The major purified protease (asclepain cI) showed a molecular mass of 23.2 kDa by mass spectrometry and a pI higher than 9.3. The N-terminal sequence showed a high similarity with those of other plant cysteine proteinases. When assayed on N-α-CBZ-aminoacid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative. Determinations of kinetic parameter (km and Kcat) were performed with PFLNA.

Two new cysteine endopeptidases obtained from the latex of Araujia hortorum fruits.

Two new endopeptidases were purified to homogeneity from the latex of Araujia hortorum fruits by a simple purification procedure involving ultracentrifugation and ion exchange chromatography. Molecular weights of araujiain h II and araujiain h III were 23,718 and 23546 (mass spectrometry), respectively. The isoelectric point of araujiain h II was 8.9, whereas araujiain h III had a pI higher than 9.3. Maximum proteolytic activity on caseine was reached at pH 8.0-9.0 for both endopeptidases, which were irreversibly inhibited by iodoacetate and E-64, suggesting they belong to the cysteine protease family. Esterolytic activity was determined on N-α-CBZ-amino acid-p-nitrophenyl esters, and the highest kcat/Km values for the both enzymes were obtained with the glutamine derivative. The N-terminal sequences of araujiain h II and araujiain h III showed a high degree of homology with other plant cysteine endopeptidases.

ACE-inhibitory peptides from bovine caseins released with peptidases from Maclura pomifera latex

In work reported here, a proteolytic extract prepared from Maclura pomifera latex was employed to hydrolyze bovine caseins. Densitograms of Tricine-sodium-dodecyl-sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) indicated that the caseins were considerably degraded after a 10-min reaction. The degree of hydrolysis determined by the 2,4,6-trinitrobenzenesulfonic-acid method was 17.1±0.7% after 180min of digestion. The concentration of small peptides increased with hydrolysis time, and analysis by reverse-phase high-performance liquid chromatography (RP HPLC) and mass spectrometry, revealed a virtually unchanged peptide profile. These results suggested that those proteases were highly specific, as only certain peptide bonds were cleaved. The hydrolysate of 180min displayed the highest inhibition of angiotensin-converting enzyme (ACE) showing an IC50 of 1.72±0.25mg/mL, and the analysis of the peptide fractionation in this hydrolysate by RP HPLC exhibited two peaks responsible for that activity. Fragmentation analysis through the use of iterated matrix-assisted-laser-desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF MS/MS) with the aid of bioinformatics tools enabled us to deduce two peptide sequences-one, YQEPVLGPVRGPFPIIV, having been previously reported as an ACE-inhibitor; the other, RFFVAPFPE, as yet undescribed. The presence of bioactive peptides in these casein hydrolysates argues for their potential use in the development of functional foods.

Biochemical and PMF MALDI-TOF Analyses of Two Novel Papain-Like Plant Proteinases

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.

Miniature cheeses made with blends of chymosin and a vegetable rennet from flowers of Silybum marianum: Enzymatic characterization of the flower-coagulant peptidase

Binary blends of S. marianum-flower extract and chymosin, as coagulant preparations, enabled the manufacture of miniature cheeses with distinctive characteristics compared to those of chymosin-renneted cheeses. The physicochemical parameters, sensory attributes of the cheeses, and in-vitro water-soluble antioxidant activity were analyzed and compared to those properties obtained from control chymosin-renneted cheeses. The preponderant proteolytic constituent in the flower extract was isolated in a two-step-purification protocol. The thus purified aspartic peptidase was maximally active at acidic pHs and exhibited a preference for peptide bonds between hydrophobic residues. Enzymologic characterization revealed differences in the kinetic parameters and specificity compared to other enzymes employed, such as rennet. S. marianum-flower extract, as a source of peptidase with distinctive characteristics, is a suitable substitute for chymosin in miniature-cheese production. The addition of vegetable rennet contributed to the development of an intense aroma and conferred antioxidant activity to the cheeses and wheys.

Preparation of soy protein hydrolysates with antioxidant activity by using peptidases from latex of Maclura pomifera fruits

A partially purified proteolytic extract prepared from Maclura pomifera latex was employed in hydrolyzing a soybean-protein isolate (4.2 mg/mL). The hydrolysis-product formation, monitored by tricine-sodium-dodecyl-sulfate-polyacrylamyde-gel electrophoresis and reverse-phase high-performance liquid chromatography, indicated that after 10 min of reaction the main soybean proteins disappeared. The maximum degree of hydrolysis was 36.2% after a 180-min digestion. The 90-min hydrolysate presented an IC50 of 31.6 ± 0.2 µg/mL, and a trolox equivalent antioxidant capacity of 157.6 and 176.9 µmoles TE per g of peptide determined by two different methods. Analysis by matrix-assisted-laser-desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS), followed by the application of bioinformatics tools, enabled the deduction of fourteen theoretical peptide sequences containing antioxidant amino acids at >60%, none of which sequences had been previously reported as antioxidants. Finally, we consider that this 90-min hydrolysate would constitute a promising ingredient in the manufacture of functional foods.

Enzymatic and chemical synthesis of new anticoagulant peptides

In this study we report the enzymatic synthesis of N‐α‐[Carbobenzyloxy]‐Tyr‐Gln‐Gln (Z‐YQQ), a new anticoagulant tripeptide. It was obtained using phytoproteases from the stems and petioles of Asclepias curassavica L. as catalyst in an aqueous–organic biphasic system formed by 50% (v/v) ethyl acetate and 0.1 M Tris–HCl buffer pH 8. The resulting peptide was compared with the analogous peptide Tyr‐Gln‐Gln (YQQ) produced by solid‐phase chemical synthesis. The in vitro anticoagulant activity of the aforementioned peptides was determined using Wiener Lab Test (Wiener, Argentina). The toxicological activity of the peptides was also determined. The enzymatically synthesized Z‐YQQ peptide acted on the extrinsic pathway of the coagulation cascade, delaying the conversion time of prothrombin to thrombin and fibrinogen to fibrin by 136 and 50%, respectively, with respect to the controls. The chemically synthesized YQQ peptide acted specifically on the intrinsic pathway of the coagulation cascade, affecting factors VIII, IX, XI, and XII from such cascade, and increasing the coagulation time by 105% with respect to the control. The results suggest that two new anticoagulant peptides (Z‐YQQ and YQQ) can be useful for safe pharmaceutical applications. Nevertheless, some aspects related to peptide production should be optimized.