Penduliflorain I: A cysteine protease isolated from Hohenbergia penduliflora (A.Rich.) Mez (Bromeliaceae).

Penduliflorain I, a new plant endopeptidase, was isolated and characterized from Hohenbergia penduliflora. Crude extract was obtained from stems. A partially purified enzyme preparation was obtained by ethanol precipitation. This preparation showed maximum activity between pH 7.5 and 8.5, was stable at ionic strength (20% decrease in proteolytic activity could be detected after 2 h in 0.4 M sodium chloride solution), and exhibited high thermal stability (inactivation required heating for 20 min at 75 degrees C). Inhibition and activation assays indicated the cysteine nature of the enzymatic preparation. Penduliflorain I was purified by anion exchange chromatography (Q-Sepharose HP) by FPLC system. Homogeneity was confirmed by mass spectroscopy. Molecular mass of the enzyme was 23 412.847 Da (MALDI-TOF-MS). Kinetic parameters were determined for PFLNA (K (m) = 0.3227 mM and k (cat) = 4.27 s(-1)). The N-terminal sequence (AVPQSIDWRDYGAVTTDKNQ) of isolated protease showed considerable similarity to other cysteine proteases obtained from stems or fruits of different Bromeliaceae species.

Philibertain g I, the most basic cysteine endopeptidase purified from the latex of Philibertia gilliesii Hook. et Arn. (Apocynaceae).

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.

Comparison of two cysteine endopeptidases from Pseudananas macrodontes (Morr.) Harms (Bromeliaceae).

The properties of two cysteine peptidases (macrodontain I and II) isolated from fruits of Pseudananas macrodontes have been compared. The enzymes showed optimum pH ranges near neutrality and were inhibited by E-64 and other cysteine peptidase inhibitors. Molecular masses were 23459 and 23703 kDa, the isoelectric points were 6.1 and 5.9, and the Km values were 13.4 and 8.9 microM (Bz-Phe-Val-Arg-AMC) for macrodontain I and II, respectively. N-alpha-CBZ-L-amino acid p-nitrophenyl esters were tested for both enzymes. The N-terminal sequences of both proteases differed slightly and showed high sequence similarity to other pineapple stem-derived cysteine endopeptidases.

Properties of a milk clotting protease isolated from fruits of Bromelia balansae Mez.

Unripe fruit extracts of Bromelia balansae Mez (Bromeliaceae), whose principal endopeptidase is balansain I (isolated for anion exchange chromatography: pI = 5.45, molecular weight = 23192), exhibit a pH profile with a maximum activity around pH 9.0 and are inhibited only by cysteine peptidases inhibitors. The alanine and glutamine derivatives of N-alpha-carbobenzoxy-L-amino acid p-nitrophenyl esters were strongly preferred by the enzyme. Enzymatic hydrolysis of milk and soy proteins yield characteristic patterns at pH 9.0. The N-terminal sequence showed a very high homology (85-90%) with other known Bromeliaceae endopeptidases.

Purification and characterization of a new plant endopeptidase isolated from latex of Asclepias fruticosa L. (Asclepiadaceae).

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.

Purification of balansain I, an endopeptidase from unripe fruits of Bromelia balansae Mez (Bromeliaceae).

A new plant endopeptidase was obtained from unripe fruits of Bromelia balansae Mez (Bromeliaceae). Crude extracts were partially purified by ethanol fractionation. This preparation (redissolved ethanol precipitate, REP) showed maximum activity at pH 8.8-9.2, was very stable even at high ionic strength values (no appreciable decrease in proteolytic activity could be detected after 24 h in 1 M sodium chloride solution at 37 degrees C), and exhibited high thermal stability (inactivation required heating for 60 min at 75 degrees C). Anion exchange chromatography allowed the isolation of a fraction purified to mass spectroscopy, SDS-PAGE, and IEF homogeneity, named balansain I, with pI = 5.45 and molecular mass = 23192 (mass spectrometry). The purification factor is low (2.9-fold), but the yield is high (48.3%), a common occurrence in plant organs with high proteolytic activity, where proteases represent the bulk of protein content of crude extracts. Balansain I exhibits a similar but narrower pH profile than that obtained for REP, with a maximum pH value approximately 9.0 and was inhibited by E-64 and other cysteine peptidases inhibitors but not affected by inhibitors of the other catalytic types of peptidases. The alanine and glutamine derivatives of N-alpha-carbobenzoxy-L-amino acid p-nitrophenyl esters was strongly preferred by the enzyme. The N-terminal sequence of balansain I showed a very high homology (85-90%) with other known Bromeliaceae endopeptidases.

Purification and characterization of macrodontain I, a cysteine peptidase from unripe fruits of Pseudananas macrodontes (Morr.) harms (Bromeliaceae).

A new papain-like cysteine peptidase isolated from fruits of Pseudananas macrodontes (Morr.) Harms, a species closely related to pineapple (Ananas comosus L.), has been purified and characterized. The enzyme, named macrodontain I, is the main proteolytic component present in fruit extracts and was purified by acetone fractionation followed by anion-exchange chromatography. Separation was improved by selecting both an adequate pH value and a narrow saline gradient. Optimum pH range (more than 90% of maximum activity with casein) was achieved at pH 6.1-8.5. Homogeneity of the enzyme was confirmed by bidimensional electrophoresis and mass spectroscopy (MS). Molecular mass of the enzyme was 23,459 (MS) and its isoelectric point was 6.1. The alanine, glutamine, and tyrosine derivatives were strongly preferred when the enzyme was assayed on N-alpha-CBZ-l-amino acid p-nitrophenyl esters. The N-terminal sequence of macrodontain (by comparison with the N-terminus of 30 plant proteases with more than 50% homology) showed a great deal of sequence similarity to the other pineapple-stem-derived cysteine endopeptidases, being 85.7, 85. 2, and 77.8% identical to comosain, stem bromelain, and ananain, respectively. It seems clear that the Bromeliaceae endopeptidases are more closely related to each other than to other members of the papain family, suggesting relatively recent divergence.