Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits

Abstract

This report describes the purification of an aspartic protease (salpichroin) from ripe fruits of Salpichroa origanifolia (Solanaceae) starting with precipitation using organic solvents and anionexchange chromatography with 32.1% recovery and 13.4-fold purification. SDS-PAGE and zymograms of this enzyme showed a single band corresponding to an apparent molecular mass of approximately 32 kDa. The biochemical and kinetic characterization of the pure enzyme showed an acidic behavior with an optimal pH value around 3.0-4.5 with hemoglobin and 5.5-6.0 with casein. Salpichroin activity was inhibited by pepstatin but not by phenylmethylsulfonyl fluoride, E-64, EDTA or 1,10-phenanthroline, thus suggesting an aspartic protease behavior. Salpichroin hydrolyzed natural substrates, such as casein and hemoglobin, with high specific activity. Kinetic studies conducted with the synthetic peptide H-Pro- Thr-Glu-Phe-p-(NO2)-Phe-Arg-Leu-OH showed lower affinity (Km 494 µM) than other representative aspartic proteases. By investigating the cleavage of oxidized insulin β-chain to establish the hydrolytic specificity of salpichroin, we found six cleavage sites on the substrate of peptide bonds similar to those of chymosin. MALDI-TOF/TOF-MS of the tryptic ingel digest of salpichroin showed that the isolated protease shared homologous sequences with other plant proteases of the A1 aspartic protease family. This is the first report concerning the isolation and biochemical characterization of an aspartic protease isolated from Salpichroa origanifolia fruits.

Antimicrobial activity of an aspartic protease fromSalpichroa origanifoliafruits

“…Tryptic digestion products of the AP salpichroin were sequenced and one of the fragments matched with the C‐terminal domain of Sl AP1‐like, where the PSI is located (Rocha et al . ).…”

“…Purification of the AP was performed as described by Rocha et al . (). Salpichroin, the purified AP showing proteolytic activity, was stored at −20°C.…”

“…The purity of salpichroin was checked by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis using 14% acrylamide gels (Laemmli ) and mass spectrometry (Rocha et al . ).…”

“…Salpichroin showed high similarity to an AP from Solanum licopersicum ( Sl AP1‐like), both belonging to the A1 family of AP and a fragment of salpichroin matched with the C‐terminal domain of SlAP1‐like where the PSI is located (Rocha et al . ). The aim of this work was to determine the cytotoxic activity of salpichroin on selected plant and human pathogens, and to evaluate if the effect was mediated by membrane disruption.…”

“…Previously, we reported the isolation of a novel AP from S. origanifolia ripe fruits (Rocha et al . ) capable to inhibit Phytophthora capsici growth (Rocha et al . ).…”