Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits

Title:Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits

Volume: 22 Issue: 4

Author(s): Gabriela F. Rocha, W. David Obregon, Fernando Munoz, M. Gabriela Guevara, Graciela Fernandez, Adriana M. Rosso and Monica G. Parisi

Affiliation:

Keywords: Caseinolytic activity, peptide mass fingerprinting, plant aspartic protease, purification, Salpichroa origanifolia, salpichroin.

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 (K_m 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.

Cite this article as:

Rocha F. Gabriela, Obregon David W., Munoz Fernando, Guevara Gabriela M., Fernandez Graciela, Rosso Adriana M. and Parisi G. Monica, Isolation and characterization of an Aspartic Protease from Salpichroa origanifolia Fruits, Protein & Peptide Letters 2015; 22 (4) . https://dx.doi.org/10.2174/0929866522666150302111059