IIB   20738
INSTITUTO DE INVESTIGACIONES BIOLOGICAS
Unidad Ejecutora - UE
artículos
Título:
Proteomic Analysis of an Aspartic Protease from Salpichroa
Autor/es:
GABRIELA F. ROCHA; GRACIELA FERNANDEZ; DAVID OBREGÓN ; FERNANDO MUÑOZ; MARIA GABRIELA GUEVARA ; ADRIANA M. ROSSO; MONICA G. PARISI
Revista:
PROTEIN AND PEPTIDE LETTERS
Editorial:
BENTHAM SCIENCE PUBL LTD
Referencias:
Año: 2015 vol. 2 p. 379 - 390
ISSN:
0929-8665
Resumen:
A novel aspartic protease was isolated from ripe fruits of Salpichroa origanifolia (Solanaceae). A simple purification procedure that combined precipitation with organic solvents and anion-exchange chromatography was performed with a 32.1% recovery and 13.4-fold purification. SDS PAGE and zymograms showed a single band of approximately 32 kDa. Biochemical and kinetic characterization of the pure enzyme were assessed. Salpichroin was active at acidic pH with an optimal value between 3.0 and 4.5 and at moderate temperatures (35-45°C). The peptidase activity was inhibited by pepstatin, whereas PMSF, E-64, EDTA and 1,10-phenanthroline had no significant inhibitory effect suggesting an aspartic behavior. Salpichroin hydrolyzes 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 peptidases. By investigating the cleavage of oxidized insulin â-chain to establish the hydrolytic specify of salpichroin, we found 6 cleavage sites on the substrate at similar peptide bonds as chymosin.  PMF and MALDI-TOF MS of the enzyme was performed and the comparison between the tryptic maps obtained for salpichroin with those deposited in databases using the MASCOT tool, showed that the isolated peptidase matched with others plant peptidases that belong to the A1 aspartic peptidase family. This is the first report concerning the isolation and characterization of a novel plant aspartic protease from Salpichroa origanifolia fruits with broad specificity on dairy products.