Isolation and characterization of an aspartic protease from salpichroa origanifolia fruits
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 enz...
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2015
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_09298665_v22_n4_p379_Rocha http://hdl.handle.net/20.500.12110/paper_09298665_v22_n4_p379_Rocha |
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paper:paper_09298665_v22_n4_p379_Rocha2023-06-08T15:52:25Z Isolation and characterization of an aspartic protease from salpichroa origanifolia fruits Caseinolytic activity Peptide mass fingerprinting Plant aspartic protease Purification Salpichroa origanifolia Salpichroin 1,10 phenanthroline aspartic proteinase benzylsulfonyl fluoride casein chymosin edetic acid hemoglobin n [n (3 carboxyoxirane 2 carbonyl)leucyl]agmatine pepstatin synthetic peptide aspartic proteinase insulin amino acid sequence anion exchange chromatography Article controlled study enzyme kinetics fruit matrix assisted laser desorption ionization time of flight mass spectrometry molecular weight nonhuman pH polyacrylamide gel electrophoresis precipitation protein analysis protein cleavage protein hydrolysis protein isolation protein purification Salpichroa origanifolia Solanaceae chemistry isolation and purification molecular genetics Salpichroa origanifolia Solanaceae Amino Acid Sequence Aspartic Acid Proteases Fruit Insulin Molecular Sequence Data Solanaceae 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. © 2015 Bentham Science Publishers. 2015 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_09298665_v22_n4_p379_Rocha http://hdl.handle.net/20.500.12110/paper_09298665_v22_n4_p379_Rocha |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Caseinolytic activity Peptide mass fingerprinting Plant aspartic protease Purification Salpichroa origanifolia Salpichroin 1,10 phenanthroline aspartic proteinase benzylsulfonyl fluoride casein chymosin edetic acid hemoglobin n [n (3 carboxyoxirane 2 carbonyl)leucyl]agmatine pepstatin synthetic peptide aspartic proteinase insulin amino acid sequence anion exchange chromatography Article controlled study enzyme kinetics fruit matrix assisted laser desorption ionization time of flight mass spectrometry molecular weight nonhuman pH polyacrylamide gel electrophoresis precipitation protein analysis protein cleavage protein hydrolysis protein isolation protein purification Salpichroa origanifolia Solanaceae chemistry isolation and purification molecular genetics Salpichroa origanifolia Solanaceae Amino Acid Sequence Aspartic Acid Proteases Fruit Insulin Molecular Sequence Data Solanaceae |
| spellingShingle |
Caseinolytic activity Peptide mass fingerprinting Plant aspartic protease Purification Salpichroa origanifolia Salpichroin 1,10 phenanthroline aspartic proteinase benzylsulfonyl fluoride casein chymosin edetic acid hemoglobin n [n (3 carboxyoxirane 2 carbonyl)leucyl]agmatine pepstatin synthetic peptide aspartic proteinase insulin amino acid sequence anion exchange chromatography Article controlled study enzyme kinetics fruit matrix assisted laser desorption ionization time of flight mass spectrometry molecular weight nonhuman pH polyacrylamide gel electrophoresis precipitation protein analysis protein cleavage protein hydrolysis protein isolation protein purification Salpichroa origanifolia Solanaceae chemistry isolation and purification molecular genetics Salpichroa origanifolia Solanaceae Amino Acid Sequence Aspartic Acid Proteases Fruit Insulin Molecular Sequence Data Solanaceae Isolation and characterization of an aspartic protease from salpichroa origanifolia fruits |
| topic_facet |
Caseinolytic activity Peptide mass fingerprinting Plant aspartic protease Purification Salpichroa origanifolia Salpichroin 1,10 phenanthroline aspartic proteinase benzylsulfonyl fluoride casein chymosin edetic acid hemoglobin n [n (3 carboxyoxirane 2 carbonyl)leucyl]agmatine pepstatin synthetic peptide aspartic proteinase insulin amino acid sequence anion exchange chromatography Article controlled study enzyme kinetics fruit matrix assisted laser desorption ionization time of flight mass spectrometry molecular weight nonhuman pH polyacrylamide gel electrophoresis precipitation protein analysis protein cleavage protein hydrolysis protein isolation protein purification Salpichroa origanifolia Solanaceae chemistry isolation and purification molecular genetics Salpichroa origanifolia Solanaceae Amino Acid Sequence Aspartic Acid Proteases Fruit Insulin Molecular Sequence Data Solanaceae |
| description |
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. © 2015 Bentham Science Publishers. |
| title |
Isolation and characterization of an aspartic protease from salpichroa origanifolia fruits |
| title_short |
Isolation and characterization of an aspartic protease from salpichroa origanifolia fruits |
| title_full |
Isolation and characterization of an aspartic protease from salpichroa origanifolia fruits |
| title_fullStr |
Isolation and characterization of an aspartic protease from salpichroa origanifolia fruits |
| title_full_unstemmed |
Isolation and characterization of an aspartic protease from salpichroa origanifolia fruits |
| title_sort |
isolation and characterization of an aspartic protease from salpichroa origanifolia fruits |
| publishDate |
2015 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_09298665_v22_n4_p379_Rocha http://hdl.handle.net/20.500.12110/paper_09298665_v22_n4_p379_Rocha |
| _version_ |
1768543759294791680 |