Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R

Background Aspartic proteases are a subfamily of endopeptidases that are useful in a variety of applications, especially in the food processing industry. Here we describe a novel aspartic protease that was purified from Peptidase R, a commercial protease preparation derived from Rhizopus oryzae. Res...

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Autores principales:	Hsiao,Nai-Wan, Chen,Yeh, Kuan,Yi-Chia, Lee,Yen-Chung, Lee,Shuo-Kang, Chan,Hsin-Hua, Kao,Chao-Hung
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2014
Materias:	Chromatography Endopeptidase Food processing industry Homogeneity Rhizopuspepsin
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582014000200006
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spelling	oai:scielo:S0717-345820140002000062014-09-02Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase RHsiao,Nai-WanChen,YehKuan,Yi-ChiaLee,Yen-ChungLee,Shuo-KangChan,Hsin-HuaKao,Chao-Hung Chromatography Endopeptidase Food processing industry Homogeneity Rhizopuspepsin Background Aspartic proteases are a subfamily of endopeptidases that are useful in a variety of applications, especially in the food processing industry. Here we describe a novel aspartic protease that was purified from Peptidase R, a commercial protease preparation derived from Rhizopus oryzae. Results An aspartic protease sourced from Peptidase R was purified to homogeneity by anion exchange chromatography followed by polishing with a hydrophobic interaction chromatography column, resulting in a 3.4-fold increase in specific activity (57.5 × 10³ U/mg) and 58.8% recovery. The estimated molecular weight of the purified enzyme was 39 kDa. The N-terminal sequence of the purified protein exhibited 63-75% identity to rhizopuspepsins from various Rhizopus species. The enzyme exhibited maximal activity at 75°C in glycine-HCl buffer, pH 3.4 with casein as the substrate. The protease was stable at 35°C for 60 min and had an observed half-life of approximately 30 min at 45°C. Enzyme activity was not significantly inhibited by chelation with ethylenediamine tetraacetic acid (EDTA), and the addition of metal ions to EDTA-treated protease did not significantly change enzyme activity, indicating that proteolysis is not metal ion-dependent. The purified enzyme was completely inactivated by the aspartic protease inhibitor Pepstatin A. Conclusion Based on the observed enzyme activity, inhibition profile with Pepstatin A, and sequence similarity to other rhizopuspepsins, we have classified this enzyme as an aspartic protease.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.17 n.2 20142014-03-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582014000200006en10.1016/j.ejbt.2014.02.002
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	Chromatography Endopeptidase Food processing industry Homogeneity Rhizopuspepsin
spellingShingle	Chromatography Endopeptidase Food processing industry Homogeneity Rhizopuspepsin Hsiao,Nai-Wan Chen,Yeh Kuan,Yi-Chia Lee,Yen-Chung Lee,Shuo-Kang Chan,Hsin-Hua Kao,Chao-Hung Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R
description	Background Aspartic proteases are a subfamily of endopeptidases that are useful in a variety of applications, especially in the food processing industry. Here we describe a novel aspartic protease that was purified from Peptidase R, a commercial protease preparation derived from Rhizopus oryzae. Results An aspartic protease sourced from Peptidase R was purified to homogeneity by anion exchange chromatography followed by polishing with a hydrophobic interaction chromatography column, resulting in a 3.4-fold increase in specific activity (57.5 × 10³ U/mg) and 58.8% recovery. The estimated molecular weight of the purified enzyme was 39 kDa. The N-terminal sequence of the purified protein exhibited 63-75% identity to rhizopuspepsins from various Rhizopus species. The enzyme exhibited maximal activity at 75°C in glycine-HCl buffer, pH 3.4 with casein as the substrate. The protease was stable at 35°C for 60 min and had an observed half-life of approximately 30 min at 45°C. Enzyme activity was not significantly inhibited by chelation with ethylenediamine tetraacetic acid (EDTA), and the addition of metal ions to EDTA-treated protease did not significantly change enzyme activity, indicating that proteolysis is not metal ion-dependent. The purified enzyme was completely inactivated by the aspartic protease inhibitor Pepstatin A. Conclusion Based on the observed enzyme activity, inhibition profile with Pepstatin A, and sequence similarity to other rhizopuspepsins, we have classified this enzyme as an aspartic protease.
author	Hsiao,Nai-Wan Chen,Yeh Kuan,Yi-Chia Lee,Yen-Chung Lee,Shuo-Kang Chan,Hsin-Hua Kao,Chao-Hung
author_facet	Hsiao,Nai-Wan Chen,Yeh Kuan,Yi-Chia Lee,Yen-Chung Lee,Shuo-Kang Chan,Hsin-Hua Kao,Chao-Hung
author_sort	Hsiao,Nai-Wan
title	Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R
title_short	Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R
title_full	Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R
title_fullStr	Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R
title_full_unstemmed	Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R
title_sort	purification and characterization of an aspartic protease from the rhizopus oryzae protease extract, peptidase r
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2014
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582014000200006
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Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R

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