Abstract
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 andhad 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.
| Original language | English |
|---|---|
| Pages (from-to) | 89-94 |
| Number of pages | 6 |
| Journal | Electronic Journal of Biotechnology |
| Volume | 17 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - 2014 Jan 1 |
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All Science Journal Classification (ASJC) codes
- Biotechnology
- Applied Microbiology and Biotechnology
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Purification and characterization of an aspartic protease from the Rhizopus Oryzae protease extract, peptidase R. / Hsiao, Nai-Wan; Chen, Yeh; Kuan, Yi Chia; Lee, Yen Chung; Lee, Shuo Kang; Chan, Hsin Hua; Kao, Chao Hung.
In: Electronic Journal of Biotechnology, Vol. 17, No. 2, 01.01.2014, p. 89-94.Research output: Contribution to journal › Article
TY - JOUR
T1 - Purification and characterization of an aspartic protease from the Rhizopus Oryzae protease extract, peptidase R
AU - Hsiao, Nai-Wan
AU - Chen, Yeh
AU - Kuan, Yi Chia
AU - Lee, Yen Chung
AU - Lee, Shuo Kang
AU - Chan, Hsin Hua
AU - Kao, Chao Hung
PY - 2014/1/1
Y1 - 2014/1/1
N2 - 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 andhad 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.
AB - 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 andhad 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=84896362750&partnerID=8YFLogxK
U2 - 10.1016/j.ejbt.2014.02.002
DO - 10.1016/j.ejbt.2014.02.002
M3 - Article
AN - SCOPUS:84896362750
VL - 17
SP - 89
EP - 94
JO - Electronic Journal of Biotechnology
JF - Electronic Journal of Biotechnology
SN - 0717-3458
IS - 2
ER -