Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract,Peptidase R |
| |
| Institution: | 1. Institute of Biotechnology, National Changhua University of Education, Changhua 500, Taiwan;2. Department of Biotechnology, Hungkuang University, Taichung 433, Taiwan;3. Department of Life Science, National Tsing Hua University, Hsinchu 300, Taiwan;4. Department of Bioagricultural Science, National Chiayi University, Chiayi 600, Taiwan |
| |
| Abstract: | BackgroundAspartic 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.ResultsAn 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.ConclusionBased 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. |
| |
| Keywords: | |
| 本文献已被 ScienceDirect 等数据库收录！ |
|
