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
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.
Chromatography; Endopeptidase; Food processing industry; Homogeneity; Rhizopuspepsin