Purification and characterization of an aspartic protease from the Rhizopus Oryzae protease extract, peptidase R

Nai Wan Hsiao, Yeh Chen, Yi Chia Kuan, Yen Chung Lee, Shuo Kang Lee, Hsin Hua Chan, Chao Hung Kao

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38 Citations (Scopus)

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 languageEnglish
Pages (from-to)89-94
Number of pages6
JournalElectronic Journal of Biotechnology
Volume17
Issue number2
DOIs
Publication statusPublished - 2014 Mar

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Applied Microbiology and Biotechnology

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