Purification and Characterization of Acidic Protease from Aspergillus oryzae

• Main Authors: Ya-Hui Chou, 周雅惠
• Other Authors: Shann-Tzong Jiang
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/27994342267188144416

碩士 === 國立臺灣海洋大學 === 食品科學系 === 92 === Abstract In order to purify and characterize the acidic protease from Aspergillus oryzae BCRC 30118, Asp. oryzae ME broth after 3 days cultivation at 25℃ was collected. After removing the cells, the crude enzyme was concentrated using Amicon ultrafiltration (cutoff: 10 kDa). The acid protease was purified after DEAE Sephacel and Sephacryl S-200 HR chromatographs. The specific activity, yield and purification fold of the resulted solutions were 121606 units/mg, 15.05% and 6.86 fold, respectively. The MW of purified acidic protease was 41 kDa estimated by SDS-PAGE. The optimal pH and temperature for the enzyme activity were 3.0 and 60℃, respectively. The purified enzyme was stable at pH 3.0-6.0 and 4-35oC, respectively. It was inhibited by Fe2+, Hg2+, Fe3+ and Pepstatin A, and slightly by Leupeptin and TPCK. According to the substrate and inhibitor specificity, it was considered to be chymotrypsin-like protease. The activation energy of purified protease was 37.46 kcal/mole. The Km, Vmax and Kcat for the hydrolysis of hemoglobin were 0.12 mM, 14.29 m mole/min and 14.55 Sec-1, respectively.