- 期刊
- OpenAccess
Purification and Characterization of Acidic Protease from Aspergillus oryzae
Aspergillus olyzae酸性蛋白酶之純化與生化特性
摘要
本研究之目的在探討自Aspergillus oryzae中分離酸性蛋白酶與其生化特性。Aspergillus oryzae BCRC 30118菌株在ME broth培養液中，於25℃、150 rpm下振盪培養3天，收集胞外酵素液，測其蛋白酶活性約為370units/mL。將粗酵素液經濃縮、透析後，進行DEAE Sephacel及Sephacryl S-200 HR管柱層析可得到具有活性之酸性蛋白酶，其比活性為121606 units/mg，回收率為15.05%，純化倍率為6.86倍。經SDS-PAGE電泳分析呈現單一色帶之純化酸性蛋白酶，其分子量為41kDa。最適酸鹼度為pH3.0，酸性蛋白酶在pH 3.0-6.0之間有較佳之安定性。最適溫度為60℃，酸性蛋白酶在40℃以下安定較佳。純化之酸性蛋白酶會受到Fe(上標 2+)、Hg(上標 2+)及Fe(上標 3+)金屬離子的抑制。純化之酸性蛋白酶會明顯的被Pepstatin A抑制，而TPCK和Leupeptin則會輕微的抑制酵素活性。酸性蛋白酶的活化能為37.5kcal/mole。酸性蛋白攁對血紅素之Vmax為14.3mol/min；Km為0.12mM；Kcat為14.6sec^(-1)。
並列摘要
In order to purify and characterize the acidic protease from Aspergillus oryzae BCRC30118, Asp. oryzae ME broth after 3 days cultivation at 25℃ was collected. After removing the cells, being concentrated by Amicon ultrafiltration (cutoff: 10 kDa), DEAE Sephacel and Sephacryl S-200 HR chromatographs, the acidic protease was purified to electrophorectical homogeneity. The MW of purified acidic protease was a monomer with a molecular mass of 41 kDa. The optimal pH and temperature were 3.0 and 60℃, respectively. It was stable pH 3.0-6.0 and ＜40℃, and moderately inhibited by 5.0 mM of Fe(superscript 3+), leupeptin and TPCK, strongly inhibited by Fe(superscript 2+), Hg(superscript 2+) and pepstatin. The apparent Km and Vmax of the purified acidic protease for hemoglobin were estimated to be 0.12 mM and 14.3 mol/min, respectively. 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/mol.