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|Title: ||Aspergillus oryzae 酸性蛋白酶之純化與生化特性|
Purification and Characterization of Acidic Protease from Aspergillus oryzae
|Authors: ||Ya-Hui Chou|
|Contributors: ||NTOU:Department of Food Science|
|Issue Date: ||2011-06-30T07:40:42Z
|Abstract: ||中文摘要 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370 units/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41 kDa。最適酸鹼度為pH 3.0，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35℃以下安定較佳。純化之酸性蛋白酶會受到Fe2+、Hg2+及Fe3+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37.46 kcal/mole。酸性蛋白酶對血紅素之Vmax為14.29 □□mole/min，Km為0.12 mM，Kcat 為14.55 Sec-1。|
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 □□mole/min and 14.55 Sec-1, respectively.
|Appears in Collections:||[食品科學系] 博碩士論文|
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