Simiduia agarivorans SA1T所產蛋白酶之純化與特性探討

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題名:	Simiduia agarivorans SA1T所產蛋白酶之純化與特性探討 Purification and Characterization of a Protease from Simiduia agarivorans SA1T
作者:	曾彥綸 Tseng, Yan-Lun
貢獻者:	生藥學研究所李宗徽
關鍵詞:	Simiduia agarivorans SA1T;蛋白酶;pH;蛋白酶抑制劑;界面活性劑;金屬離子 Simiduia agarivorans SA1T;serine protease;pH value;protease inhibitor;surfactant;metal ion
日期:	2013-07-05
上傳時間:	2018-10-05 09:33:59 (UTC+8)
摘要:	Simiduia agarivorans SA1T 具良好蛋白酶活性，本研究對此菌株所產生的蛋白酶進行分離、純化並探討該酵素的一般性質。探討最佳化培養條件顯示，使用250 mL具凹槽錐形瓶，添加 0.75% tryptone peptone、pH 7.8、培養液 50 mL、25 °C 下，震盪培養 72 小時後，蛋白酶的活性最佳。粗酵素液經由硫酸銨沉澱、透析、並以SDS-PAGE單離出分子量約 120 kDa 的蛋白酶，其最適反應溫度和 pH 分別為 60 °C，和 pH 9；在溫度 4-37 °C 及 pH 8下，酵素有良好穩定性；當超過 37 °C，則蛋白酶無法穩定維持活性。此蛋白酶在 5 mM 酵素抑制劑 phenylmethanesulfonylfluride (PMSF) 的作用下活性明顯受到抑制，由此推測此酵素為絲胺酸型蛋白酶 (serine type protease) 。在界面活性劑影響方面，此蛋白酶在 SDS 含量超過 0.5% (g/mL) 時活性開始降低，SDS 濃度增至 2% 時活性完全受到抑制。在金屬離子影響方面， 5 mM 的 Mg2+ 和 Ca2+ 會提高蛋白酶的活性，而 Hg2+、Ni2+ 和 Cu2+ 則會降低蛋白酶活性。蛋白酶活性表現在 1.25% (w/v) NaCl下活性最佳，在 2.5% NaCl 下則不具耐受性；蛋白酶與基質液 (酪蛋白) 反應的最佳濃度為12.5 mg/mL。 A protease of Simiduia agarivorans SA1T was purified and characterized in this study. Optimal protease yielding conditions for the culture were determined. For the best protease production, the culture was incubated at 25 °C with 50 mL PY broth (pH 7.8) in 250 mL flask and shaken for 72 hours (75 rpm). Tryptone peptone (0.75%) was added as an additional nutrition in PY broth. The protease was purified by SDS-PAGE after ammonium sulfate precipitation, dialysis, and desalting. The molecular mass of the protease as determined by SDS-PAGE was approximately 120 kDa. The protease showed optimum activity at alkaline pH 9 and temperature 60 °C, and was stable at pH 8 and 4-37 °C, respectively. The protease was characterized as a serine protease, since it was inactivated by 5 mM phenylmethanesulfonylfluride (PMSF). When the protease was treated with various surfactants, the activity was completely inhibited by 2% (g/mL) SDS. Among the metal ions tested, the enzyme activity was found to be enhanced by 5 mM Mg2+ and Ca2+, and was inhibited when treated with 5 mM Hg2+, Ni2+ and Cu2+. The protease showed optimum activity at salt (NaCl) concentration 1.25% (g/mL). However, when salinity was increased to 2.5%, the protease lost 80% of its activity. The activity of this protease was increased with increases of substrate concentrations up to 12.5 mg/mL and was inhibited as the concentrations of substrates were more than 50 mg/mL.
描述:	碩士指導教授-李宗徽委員-謝文陽委員-林俊茂
資料類型:	thesis
顯示於類別:	[生藥學研究所] 博碩士論文

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