重組地衣芽孢桿菌鹼性絲胺酸蛋白酶之特性及其於米蛋白之應用

Characteristics of the Recombinant Alkaline Serine Protease from Bacillus licheniformis and its Application on Rice Proteins

10.6342/NTU201602302

蘇香萍

地衣芽孢桿菌 ； 鹼性絲胺酸蛋白酶 ； 枯草桿菌蛋白酶 ； 原胜肽 ； 選殖 ； 米蛋白 ； 蛋白水解 ； Bacillus licheniformis ； alkaline serine protease ； subtilisin ； pro-peptide ； cloning ； rice protein ； protein hydorlysis

國立臺灣大學園藝暨景觀學系學位論文

2016年

碩士

許輔

繁體中文

微生物鹼性絲胺酸蛋白酶廣泛應用於食品業、洗滌業及飼料業，又以 Bacillus sp. 所產之枯草桿菌蛋白酶最為重要。本研究成功將 Bacillus licheniformis ATCC 14580 之鹼性絲胺酸蛋白酶基因 (apr) 選殖於 Escherichia coli 中，此序列全長為 1,140 bp，具 379 個胺基酸。經胺基酸序列比對後，與 subtilisin Carlsberg 具高度同源性，因此，推測 apr 亦為 pre-pro-mature 之酵素結構。透過選殖去除本身 pre-peptide，為 pro-mature-peptide 之 aprO 及僅含 mature peptide 之 aprM 於表現載體 pET-22b(+)，並轉型至 E. coli BL21 (DE3)。依據 SDS-PAGE、Western blot、蛋白酶活性測試及質譜定序之結果，可知 pro-peptide 為蛋白酶形成活性結構之必要片段。將 E. coli BL21 pET-22b-aprO 之粗萃液純化後，可得具有活性且分子量約為 28.4 kDa 之重組蛋白 rHis-aprO。rHis-aprO 之最適溫度及 pH 值為 60℃ 及 pH 10，並於 50℃ 下具熱安定性及中、鹼性環境仍保持穩定活性。rHis-aprO 之蛋白酶活性會受 phenylmethylsulfonyl fluoride 影響而所抑制，判定其為一絲胺酸蛋白酶。另外，rHis-aprO 之活性會被 5 mM Ca2+、Mn2+ 及 Mg2+ 所提升，且於界面活性劑、ethylenediaminetetraacetic acid 及多種有機溶劑中具安定性。rHis-aprO 可水解牛血清白蛋白、酪蛋白、膠原蛋白及米蛋白。且 rHis-aprO 於米蛋白之水解率可達 11%。因此，rHis-aprO 具有應用於生產米蛋白水解物之潛力。

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