Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/95991
標題: Expression optimization, enzyme characterization and product investigation of recombinant levansucrsae produced by Bacillus subtilis and surface-displayed spore
以枯草桿菌及孢子表層展示技術表現重組果聚糖蔗糖酶之最佳化及其酵素特性與產物探討
作者: Szu-Ni Chen
陳思霓
關鍵字: 益生質;孢子表層展示;乳果寡醣;重組果聚醣蔗糖酶;prebiotics;spore surface diplay;lactosucrose;recombinant levansucrase
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摘要: 
益生質(prebiotics)在食品工業中為一種功能性成分,使腸道內菌叢組成產生明顯變化,選擇性促進乳酸菌或雙叉桿菌的增加,以及降低病原菌和腐生菌,進而有益於宿主之健康狀態。功能性寡糖為益生質的其中一種,而果寡醣及乳果寡醣皆歸類於功能性寡糖之中,目前最具前景之制備功能性寡糖方式為酵素催化之糖基轉移反應合成。枯草桿菌(Bacillus subtilis)為 GRAS (generally recognized as safe)級之菌種,枯草桿菌在惡劣的環境下(例如:UV照射、溶解酶、加熱)生成孢子。先前實驗利用B. subtilis(pSECS-3sacB)表現重組蛋白,本實驗中將重組果聚醣蔗糖酶加入含有22.5%蔗糖及22.5%乳糖之pH=3之緩衝溶液、於50℃反應,分別在0、1和5小時添加25ug / ml之重組果聚糖酶,隨後於反應的3小時將pH值調整為6、於30℃下反應,得結果於反應第2小時乳果寡糖生成量約為298g/L為目前最高。另外,本實驗利用孢子表層展示的技術將目標蛋白錨定於孢子表面,使用枯草桿菌孢子Crust層之CotZ蛋白進行構築,並設計Linker (GGGGGG)以增加其暴露於表層。成功構築出質體型Bacillus subtilis WB800 pCotZ-L-SacB以及Bacillus subtilis WB800 pCotZ-SacB。利用質體型孢子之重組果聚醣蔗糖酶進行酵素特性探討,最適作用pH值為5,在pH 3-pH 5之下酵素活性皆有80%以上。最適作用溫度Linker組孢子為40℃、無-Linker組為50℃,而熱穩定性Tm值皆為55℃。其作用的條件皆比Free form ( Bacillus subtilis WB800 (pSECS-p3sacB))來的優化,後續希望能利用孢子可重複使用的特性,作為固定化酵素模式生成乳果寡醣
URI: http://hdl.handle.net/11455/95991
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