Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3177
標題: 以酵素法生產海藻糖與製程純化之研究
Enzymatic process for the Production and Purification of Trehalose
作者: 吳宗達
Wu, Tsung-Ta
關鍵字: 海藻糖的純化;trehalose synthesis;固定化海藻糖合成酶;固定化澱粉葡萄糖化酶;固定化葡萄糖氧化酶;trehalose purification;immobilized trehalose synthase;immobilized glucoamylase;immobilized glucose oxidase
出版社: 化學工程學系所
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摘要: 
海藻糖為一種多功能的非還原雙醣,具低卡路里與新風味之優點,已逐漸成為可取代蔗糖與麥芽糖的食品調味劑。因海藻糖具有保濕性與穩定蛋白質之功能,又可做為化妝品保濕劑與食品抗凍劑。雖然海藻糖廣泛存在於自然界生物中,但其含量稀少無法直接取得。文獻提及合成海藻糖的方法有數種,僅有酵素合成法較符合生產成本,其中以海藻糖合成酶直接催化麥芽糖轉成海藻糖的製程較具有工業化量產之潛力。源自Picrophilus torridus海藻糖合成酶轉殖於大腸桿菌後,可經由大腸桿菌大規模生產海藻糖合成酶。因海藻糖合成酶在進行基因重組時,植入了poly-His tag的基因序列,其基因重組海藻糖合成酶的poly-His tag可與含有金屬離子的螯合吸附基材擔體進行配位共價鍵固定化反應。比較六種金屬離子的金屬螯合吸附材之擔體可知,雖然Cu(II)-loaded adsorbent吸附蛋白質的能力最高,蛋白質承載量為5.39 ± 0.03 mg/g gel,但其吸附海藻糖合成酶的專一性卻不如其他種金屬離子。然而,Co(II)-loaded adsorbent吸附海藻糖合成酶呈現出高度的專一性,其純化倍率為Cu(II)-loaded adsorbent的四倍以上。海藻糖合成酶經由固定化反應後,其最適反應的pH值、反應溫度與穩定性與自由態海藻糖合成酶相較並無顯著差異。固定化海藻糖合成酶於最適的反應條件之下進行麥芽糖的轉換反應6 h後,可獲得64%的海藻糖轉換率。固定化海藻糖合成酶經由24次重複操作後,尚有80%的殘存活性,經由蛋白質活性分析可知,酵素活性的下降係由海藻糖合成酶的脫附所致。然而,此製程中尚有26 mM麥芽糖與20 mM葡萄糖的存在,需進行麥芽糖與葡萄糖的移除或分離,增加海藻糖之純度。
為了簡化海藻糖的純化製程,本研究接續發展一套結合固定化澱粉葡萄糖化酶水解麥芽糖與固定化葡萄糖化酶氧化葡萄糖的製程,有效地將殘存的麥芽糖與葡萄糖轉換成葡萄糖酸。在固定化澱粉葡萄糖化酶的部分,因CM Sepharose的蛋白質承載量(49.35 ± 1.43 mg/g gel)與固定化酵素的酵素總活性(1141.89 ± 31.89 U/g gel)高於EupergitR C,故選用CM Sepharose做為澱粉葡萄糖化酶之固定化擔體。澱粉葡萄糖化酶經由固定化反應後,其最適反應pH值和溫度與自由態澱粉葡萄糖化酶相同,無顯著差異。當固定化澱粉葡萄糖化酶於最適的反應條件之下進行麥芽糖的水解作用,反應40 min後即可完全地將殘餘麥芽糖水解轉換成葡萄糖。在固定化澱粉葡萄糖化酶的穩定性方面,經過80個批次反應後,酵素活性仍含有92%的殘存活性;根據此趨勢推算,固定化澱粉葡萄糖化酶可於操作110個批次反應後,尚有80%的的殘存活性,此結果說明固定化澱粉葡萄糖化酶具有高度的穩定性。在固定化葡萄糖氧化酶的部分,使用環氧基聚丙烯酯Immobead 150做為葡萄糖氧化酶之擔體。當葡萄糖氧化酶進行共價鍵固定化反應時,提升離子濃度有助於酵素承載量的增加。葡萄糖氧化酶經由固定化反應後,其最適反應的pH值、反應溫度以及穩定性與自由態葡萄糖氧化酶相較並無顯著差異。固定化葡萄糖氧化酶於最適的反應條件之下,反應90 min後可完全地將殘存的葡萄糖轉換成gluconolactone,再水解成葡萄糖酸。在操作穩定性的部分,固定化葡萄糖氧化酶於第12個批次反應後活性驟然下降,推測應為過氧化氫引起的不可逆之抑制作用所致。最後再以陰離子交換樹酯,移除海藻糖液體中的葡萄糖酸,獲得高純度的海藻糖。
URI: http://hdl.handle.net/11455/3177
其他識別: U0005-0608201317050900
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