| Summary: | 在近年來發展的純化技術中，固定化金屬親和薄膜(immobilized metal ion affinity membrane, IMAM)被廣泛應用在酵素純化上，其原因是由於比起傳統管柱層析系統來說，薄膜系統具有低成本、低壓降、易放大製程的優點。 南極假絲酵母(Candida antarctica)能夠生產兩種不同的脂肪酶，分別為脂肪酶A及脂肪酶B。關於脂肪酶B的研究已不勝枚舉，卻鮮少有對脂肪酶A的研究，但脂肪酶A在有機溶劑中有著比脂肪酶B更佳的水解活性、耐溫性，及更廣泛的pH耐受性，因此值得對脂肪酶A進行研究。 在本研究中，脂肪酶A經由大腸桿菌大量表現，並透過螯合Cu2+或Ni2+的固定化金屬親和薄膜來純化，且將純化效果與商業樹脂比較。另外，純化過程中緩衝溶液的pH值及NaCl的添加濃度也將一併探討。結果表明，最適於破菌及保存活性的緩衝溶液pH值為7，且添加NaCl對於抑制非特定吸附並無效果。評估Cu2+及Ni2+對於純化脂肪酶A的效果可以發現Cu2+具有較高的蛋白質吸附量，但選擇性則是Ni2+較好。最後，當使用IMAM-Ni2+純化脂肪酶A時可以得到61.32%的回收率及20.89倍的純化倍率；而使用商業樹脂則是69.24%及27.08倍。雖然IMAM-Ni2+的純化效能仍不及商業樹脂，但其最大的優勢是成本遠低於商業樹脂。 本研究亦嘗試將脂肪酶A以物理吸附、配位鍵結、共價鍵結等不同方式將其固定化於修飾再生纖維素薄膜表面；然而無論以何種方式固定化脂肪酶A於薄膜表面皆無法測得活性。 Among the recently developed purification techniques, immobilized metal ion affinity membrane (IMAM) has been widely applied in the enzyme purification processes with advantages such as low cost, low pressure drop and easier scale up, which limited the conventional packed-column systems. The yeast Candida antarctica produces two different lipases, lipases A and B. Many studies have focused on lipase B(CALB), whereas, rare about lipase A(CALA) was mentioned. It is worth to put effort on CALA owing to its hydrolytic activity in organic solvents, high temperature endurance and broad pH range in operation. In this study, CALA was overexpressed in Escherichia coli and purified via an IMAM chelated with Cu2+ and Ni2+, and the performance of IMAM was compared to the commercial resin packed in the column. The conditions for purification process such as the pH of buffer, concentration of NaCl, were also investigated. The results show that the best pH for lysis and storage was set as 7, and the addition of NaCl is not helpful for reducing nonspecific adsorption. As compare the efficacy of Cu2+ and Ni2+ on purifying CALA, we found that Cu2+ has a higher adsorption capacity of protein but a lower selectivity than Ni2+. The recovery and purification fold were 61.32% and 20.89 for IMAM-Ni2+, and 69.24% and 27.08 for commercial resin. IMAM-Ni2+ is not as efficient as the commercial resin, however, its low cost is much advantageous over the commercial resin. In this study we also tried to immobilize CALA on the modified regenerated cellulose membrane by physical adsorption, coordination bonding, covalent bonding. However, no CALA activities were found regardless of the immobilized methods. 摘要 i Abstract ii 圖目錄 vi 表目錄 viii 第一章、緒論 1 1.1前言 1 1.2研究動機與目標 2 第二章、文獻回顧 3 2.1脂肪分解酶 3 2.1.1簡介 3 2.1.2南極假絲酵母脂肪酶A(Candida antarctica lipase A) 7 2.2固定化金屬親和層析法 8 2.2.1簡介 8 2.2.2固體載體 9 2.2.3活化劑 10 2.2.4螯合劑 10 2.2.5金屬離子 10 2.3固定化金屬親和薄膜 12 2.4酵素固定化 13 第三章、實驗材料與方法 16 3.1實驗材料 16 3.1.1再生纖維素薄膜 16 3.1.2菌種 17 3.1.3實驗儀器及設備 18 3.1.4實驗藥品 19 3.1.5本實驗使用藥品之配製 20 3.2實驗方法 24 3.2.1固定化金屬親和薄膜之製備 24 3.2.2菌種培養及CALA酵素液之製備 24 3.2.3 CALA之酵素活性分析 26 3.2.4蛋白質定量分析 27 3.2.5固定化金屬親和薄膜純化CALA 28 3.2.6管柱層析純化CALA 30 3.2.7不同官能基之再生纖維素薄膜製備及固定化CALA 32 3.2.8固定化CALA之酵素活性分析 34 3.2.9蛋白質電泳分析(SDS-PAGE) 35 3.2.10傅立葉轉換紅外線光譜儀 36 3.2.11接觸角量測儀 36 第四章、結果與討論 38 4.1再生纖維素薄膜之改質 38 4.2不同pH緩衝溶液對於破菌後保留CALA活性及蛋白量之影響 40 4.3不同pH保存條件對於CALA活性之影響 42 4.4不同金屬離子對於CALA吸附量之影響 43 4.5使用Cu2+離子純化CALA 45 4.5.1時間對於IMAM-Cu2+吸附CALA及蛋白量之影響 45 4.5.2不同濃度NaCl對於IMAM-Cu2+吸附CALA及蛋白量之影響 46 4.5.3不同濃度(5 mM~25 mM)imidazole梯度洗脫效果 48 4.5.4不同濃度(25 mM~40 mM)imidazole洗脫效果 50 4.5.5不同濃度(40 mM~70 mM)imidazole洗脫效果 51 4.6使用Ni2+離子純化CALA 53 4.6.1不同濃度(20 mM~50 mM)imidazole梯度洗脫效果(IMAM-Ni2+) 53 4.6.2不同濃度(20 mM~50 mM)imidazole梯度洗脫效果(Resin-Ni2+) 55 4.6.3 IMAM-Ni2+與Resin-Ni2+純化效果之比較 56 4.7固定化CALA於修飾再生纖維素薄膜 58 4.7.1矽烷化對於再生纖維素薄膜親疏水性之影響 58 4.7.2不同固定方法對於CALA固定量及活性之影響 60 第五章、結論及未來展望 62 參考文獻 63 附錄 73
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