| 摘要: | 本研究之主要目的在探討巨大孔徑幾丁聚醣膜的製備及其表面特性改質對酵素親和性之影響並藉由薄膜上金屬的摻合作為架橋，再將其固定於具特定生化功能的酵素以了解固定化酵素再利用之可行性。此外本研究亦針對幾種於生化和環境污染物降解上應用廣泛之酵素進行固定化基礎和應用研究，以解決目前固定化基材孔洞過小，易碎無法成形，不耐酸鹼以及再利用性和親水性不佳等缺失。
本研究以矽膠為固定支撐物並利用相轉化的製備方法將幾丁聚醣交聯於矽膠表面以形成巨大孔徑的幾丁聚醣薄膜。幾丁聚醣膜作為固定基材之前其表面必須先以高分子化合物進行交聯以使幾丁聚醣膜表面功能化。實驗結果顯示在製備巨大孔徑之幾丁聚醣膜時所利用之幾丁聚醣與粒徑為60-200 µm矽膠比例為1:25時為最佳製備條件，利用PMI測得平均孔洞大小為0.1623 µm，推論係因幾丁聚醣分子結構交互作用而使分子內之氫鍵產生作用，當矽膠含量增加則Si-O-Si 的吸收更加強烈，隨著矽膠含量的增加，孔洞強度明顯增加。利用不同之交聯劑(1,4 butanediol diglycidyl ether、Glutaraldehyde及 Epichlorohydrin)交聯幾丁聚醣膜，1,4 butanediol diglycidyl ether與 Epichlorohydrin需先開環以利與幾丁聚醣產生交聯，利用 FTIR測得之-OH、-NH2等官能基伸縮強度為最強，推斷此三種交聯劑皆有多個線性相互鍵結形成網絡結構，因此可以更有效地使幾丁聚醣膜上之官能基發揮到最佳效果。以13C NMR分析鑑定，可以確定改質後幾丁聚醣膜本身的官能基增加，以未改質之幾丁聚醣膜為基準，利用交聯劑1,4 butanediol diglycidyl ether之胺基為4.88 ，Glutaraldehyde之胺基為0.14 ，Epichlorohydrin之胺基為3.20。羧基的部分以1,4 butanediol diglycidyl. ether改質後之羧基為2.73 ，Glutaraldehyde改質後之羧基為1.34 ，Epichlorohydrin改質後之羧基為4.43，推論 1,4 butanediol diglycidyl ether及Epichlorohydrin開環後大部分接枝於幾丁聚醣膜矽膠上，因此可測得大量的胺基，但 Glutaraldehyde僅測得少數胺基，可能係由於大部分的胺基與幾丁聚醣膜相互反應所造成之結果。
游離與固定化酵素的活性實驗發現，游離Laccase之最適活性為pH5(8.01 %)，在摻合不同交聯劑後最適活性以 Epichlorohydrin為pH 7(4.46 %)之效果最佳，推論是因為其於Epichlorohydrin交聯時的官能基羧酸增加而無法有多的空間固定化Laccase所致。游離Tyrosinase之最適活性為pH 5(1.74 %)，在摻合不同交聯劑後最適活性1,4 butanediol diglycidyl ether為pH 7(0.45 %)之效果最佳，可能是因為幾丁聚醣會吸附H＋離子，使固定化酵素對於H＋離子產生斥力，若Tyrosinase與幾丁聚醣膜之距離愈近，則固定化酵素對於H＋離子之斥力越大，因此需要更高之H＋離子濃度。
固定化酵素再利用實驗，藉由FTIR觀察幾丁聚醣膜交聯改質前後官能基的變化，證明交聯劑與幾丁聚醣膜上的胺基產生鍵結。當金屬溶液pH值愈小，胺基質子化程度愈高，可架橋金屬離子的胺基數目就愈少使得金屬離子的吸附能力降低，但固定Tyrosinase後於低pH值(pH3)時的效果最佳，推測經固定Tyrosinase的幾丁聚醣膜其胺基減少而羧酸官能基增加。;The main objective of this study was to evaluate the preparation and modification of macroporous chitosan membrane affecting the surface properties of membrane and the affinity for the specific biochemical function enzymes. In this study, silica gel was used as support for the preparation chitosan crosslinked surface to form a macroporous of chitosan membrane by phase inversion methed. Chitosan membrane surface as a substrate before fixing must be first crosslinked to make surface functionalized on the chitosan membrane. Experimental results show that silica particle size of 60-200 μm at a ratio of 1:25 was optimum conditions to prepare the macroporous chitosan membrane. The measurment of PMI average pore size 0.1623 μm indicated that the molecular structure of chitosan with hydrogen bonding interactions within the molecule which effect increasing with the content of silica increased. From SEM (Scanning Electron Microscopy) results, the surface structure, the roughness and pore distribution arrangement of the chitosan membrane could be revealed. The use of different cross-linking agents (1,4 butanediol diglycidyl ether, Glutaraldehyde and Epichlorohydrin) to form crosslinked chitosan membranes revealed that 1,4 butanediol diglycidyl ether and Epichlorohydrin must be first open-ring to facilitate the cross-linking of chitosan. This is confirmed by the reserved peak shift of both OH and NH2 group in the FT-IR spectra. In 13C NMR analysis indicated that amine and carboxyl group are significantly increased after membrane surface modification.
The effect of various parameters such as temperature and pH on the relative activity of both free and immobilized enzymes was also studied in details. The relative enzyme activity upon immobilization was greatly enhanced several folds of its original activity. The stability of enzymes over a range of pH was significantly improved by immobilization. The immobilized enzyme possessed good operational stability and reusability properties that support its potentiality for practical applications. This decreased activity is probably due to the repulsion between positive surface of chitosan membrane surface and Tyrosinase.
The results of FT-IR, NMR, UV–vis, and SEM analyses revealed the effect of the presence of silica gel as a support could provide a large surface area, and therefore, the enzyme could be immobilized only on the surface, and thus minimized the diffusion limitation problem. The resultant enzyme immobilized membranes were also characterized based on their activity retention, immobilization efficiency, and stability aspects.The immobilization process increased the activity of immobilized enzyme even higher than that of total (actual) activity of native enzyme. Thus, the obtained macroporous chitosan membranes in this study could act as a versatile host for various guest molecules. |
