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標題: Application of Immobilized HRP in Industrial Wastewater Treatment
固定化 HRP 於工業廢水處理之應用
作者: 賴奕成
Lai, Yi-Cheng
關鍵字: covalent immobilization;共價鍵結法;HRP;辣根過氧化酵素
出版社: 化學工程學系
Immobilized HRP was developed to remove the 4-chlorophenol from industry wastewater. The optimal conditions for immobilization and for enzyme activity were determined. Porous aminopropyl glass bead (APG) was selected as the support for the covalent bonding of HRP. The optimal conditions were determined as: conjugation with 2.5% glutaraldehyde at 25℃ for 8 hours followed by 2.5mg immobilization with HRP/mL at 4℃ for 12 hours. The optimal reaction pH for the immobilized HRP ranged from 7~9, one unit pH wider than the free HRP. The optimal reaction temperature was raised to 70℃. The thermo stability (50~80℃) of the immobilized HRP was also improved significantly.
The removal efficiency of 4-chlorophenol from waste water by immobilized HRP was also investigated in our research. The optimal 4-chlorophenol removal efficiency was observed at pH 7.5. The results represented that at low concentration of HRP, the removal efficiency of 4-chlorophenol was increased, about 2.7 fold by the addition of PEG. Meanwhile, the removal efficiency of 4-chlorophenol was also enhanced by the inclusion of PEG in the muti-cycle operation. This study has successfully demonstrated that combining immobilization of HRP and PEG addition can increase operation stability.

本研究主要目的是利用固定化辣根過氧化酵素 (horseradish peroxidase, HRP),進行廢水中4-氯酚之去除。取具丙基胺基之孔洞玻璃珠 (aminopropyl glass beads, APG) 為擔體,利用共價鍵結法固定HRP,探討最適固定化反應條件與固定化酵素性質,結果顯示:固定化酵素程序中,以APG加入2.5 % 戊二醛溶液於25℃下反應8小時後,與 2.5 mg/mL 酵素液於4℃鍵結12小時,可得最佳酵素鍵結率及活性。在固定化酵素性質上,最適反應pH值為7~9與自由態酵素(pH 8~9)相比,往酸性方向增加一個單位;最適反應溫度與自由態酵素相比可提高至70℃;至於熱穩定性方面,固定化酵素自50~80℃皆比自由態酵素有較高之活性。
在去除廢水中4-氯酚試驗上,分別探討反應pH值、反應時間及酵素濃度等,本研究中去除廢水中4-氯酚之最佳條件為固定化HRP濃度為 0.5 U/mL在pH 7.5下,反應3小時,而4-氯酚去除率在重複批次反應上,可維持8個循環,操作時間長達24小時。同時本研究亦進行添加聚乙二醇 (polyethylene glycol, PEG) 於4-氯酚廢水中,結果顯示在低固定化HRP濃度並加入PEG,4-氯酚之去除率增加至2.7倍,且添加PEG於重複批次反應的探討,在長時間下4-氯酚之去除率亦明顯的增加。因此本研究結合固定化酵素及加入PEG之特性,可大幅延長操作時間並減少酵素用量。
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