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標題: Purification and immobilization of penicillin G acylase using multifunction membrane
作者: 黃振翁
Huang, Cheng-Weng
關鍵字: purification;純化;immobilization;multifunction membrane;penicillin G acylase;固定化;多官能基薄膜;盤尼西林醯胺酵素
出版社: 化學工程學系
This research is constituted of two parts. The first part was to study the preparation of surface modification of the regenerated cellulose membrane to yield an immobilized metal affinity membrane(IMAM), and the second part was to study the application of the IMAM to enzyme immobilization. For the preparation of IMAM, immobilized metal ion(Cu2+)was used to determine the proper conditions for enzyme covalently immobilized, and to construct a multifunctional membrane(MFM) for enzyme immobilization. In this work, the MFM was used to study the effect of penicillin G acylase(PGA)purification and immobilization.
It is shown that when the iminodiacetic acid(IDA)was increased on the multifunctional membrane, the immobilized copper ions were increased, and the maximum content of copper ions adsorption could reach 57 mmol∕disk. The results of copper ions adsorbed amount indicated that IDA and epoxy were coexist on the membrane. The results showed that when using 0.05 M IDA reacted with membrane at 24℃ for 12 h, then incubating for 120 h to immobilize PGA. The resulting immobilized membrane could be reused 38 times at 37℃ for 60 days, and even the copper ions were eluted from the membrane with ethylenediamine tetraacetic acid disodium salt dihydrate (EDTA), still 80% residual activity could be maintained.

本研究分為二部份,第一部分探討薄膜表面改質之製備條件,第二部份探討薄膜改質後,在酵素固定化上之應用。首先,利用固定化金屬離子(Cu2+)分別探討薄膜改質之條件,以達到適合酵素共價固定之條件,並完成多功能基薄膜之製備。然後利用此多功能基薄膜,其表面同時具備有環氧基(epoxy)及螯合銅離子(immobilized copper ion)兩種官能基之性質,來進行盤尼西林醯胺酵素(penicillin G acylase, PGA)純化及固定化的探討。
當使用高濃度螯合劑(iminodiacetic acid, IDA)於製作多功能基薄膜時,有較高之銅離子吸附量,其最大銅離子吸附量可達57 mmol∕disk。以銅離子的吸附結果,可以確認當使用0.05 M IDA時,多功能基薄膜表面尚有環氧基存在。經由實驗結果可知,當多功能基薄膜之製作條件在溫度24℃、IDA濃度為0.05 M,反應12小時後,可用來進行PGA酵素純化,然後,經由培養120小時來固定化PGA酵素,所得固定化薄膜可在二個月中,於37℃中,重複使用38次,甚至利用0.1 Methylenediamine tetraacetic acid disodium salt dehydrate(EDTA)將銅離子脫附,其PGA的最高活性殘存率仍可達80%以上。
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