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標題: 硼酸官能基薄膜之製備及其在純化盤尼西林醯胺酵素上之應用
Preparation of Boronate Ligands Membrane and Its Application on Penicillin G Acylase Purification
作者: 馮皓翔
Feng, Hao-Hsiang
關鍵字: 間胺基苯硼酸;boronate affinity chromatography;盤尼西林醯胺酵素;固定化金屬親和薄膜;純化;m-aminophenylboronic acid;penicillin G acylase;immobilized metal affinity membrane;purification
出版社: 化學工程學系所
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本研究應用再生纖維薄膜(Regenerated cellulose membrane, RCM)為固體基材,使用前以epichlorohydrin (EPI)修飾RCM,再以化學合成方式接上間胺基苯硼酸官能基(m-aminophenylboronate ligand, m-APB),即為硼酸官能基薄膜(Boronate affinity chromatography, BAC),並利用BAC膜來純化盤尼西林醯胺酵素(Penicillin G acylase, PGA)。
首先探討在膜上反應m-APB之最佳條件,其結果為:3% m-APB (0.75 g)添加於25 ml, 20% acetonitrile中,反應條件為60℃, 100 rpm, 24 h, pH 8。使用SEM分析BAC表面型態,發現RCM長時間的化學反應會造成BAC薄膜的結構和孔洞分佈損壞。
以此最適化條件製作出的BAC膜,探討純化PGA之條件,其結果為: PGA (活性0.1 IU/ml)與BAC膜浸泡於25 ml loading buffer( 3 M NaCl, 10 mM phosphate buffer, pH 6)吸附12小時,接著使用10 ml elution buffer(1.5 M NH4Cl, 50 mM acetate buffer, pH 4.5),將BAC膜上PGA脫附,此操作之PGA純化倍率為9.63倍。
使用BAC膜將PGA粗酵素液進行第一步純化,再使用本實驗室研發之固定化金屬親和薄膜進行第二階段純化PGA酵素,經由二階段純化步驟,PGA純化倍率可達32.93倍,回收率為64.06%。使用SDS-PAGE分析蛋白質,可看出PGA之α-Chain及β-Chain在23 kDa與62 kDa顯示。

The regenerated cellulose (RC) membrane as solid matrix was modified by epichlorohydrin (EPI), followed by constructing the m-aminophenyl boronate ligand (m-APB). The modified membrane named as boronate affinity chromatography membrane (BAC) was used to apply on penicillin G acylase (PGA) purification.
The optimal preparation conditions were concluded as follows: for one RC-EPI membrane, 3% m-APB (0.75 g) and 25 ml 20% acetonitrile were added and the reaction conditions were carried out at 60°C, 100 rpm, pH 8 for 24h. From the surface morphologies scanned by SEM, the modified membrane had the structure and pore destruction for a long time chemical reaction.
In order to evaluate the BAC for purification of PGA, the crude enzymes were adsorbed to one piece of BAC in the presence of 25ml loading buffer ( 3 M NaCl, 10 mM phosphate buffer, pH 6). After that, Elution buffer (1.5 M NH4Cl, 50 mM acetate buffer, pH 4.5) were used to elute PGA and a 9.63-fold PGA purification was obtained.
Two-stage purification was presented by combining the BAC and IMAM (immobilized metal affinity membrane) for PGA purification. Under the elution conditious, a 32.93-fold PGA purification and 64.06% recovery were obtained.
其他識別: U0005-0402200913091200
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