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Investigation on the stability and regeneration of penicillin G acylase immobilized in immobilized metal affinity membrane
|關鍵字:||immobilized metal affinity membrane|
penicillin G acylase
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|摘要:||本研究以再生纖維素薄膜（regenerated cellulose-based membrane）為固體載體，先使用EPI（epichlorohydrin）活化再生纖維素薄膜，使其表面帶有高反應性之環氧基，再利用化學合成的方式接上IDA（iminodiacetic acid）螯合劑，即為固定化金屬親和薄膜（immobilized metal affinity membrane, IMAM），並利用所得之金屬親和薄膜純化醱酵液中之盤尼西林醯胺酵素（Penicillin acylase, 簡稱PGA）。
第一部分探討再生纖維素薄膜表面改質之最適化條件及純化PGA之效果；在使用EPI活化薄膜過程中，NaOH濃度提高有利反應進行，但NaOH濃度在高於1.6M後，易造成再生纖維素薄膜變質，故EPI活化薄膜最適條件為20ml 1.4M NaOH, 5ml EPI, 24℃, 150rpm, 14h；而在使用IDA螯合劑之最適條件為25ml 1M IDA, 1M Na2CO3, pH 11, 24℃, 120rpm, 12h。以此條件改質之薄膜，銅離子螯合量為75μmol/disc、吸附PGA為1.8 IU/disc。所得之固定化酵素膜不需經長時間培育步驟，即可達到配位鍵結固定化酵素，此薄膜可在40天內，37℃重覆使用16次，不使用時保存含0.1﹪NaN3之PB（potassium phosphate buffer, pH 8）中，其PGA殘存活性可維持99﹪以上。而將10片固定化酵素膜置於流動式反應器純化PGA， PGA之回收率則可達97.1%，純化倍率為21.31倍。
在固定化酵素膜再生清洗步驟為，先使用Strip buffer（300mM NaCl, 100mM EDTA, 20mM Sodium phosphate buffer, pH8）浸泡30min去除膜上銅離子，接著0.5M HCl、0.5M NaOH分別浸泡10min去除膜上化合物、疏水性和親脂性蛋白質，經再生5次後，膜上PGA酵素活性殘存率仍有99﹪以上。|
The regenerated cellulose-based membrane (RC membrane) was employed to construct the immobilized metal affinity membrane (IMAM) for purification and immobilization of penicillin G acylase (PGA). This research is constituted of two parts. The first part was the preparation of IMAM on the RC membrane and the purifications of the PGA by using IMAM. For the preparation of IMAM, factors such as chelator density on the surface, epoxy functional group, NaOH concentration and elution solutions were investigated. The epichlorohydrin (EPI) reaction was enhanced as increasing NaOH concentration. However, the RC membrane was deteriorated when the NaOH concertation was higher than 1.6M. The optimal reaction conditions were concluded as follows: for one RC membrane, 5ml EPI and 20ml 1.4M NaOH was used and the reaction conditions were 24℃, 150rpm for 14h. After that, the membrane was immersed in 25ml of 1M IDA (dissolved in 1M sodium carbonate) and allowed to react at 24℃ for 12h. The amount of Cu2+ chelated on IMAM under this condition was 75μmol/disc and the adsorbed PGA was 1.8 IU/disc. By applying this conditions to the flow experiments in a cartridge, a 21.31-times of purification in specific activity with 97.1﹪recovery for PGA purification was obtained. Meanwhile, the activity of immobilized PGA could be retained for more than 40 days under 16-times repeated use at 37℃. The second part was to discuss the regeneration procedures of immobilized enzyme membrane. The regeneration was conducted by immersing the immobilized enzyme membrane in the strip buffer (300mM NaCl, 100mM EDTA, 20mM Sodium phosphate buffer, pH8)for 30 min to elute Cu2+ ion on the membrane. Then the membrane was immersed in 0.5M HCl and 0.5M NaOH respectively for 10 min to wash out the residual reactants, hydrophobic protein, etc. If was found that even under regeneration 5 times, the activity of the immobilized PGA membrane still could be maintained above 99%. This means a repeated use of the PGA immobilized on the membrane is feasible with a marked stability on the immobilized PGA activity.
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