製備固定化金屬親和薄膜對盤尼西林醯胺酵素之純化與固定化探討

陳志義

doi:10.6845/NCHU.2009.00957

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製備固定化金屬親和薄膜對盤尼西林醯胺酵素之純化與固定化探討

Preparation of the Immobilized Metal Ion Membrane for Penicillin G Acylase Purification and Immobilization

陳志義 , Ph.D　　Advisor：劉永銓　　

繁體中文 DOI： 10.6845/NCHU.2009.00957

盤尼西林醯胺酵素；固定化金屬親和薄膜；最適化條件；製備程序；雙官能基之EPO/Cu膜；再生程；固定酵素膜； Penicillin G acylase ； Immobilized metal affinity membrane ； Enzymes purification ； regeneration process ； immobilized PGA membrane ； bifunctional membrane

Abstract │ Reference (77) │ Times Cited (1) │ Altmetrics

Abstract 〈TOP〉

盤尼西林醯胺酵素（penicillin G acylase, PGA）是一重要生化觸媒，使盤尼西林G（penicillin G, PG）進行水解反應生成六青黴素酸（6-aminopenicillanic acid, 6-APA），它和一些有機物以有機合成反應形成β-lactam抗生素。在全球巿場，每年有數千噸β-lactam抗生素需求量應用於治療方面。因此製備出高活性與穩定性之PGA對於6-APA生產是很重要。近年來純化技術發展，固定化金屬親和薄膜（immobilized metal affinity membranes, IMAM）己被應用於酵素純化領域，因為它較一般填充床系統無顆粒內部擴散問題，且具有短軸擴散途徑、壓力降低和易規格化等優點，因此完整探討最適化之IMAM製備程序和再生程序以得到更佳IMAM之功能是有必要性。夲研究共分三個部份。
第一部份針對製備IMAM具高量金屬離子和蛋白質吸附能力之方法。NaOH濃度是再生纖維素膜 (RC膜) 與Epichlorohydrin (EPI) 之間接合反應的最重要關鍵步驟，在最適化反應條件: 一片RC膜浸泡於20 ml、 1.4 M NaOH及5 ml EPI溶液，以24 °C、150 rpm 反應14 h進行製備。製備完成之IMAM膜有高吸附75.5 ± 0.25 μmol Cu2+/disc 、1044 μg lysozyme/cm2 and 1.8 U PGA/disc之量，這些值高於相關文献[1]之研究結果。使用10片IMAM裝置於流動式層析之筒匣內，在18℃下，以10 mM、 pH 6 之PB buffer為負載液，1.5 M NH4Cl為洗滌液，以流動式層析進行PGA純化試驗，有97% PGA 回收率和21.31 倍純化倍率之結果。
      第二部份針對雙官能基之EPO/Cu膜 (epoxy and immobilized copper ion membrane，簡稱EPO/Cu)對PGA 純化與固定化之吸附機制探討。Iminodiacetic acid disodium (IDA)濃度是製備雙官能基之EPO/Cu膜的最重要關鍵地方，當使用0.05M IDA製備之EPO/Cu膜，僅螯合29.9 
μmol Cu2+/disc, 是螯合飽和量之58% ，但此EPO/Cu膜歷時2個月26次純化與固定化測試，有96.3% PGA固定在EPO/Cu膜。
       第三部份針對固定酵素之IPM膜(the immobilized PGA membrane ，簡稱IPM)之再生方法。由製備完成之IMAM膜吸附PGA成IPM膜，探討IPM膜之PGA穩定性與再生方法。IPM膜放入4 &ordm;C之DI water內，經10天連續測試後IPM之PGA活性剩下19%，同樣地，若放入10mM PB buffer (含0.1% NaN3, pH8) 經10天連續測試後，IPM之PGA活性維持在99%以上，非常穩定。在再生方面，使用100mM EDTA當刮除液，經連續4次再生步驟後，IPM固定PGA能力只剩下52%，同樣地，若使用各刮除溶液浸泡，如100mM EDTA (300mM NaCl, 20mM PB, pH8)浸泡30 min， 接著浸於25ml、0.5 M HCl與25ml、 0.5 M NaOH各10 min等為再生程序，經連續5次再生程序後， IPM固定PGA活性仍維持在99%以上，非常穩定。

Parallel Abstract 〈TOP〉

Penicillin G acylase (PGA) is an important bioenzyme to hydrolyze penicillin G into 6-aminopenicillanic acid (6-APA) which could react with some organic chemicals by organic synthetic process to form β-lactam antibiotics. The global market demand of β-lactam antibiotics reaches thousand of tons annually for medical treatment. However, the preparation of highly active and stable PGA is thereby critical for 6-APA production. Among the recently developed purification techniques, immobilized metal ion affinity membrane (IMAM) has been widely applied in the enzyme purification processes with advantages such as no intra-particle diffusion, short axial-diffusion path, low pressure drop and easier scale up, which limited in conventional packed-column systems. To achieve better IMAM performance, a thorough study for optimizing the IMAM-preparation procedures and regeneration process is required. In this paper, it contains three parts.
In the first part, an approach to prepare IMAM with high adsorption capacities of metal ions and protein was developed. NaOH concentration is found to be the most influential one for the reaction of coupling epichlorohydrin (EPI) to the regenerated cellulose membrane (RC membrane). According to the criterion, the optimal reaction conditions were found as follows: a RC membrane immersed in 20 ml, 1.4 M NaOH, 5 ml EPI and operated at 24 °C, 150 rpm for 14 h. Under this condition, the copper ions and PGA in IMAM were significantly increased to 75.5 ± 0.25 μmol/disc and 1.8 U/disc respectively. The adsorption amount for lysozyme on the prepared IMAM reached 1044 μg/cm2, which was the highest in the literature. We could get the result of 97.1% recovery for PGA purification, which is 21.3-fold than before, by loading 10 pieces of IMAM into the cartridge, conducting the flow chromatography at 18 &ordm;C with 10 mM, using pH 6 sodium phosphate buffer as loading buffer and 1.5 M NH4Cl as elution buffer, a 21.3-fold purification with 97.1% recovery for PGA purification was obtained.
In the second part, the bifunctional membrane (epoxy and immobilized copper ion) was constructed to study the effect on PGA purification and immobilization. It is found that iminodiacetic acid disodium (IDA) concentration has the critically effect on the bifunctional group ratio in the membrane. When IDA concentration was 0.050 M, the chelated copper ion would reach 29.9 μmol/disc, nearly 58% of its saturation. After purification and immobilization cycling process 26 times within two month, 96.3% of the PGA activity could be still retained.
In the third part, the stability and regeneration of immobilized PGA membrane (IPM) by using pre-IMAM to immobilize PGA reversibly is investigated. IPM was stored in deionized water (DI) at 4 &ordm;C, its residual activity was 19 % left of the original one after 10 days. However, when stored in 10mM phosphate buffer (PB, pH8) with 0.1% NaN3, the IPM can be more stable which retains 99% of its activity after 10 days testing. In regeneration, the re-immobilization capacity for PGA is only 52% left of the original one after 4 times regeneration by using 100mM EDTA as the stripping buffer. When the regeneration process was modified by immersing in 100mM EDTA (300mM NaCl, 20mM PB, pH8) for 30 min, in 25ml, 0.5 M HCl for 10 min, then rinsed twice with DI water and finally immersed in 25ml, 0.5 M NaOH for 10 min. The resulting IMAM can reserve 99% PGA immobilized activity even after 5-times regeneration.

Reference ( 77 ) 〈TOP〉

[1] H. L. Hu, M. Y. Wang, C. H. Chung, S. Y. Suen, Purification of VP3 protein of infectious bursal disease virus using nickel ion-immobilized regenerated cellulose-based membranes, J Chromatogr B (2006) 840 76-84.
連結：
[2] P. Knight, Downstream Processing, Nat Biotechnol (1989) 7 777-82.
連結：
[3] R. Ghosh, Protein separation using membrane chromatography: opportunities and challenges, J Chromatogr A (2002) 952 13-27.
連結：
[4] S. Y. Suen, Y. C. Liu, C. S. Chang, Exploiting immobilized metal affinity membranes for the isolation or purification of therapeutically relevant species, J Chromatogr B (2003) 797 305-19.
連結：
[5] R. Freitag, H. Splitt, O.-W. Reif, Controlled mixed-mode interaction chromatography on membrane adsorbers, J Chromatogr A (1996) 728 129-37.
連結：

Times Cited (1) 〈TOP〉

林宜萱(2012)。磁性奈米粒子製備方法、特性及固定化應用之評估分析。中興大學化學工程學系所學位論文。2012。1-102。

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