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dc.contributor.authorChen, Chin-Wenen_US
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M., A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 1976. 72 248. [54] Hu H.-L., Wang M.-Y., Chung C.-H. and Suen S.-Y., Purification of Vp3 Protein of Infectious Bursal Disease Virus Using Nickel Ion-Immobilized Regenerated Cellulose-Based Membranes. Journal of Chromatography B, 2006. 840(2) 76-84. [55] Pavia D. L., Lampman G. M. and Kriz G. S., Introduction to Spectroscopy : A Guide for Students of Organic Chemistry. Third Edition ed. 2001, USA. Appendix 1. [56] van Oss C. J., Good R. J. and Chaudhury M. K., Nature of the Antigen-Antibody Interaction: Primary and Secondary Bonds: Optimal Conditions for Association and Dissociation. Journal of Chromatography: Biomedical Applications, 1986. 376 111-119. [57] Melander W. R., El Rassi Z. and Horvath C., Interplay of Hydrophobic and Electrostatic Interactions in Biopolymer Chromatography : Effect of Salts on the Retention of Proteins. Journal of Chromatography A, 1989. 469 3-27. [58] Product Note: Mep Hypercelzh_TW
dc.description.abstract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吡啶衍生物(Pyridine derivatives, PDs),即為疏水官能基薄膜(Hydrophobic ligands membrane,HLM),並利用HL膜來純化盤尼西林醯胺酵素(Penicillin G acylase,PGA)。 首先挑選三種含疏水官能基之PDs:2-mercaptopyridine (2-MP)、3-picolylchloride (3-PC)及4-(mercaptoethylmpyridine (4-MEP)來製備HL膜,由實驗發現4-MEP對PGA有較佳之特異性吸附(純化倍率為1.91)。因此進一步探討在膜上反應4-MEP之最佳條件,其結果為:2.54 g 4-MEP添加於25 ml, 1 M Na2CO3中,反應條件為60°C, 24 h, 150 rpm。接下來的研究發現修飾有1,8-diaminoctane延伸臂可增加4-MEP對PGA親和力,得到最佳之PGA純化倍率為2.80。使用SEM分析薄膜表面型態,改質前後的薄膜有相似的結構和孔洞分佈。 以此最適化條件製作之HLM探討PGA純化之最佳條件,其結果為: 25 ml, 50 mM Phosphate buffer (pH 6)溶液中含PGA(活性0.2 IU/ml)和2 M (NH4)2SO4,與一片HL膜吸附12小時,接著再使用10 ml, 50 mM Acetate buffer (pH 4),內含1 M NH4Cl,將膜上之PGA脫附,此操作之PGA純化倍率為3倍。 使用本實驗室研發之固定化金屬親和薄膜,將PGA粗酵素液進行第一步純化,再使用HLM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50.62倍,回收率為45.4%。使用SDS-PAGE分析蛋白質,可看出許多雜蛋白已被分離,證明二階段式純化PGA之可行性。zh_TW
dc.description.abstractThe regenerated cellulose (RC) membrane as solid matrix was modified by epichlorohydrin (EPI), followed by constructing the pyridine derivatives (PDs) as the hydrophobic ligand. The modified membrane named as hydrophobic ligands membrane (HLM) was used to apply on penicillin G acylase (PGA) purification. Three kinds of PDs: 2-mercaptopyridine (2-MP), 3-picolylchloride (3-PC) and 4-mercaptoethylpyridine (4-MEP) was selected as hydrophobic ligands to construct the HLM. The HLM with 4-MEP ligand for PGA purification having the highest purification fold (1.91 times) was selected in this study.The optimal preparation conditions were concluded as follows: for one RC-EPI membrane, 2.54 g 4-MEP and 25 ml 1 M Na2CO3 were added and the reaction conditions were carried out at 60C, 150 rpm for 24h. Further study showed that adding a spacer arm in the modification step could incrase the HLC affinity to PGA. From the surface morphologies scanned by SEM, the original membrane and the modified membrane had the similar structure and pore distribution. In order to evaluate the HLM for purification of PGA, the crude enzymes were adsorbed to the HLM in the presence of 25ml, 50 mM phosphate buffer (pH 6) containing 2 M (NH4)2SO4, After that, 10 ml, 1 M NH4Cl and 50 mM acetate buffer (pH 4) were used to elute PGA and a 3.03-fold PGA purification was obtained. A two-stage purification was presented by combining the immobilized metal affinity membrane and HLM for PGA purification. Under the elution conditious, a 50.62-fold PGA purification and 45.4% recovery were obtained.en_US
dc.description.tableofcontents摘要 I Abstract II 總目次 III 圖目錄 VI 表目錄 VII 縮寫符號查詢表 VIII 壹、緒論 1 ㄧ、前言 1 二、研究目的 2 貳、文獻回顧 4 ㄧ、疏水作用層析簡介 4 (一) 基材的種類 5 1. 親水性吸附劑 5 2. 疏水性吸附劑 5 (二) 疏水官能基的選擇及其對蛋白質之選擇性 6 (三) 其他層析技術之發展 9 1. 親硫層析(TIC) 9 2. 疏水電荷誘導層析(HCIC) 10 3. 固定化金屬親和層析法(IMAC) 12 二、HIC吸附機制及其影響因素 12 (一) 蛋白質結構及其穩定度 13 (二) 吸附基材、延伸臂的種類 13 (三) 鹽類種類及濃度 14 (四) 溫度 16 (五) 其他因素 16 三、PGA酵素純化 16 (一) 固定化金屬親和層析法 (IMAC) 18 (二) 疏水電荷誘導層析(HCIC)之蛋白質純化 19zh_TW
dc.subjecthydrophobic ligandsen_US
dc.subjectpenicillin G acylaseen_US
dc.subjectimmobilized metal affinity membraneen_US
dc.titlePreparation of Hydrophobic Ligands Membrane and Its Application on Penicillin G Acylase Purificationen_US
dc.typeThesis and Dissertationzh_TW
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item.openairetypeThesis and Dissertation-
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