蛋白質在黏土奈米混成材中之吸脫附行為的研究

Study on the Adsorption and Desorption Behavior of Proteins in the Nano-hybrid Clay Materials

徐兆稹(Chao-Chen Hsu)

指導教授 ： 蔡宗燕

中原大學/理學院/化學研究所/博士(2014年)

https://doi.org/10.6840/cycu201400545

摘要

不同的蛋白質通常具有不同的等電點，因此改變蛋白質水溶液的酸鹼值可以改變蛋白質的帶電性質。本研究選用天然黏土及改質型黏土作為吸脫附蛋白質的載體，因此能夠透過載體與不同蛋白質吸脫附能力的差異性進行蛋白質吸脫附行為探討進而達到分離之應用。
無機層狀材料因層板晶格缺陷帶有電荷，在蒙脫土層板帶負電，層間為了維持電荷平衡帶有正電，則可藉由離子交換進行蛋白質吸脫附，利用蛋白質等電點在不同pH值的電性轉換的特性，當蛋白質在環境pH值小於等電點時，蛋白質帶正電，因電荷吸引而吸附在層板間，則當環境pH值大於等電點，蛋白質為負電，與無機層板之電荷互斥進行脫附行為。
天然蒙脫土以有機改質劑椰油醯胺丙基羥基磺基甜菜鹼(Cocamidopropyl Hydroxysultaine, CS)和椰油醯胺丙基甜菜鹼 (Cocamidopropyl Betaine, HP)製備改質型黏土，以離子交換法將上述兩種改質劑分別與天然黏土表面進行有機化改質，利用X光繞射儀（X-ray Diffraction, XRD）觀察無機層材之層間距變化，傅立葉轉換紅外線光譜儀（Fourier Transform Infrared, FT-IR）鑑定改質蒙脫土層間之有機與無機的官能基，證明CS、HP的長碳鏈改質於蒙脫土層間，層間距最高可增加至21.71 Å。以熱重分析儀（Thermogravimetry Analyzer, TGA）定量分析改質蒙脫土中改質劑的插層量皆控制在50 % ± 5 %後再藉由界面電位分析儀 (Zeta Potential Analyzer)分析改質前後無機層材之電性差異，證明改質前後之無機層材皆具有負電荷。
將其添加於牛血清蛋白(Bovine Serum Albumin, BSA)溶液後以紫外光-可見光光譜儀(UV-Visible Spectrophotometer, UV-Vis)檢測，無機層材改質後能吸附的BSA 量分別為259.7 mg/ g及187.3 mg/ g，脫附量分別為95.1 mg/ g及106.0 mg/ g，脫附率分別為36.6 %及56.6 %，並利用等溫吸附方程式及吸附動力學方程式做理論計算。再將BSA 和雞蛋白溶菌酶 (Lysozyme) 混合後測試，並藉由十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (SDS-PAGE electrophoresis) 檢測，利用磷酸緩衝溶液脫附後也能將BSA 脫出，因此說明無機層材具蛋白質純化的應用潛力。
接著將無機層材對紅螢光蛋白進行吸脫附之探討，其結果利用螢光光譜儀 (Fluorescence Spectrophotometer, FL) 檢測，以CL111-HP改質型黏土吸附率可以達到94.2 %，而脫附率可達到84.7 %，為最佳脫附之效果，而CL111-HP飽和吸附度為紅螢光蛋白水溶液濃度75 abs.，吸附時間約為10 min時可達到最佳吸附效果，然而CL111-HP生命週期只能使用一次，最後藉由SDS-PAGE檢測，可得知已成功將改質型黏土應用於紅螢光蛋白之分離上。

關鍵字

黏土 ； 牛血清蛋白 ； 紅螢光蛋白 ； 吸附

並列摘要

The natural clay Montmorillonite is inorganically layered material and possesses lattice defects with negative charges. Neutralization of the positively charged protein molecules by the negative charges of the layered lattices in the clay can be applied to achieve the adsorption and desorption of these protein molecules. Protein solution can be prepared at an optimal pH value comparing its respective isoelectric point (pI). As soon as the environmental pH is less than the pI value, the protein is to be positively charged, and will be adsorbed within the lattice formed template; as soon as the environmental pH is adjusted higher than pI, the protein becomes negatively charged and will be easily desorbed.
Montmorillonite as natural clay was organically modified into “Cocamidopropyl Hydroxysultaine (CS)” and “Cocamidopropyl Betaine (HP)” by two different ion exchange methods, respectively. Change of the space of the lattice layers in the inorganic natural and modified clays can be observed by X-ray diffraction (XRD). Organic and inorganic functional groups of the long carbon chains modified into the montmorillonite layers as CS and HP can be well identified by Fourier Transform Infrared (FT-IR). The quantitative analysis of the intercalation capacity of CS and HP as well as the modified natural clay can be processed by Thermogravimetry analyzer (TGA). The surface charge densities as well as zeta potentials of the natural and modified lattice surfaces can be analyzed. Preparation of CL111-HP was also described here.
Selective adsorptions and desorptions of proteins ─ BSA, Lysozyme, and RFP by means of their characteristic isoelectric points have been investigated using natural (montmorillonite) and modified (CS, HP and CL111-HP) clays. Separation of these proteins has been successfully demonstrated by Fluorescence spectrometry, UV, and SDS PAGE.
BSA adsorption capacities of 259.7 mg/g and 187.3 mg/g, desorption of 95.1 mg/g and 106.0 mg/g in gram of either CS or HP, as well as 46.6 % and 56.6 % on the lattice templates of CS and HP, respectively, have been measured by UV with wavelength 280 nm. The adsorption isotherm Langmuir equation was used for the theoretical comparison. Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis of CS and HP mixed with both BSA and lysoyzme solutions was used to demonstrate the desorption ability of BSA comparing that of lysoyzme from both modified clay lattices. Such inorganic layer or lattice materials are well applicable for the protein purification has been demonstrated.
Furthermore, red fluorescent protein (RFP) and CL111-HP also modified from natural clay were used instead of either BSA or lysozyme combined with CS and HP for the fluorescence experiment. Rates of adsorption and desorption were reached and optimized to 94.2 % and 84.7%, respectively. 75 abs./mL was achieved as the best degree of saturation adsorption of RFP. It takes only 10 mins for the optimal adsorption. However, the modified CL111-HP to purify RFP can be used for only once.

並列關鍵字

red fluorescent protein ； adsorption ； clay ； bovine serum albumin

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