역미셀을 이용한 단백질의 역추출과 퍼콜레이션 프로세스

이성식, 홍동표*, 료이치 쿠보이*, 이봉국

동아대학교 화학공학과

오사카대학교 화학공학과*

Back-Extraction of Proteins Using Reverse Micelles and Percolation Processes

Sung-Sik Lee, Dong-Pyo Hong*, Ryoichi Kuboi*, Bong-Kuk Lee

Dept. of Chem. Eng., Dong-A Univ., 840 Hadan-dong, Pusan 604-714, Korea

Dep. of Chem. Sci. & Eng., Osaka Univ., Toyonaka Osaka 560-8531, Japan*

1. INTRODUCTION

Reverse micelles are spherical aggregates of surfactant molecules embracing micro-water pools in the organic solvent [1], and have a capability to solubilize a variety of biomolecule into the nanometer-size water pools. Therefore, the reverse micellar systems (RVMS) have a variety of potential such as extraction systems of proteins, hydrophobic reaction media for enzymes and preparation media for functional materials.

Using RVMS for extraction systems, the back-extraction is more difficult compared with the forward extraction, because proteins are denatured and the back-extraction fraction decreases significantly when the protein-micellar interactions are too strong. Selective extraction is achieved by the control of the interactions such as electrostatic, hydrophobic, steric and affinity interactions [2]. Therefore, these interactions must be studied thoroughly to explain the dependence of pH in the back-extraction and the stability of RVMS solubilizing proteins.

A percolation process reflects these interactions and it can be quantified by the measurement of the electrical conductivity of the RVMS. The electrical conductivity measurements have been used to assess RVMS formation and probe the structural changes occurring in such systems [3,4]. The shape increase in conductivity caused by the percolation phenomenon well demonstrates the interaction between the micelles. It is generally accepted that the conductivity percolation in RVMS with a spherical droplet structure is a result of reverse micellar droplet clustering [4,5,6]. The cluster formation of micelles increases the electrical conductivity after the percolation threshold which indicates the starting point of the reverse micellar droplet clustering.

Different additives such as synthetic and natural polymers can vary the percolation threshold. It has also been demonstrated that the solubilization of proteins favors the percolation process with an increase the conductivity at lower or higher water content and temperature, suggesting stronger or weaker attractive interaction between reverse micelles in the presence of protein [6]. In the present paper, back-extraction of the various proteins solubilized into AOT/isooctane reverse micelles and their percolation phenomena have been studied focusing the formation of micellar cluster via protein-micellar and micellar-micellar interactions by the solubilization of proteins and pH.

2. EXPERIMENTAL

2.1. Materials

AOT (sodium di[2-ethylhexyl] sulfosuccinate) of purity 95% from Tokyo Kasei Co. (Tokyo, Japan) was used without further purification. 2,2,4-Trimethylpentane (Isooctane) was purchased from Ishizu Seiyaku (Osaka, Japan). Bovine serum albumin (BSA, pI 4.9), bovine carbonic anhydrase B (CAB, pI 5.9), □-lactoglobulin (□-LG, pI 5.2), hemoglobin (HEMO, pI 6.8) from bovine, cytochrome c (CYT, pI 10.1) from horse heart, and lipase (pI 4.3) from Candida cylindrasea were purchased from Sigma (St. Louis, Mo, USA). Catalase (pI 5.5) from bovine liver and lysozyme (LYS, pI 11.1) from hen egg white were obtained from Wako Pure Chemical (Osaka, Japan).

2.2. Back-extraction of proteins

The proteins were solubilized into 200mM AOT/isooctane solution by the injection method [5,6]. The value of Wo (=[H2O]/[AOT]) of the reverse micellar solution was kept at 20 in all the back-extraction experiments. Back-extraction of proteins from the reverse micelles was carried out by contacting reverse micellar solution containing proteins with new aqueous phase (pHaq) containing 0.1 M KCl. The pH value of feed solution injected into the reverse micelles, pHinj, varied in these experiments. Protein concentrations were determined by using an UV spectroscopy (UV-1600A Shimadzu). The back-extracted fraction of protein, Eb, was determined from the concentration of the protein back-extracted into the aqueous phase and the concentration of protein solubilized in the reverse micelle.

2.3. Percolation processes

The conductivity of RVMS was measured as a function of water content (□aq) with a conductivity meter CM-40V (TOA Electronics Ltd.). The electrode was inserted into the test tube containing the reverse micellar solution and the tube was placed in a thermostated water bath. Electrical conductivity measurements were performed with dropwise addition of an aqueous phase to 0.2 M AOT/isooctane solution until the percolation phenomenon was observed. As shown schematically in Fig.2, individual droplets maintain a low conductivity, while the conductivity increases as droplets start to assemble in clusters. The percolation threshold with and without proteins defined as the starting point of the sharp increase in conductivity is abbreviated as □p or □p.p according to the previous paper [5, 6].

Fig.1 Effect of pHinj on the back- extracted fractions of various proteins. [AOT] = 0.2M, [KCl] = 0.1M

3. RESULTS AND DISCUSSION

3.1. Back-extraction of proteins

After the protein was solubilized at various pHinj, back-extraction was carried out under the same condition of pHaq. The back-extracted fraction of protein into aqueous phase, Eb, is plotted against the pH deviation from pI (isoelectric point) of the protein as shown Fig.1. In the case of some proteins such as BSA, catalase, lipase and □-LG were easily back-extracted into the new aqueous phase. On the other hand CYT, HEMO and LYS were comparatively difficult to back-extract. These results can be explained by the influence of the protein-micellar interaction. In general, it is known that CYT have a localized plus charged site as a membrane protein. Therefore, the low Eb of CYT in the contrast with those of other proteins can explain the result of the electrostatic strong interaction between the localized plus charged site of CYT and the anionic surfactant. The back-exacted fractions were strongly influenced by the pHinj values in the feed protein solution. The effect of pH on back-extraction of proteins could also be explained by the electrostatic interaction between the solubilized proteins and anionic surfactants, AOT. Therefore, the control of protein-micellar interaction may become a very important factor for the success of the back-extraction processes of proteins.

3.2. Effect of solubilized proteins on percolation processes

Specific interfacial properties, which affect micellar-micellar interaction in the AOT RVMS, have been studied by using percolation process of micelles. Fig.2 show the variation of electrical conductivity (□) as a function of the volume fraction □aq of the aqueous phase solubilized into the AOT reverse micelles in the presence of proteins.  In the case of CYT, the electrical percolation threshold (□p.p) relatively decreased more than the percolation threshold (□p) of protein-free systems with the increase of the concentration of the solubilized CYT into the reverse micelles. These results indicate an increase in the clustering formation of reverse micelles by attractive interaction between micelles. On the other hand, the solubilization of BSA shows a decreasing effect of the clustering formation of reverse micelles by attractive interaction between micelles. These results suggest that the micellar-micellar interactions are notably influenced by the concentration and species of proteins solubilized in the reverse micelles.

Fig.2 Effect of CYT (open key) and BSA (closed key) solubilization on the percolation process of RVMS. Protein concentrations are 0 mg/ml (□), 3 mg/ml (△,▲) and 5 mg/ml (○,●), respectively at pH8.

The difference, □□p(=□p.p-□p ), show the effect of the protein concentration on the percolation process. Here, □p.p and □p are the values of the percolation threshold with and without proteins, respectively. This shows a linear relationship between the relative percolation threshold and solubilized protein concentration, Cpr (data not shown). The slope, □pr, is a measure of the effect of protein solubilization on the attractive interaction between micelles. The values of □pr means the structural change of reverse micelles that become the formation of micellar cluster by the micellar-micellar interaction.

3.3. Correlation of the back-extraction and percolation processes

The values of □pr are plotted in Fig.3 against the pH deviation of the injected solution from pI of each proteins. The values of □pr are affected by pH of the solution, that is by some electrostatic interactions between the micelles and proteins. The positive and higher values of □pr means the suppression of the micellar cluster formation with attractive interaction between micelles by solubilized proteins. We define that these proteins are of type A such as catalase, lipase, □-LG and BSA. On the contrary, the negative or lower of □pr promote the micellar cluster formation. We define that these proteins are of type B. CYT, LYS and HEMO have the negative or lower values of □pr. HEMO shows either increase of the micellar cluster formation or decrease of the micellar cluster formation depending on the solution pH of protein. These results show that the values of □pr reflect the micellar cluster formation depending on the pH of the protein solution. It is assumed that the micellar cluster formation is strongly affected by the properties of proteins such as the molecular weight, charge density and surface hydrophobicity of the proteins [5,6]. The percolation processes with proteins can be utilized to evaluate and understand the protein-micellar interaction and structural changes of the reverse micelles.

Fig.3 Effect of pHinj on the percolation processes of various proteins. [AOT] = 0.2M

There were also two groups in the back-extraction behaviors of proteins (Fig.1). When the comparison between □pr and Eb, the proteins belong to the type A with the high values of □pr have relatively the high back-extracted fraction (Eb). On the contrary, the proteins belong to the type B have the low Eb. It was found that a strong interaction between the solubilized protein and micelles induces the micellar-micellar interaction or micellar cluster formation, resulting in the decrease of back-extracted fractions. Therefore, the percolation process induced by the solubilization of proteins is very useful to explain the behaviors of back-extractions as well as the protein-micellar interactions.

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