알코올과 지방산이 첨가된 AOT/isooctane 역미셀 시스템을 이용한

단백질의 역추출 프로세스

이봉국, 홍동표*, 료이치 쿠보이*, 이성식

동아대학교 화학공학과

오사카대학교 화학공학과 *

Protein Back-extraction Process Using Alcohol and Carboxylic acid Mediated AOT/isooctane Reverse Micellar Systems

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

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

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

1. INTRODUCTION

The reverse micellar systems (RVMS) have been widely used for protein extraction systems composed of the bulk aqueous and micro aqueous phase, which is surrounded by surfactant and dispersed in the organic phase. In general, using RVMS for extraction systems, the distribution of proteins between a micellar organic phase and an aqueous phase is largely determined by the conditions in the aqueous bulk phase, namely pH, ionic strength and types of salt. The parameters related to the organic phase also influence the partition of protein, such as the concentration and type of surfactant, presence of cosurfactant, type of solvent and so on. By controlling these parameters, the extracted fraction can be varied via variations of protein-micellar electrostatic, hydrophobic and steric interactions. However, since the proteins are subjected to denaturation, the back-extracted fraction decreases significantly when the protein-micellar interactions are so strong.

In order to improve the back-extraction process, many studies have been reported using various methods [1-4]. Although the back-extraction process on RVMS is a difficult process, it may be carried out successfully to control the properties or structures of reverse micelles. Dungan et al.[5] explain that in the reverse micellar extraction processes, particularly in the back-extraction process, the micellar-micellar interaction has to be considered to be one of the important factors. We expect that the alcohol and carboxylic acid molecules could be a good modifying agent to control the micellar-micellar interaction. In our previous papers [6, 7] we have shown that the interaction between micelles, reflecting the properties or structures of reverse micelles, could be easily evaluated by a percolation phenomenon of RVMS. We have also examined the effect of various alcohols on the RVMS using the percolation process [8]. The percolation processes can be varied by different additives. It has been also demonstrated that the solubilization of proteins clearly affects the percolation process with a rapid increase in the conductivity at lower or higher water content, suggesting that stronger or weaker attractive interactions between micelles in the presence of proteins [6, 7].

In the present paper, we have examined the micellar-micellar and protein-micellar interaction as a key factor governing the protein back-extraction processes and its control systems by using various alcohols and carboxylic acids. We also measured the change of the back-extracted fraction and activity yield of CAB by the addition of a small amount of alcohol and carboxylic acid molecules.

2. EXPERIMENTAL

2.1. Materials

AOT(sodium di[2-ethylhexyl] sulfosuccinate) of purity 95% was purchased from Tokyo Kasei Co. and used without further purification. Bovine carbonic anhydrase B (CAB, pI 5.9) were purchased from Sigma Chemical Company. Butanol (BuOH), pentanol (PenOH), hexanol (HexOH), octanol (OctOH), butanoic acid (BuCOOH), pentanoic acid (PenCOOH), heptanoic acid (HepCOOH) and octanoic acid (OctCOOH) were purchased from Wako Pure Chemical. p-Nitrophenyl acetate (p-NPA) was supplied from Wako Pure Chemical.

2.2. 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 with and without alcohols or carboxylic acids solution until the percolation phenomenon was observed.

2.3. Back-extraction of proteins

The proteins were solubilized into AOT (0.1 M)/isooctane solution by the injection method following the description in the previous paper [6, 7]. The buffer solution containing the protein (1×10-4 M) was injected into AOT/isooctane solution (Wo = 20) and the mixture as shaken vigorously until a clear solution was obtained. Back-extraction of the protein from the reverse micelles was carried out by contacting reverse micellar solution containing the protein with buffer solution (pHaq) containing 0.1 M KCl for 4 hours reached an equilibrium state. Similar experiments were also carried out using a system containing alcohol or carboxylic acid. The activity of CAB was determined by measuring the ester hydrolysis rate using p-NPA as the substrate [9]. CAB-containing sample (50□L) was added to 1mL of 50 mM Tris-HCl buffer solution (pH 7.5) containing 5 mM EDTA and 1 mM p-NPA. After mixing, the increase in absorbance at 348 nm, which reflects the formation of p-nitrophenol, was monitored continuously with a UV spectrophotometer at room temperature. The initial rate of hydrolysis (measured for the first 25 s after a 5 s lag time) was taken as a measure for the activity of CAB.

3. RESULTS AND DISCUSSION

3.1. Control of micellar clustering formation by alcohol and carboxylic acid

The percolation processes are effective for the evaluation of the micellar-micellar interactions [6, 7]. The electrical percolation threshold decreases with the addition of BuOH into the RVMS as shown in our previous study [8]. However, the addition of PenOH shows a decreasing effect on the attractive interaction between micelles. These results suggest that the micellar-micellar interactions are notably influenced by the alcohol species and concentration. We also examined the effect of carboxylic acids in order to compare with alcohols.

Carboxylic acids with long alkyl chains than PenCOOH suppressed the micellar clustering better than PenCOOH, and carboxylic acids with short alkyl chains than BuCOOH enhanced the micellar clustering better than BuCOOH. These results are similar to the case of alcohol in our previous study [8]. We also examined the effect of the various alcohols such as diols and halogenols on the suppression of micellar clustering, indicating that the alcohol effects can be explained by the additive contribution of each group where hydrocarbon and halogen groups contribute positively and OH groups contribute negatively [8].

In the case of carboxylic acids, hydrocarbon groups also contributed positively and COOH groups contributed negatively (data not shown).

3.2. Difference between alcohol and carboxylic acid on percolation processes

Fig.1 Effect of CAB solubilization of the percolation processes of (a) AOT (0.2M)-OctOH and (b) AOT-OctCOOH RVMS: [CAB] = 1×10-4M

The effect of CAB solubilization on the percolation processes of RVMS added OctOH and OctCOOH was shown in Fig.1. In the case of OctOH, the protein solubilization shifted the percolation thresholds (□t.p) to a higher value of □aq than that of the protein-free systems (Fig.1 (a)). This indicates that the solubilization of CAB contribute to the suppression of micellar-micellar interactions or micellar clustering in the OctOH mediated RVMS. On the other hand, for the system containing OctCOOH, the values of □t.p decreased to the lower values of □aq rather than □t as shown in Fig.1 (b). This result shows that the solubilization of CAB into the RVMS containing OctOH increases in the protein-micellar interaction inducing the formation of micellar clustering. The reason is not known clearly, but it is considered probably as one reason that the OctCOOH have the higher factor of denaturation than the factor of denaturation by OctOH.

Therefore, this result indicates that the effect of carboxylic acid on the micellar clustering could be evaluated quantitatively by using the percolation process of RVMS as found for alcohol molecules, suggesting carboxylic acids may also be a good modifying agent to control the micellar-micellar interactions.

3.3. Effects of alcohols and carboxylic acids addition on back-extraction behaviors of protein

We have also examined the effect of alcohols and carboxylic acids addition on the back-extraction of CAB. Fig.2 shows the changes of back-extracted fraction and activity yield by the addition of alcohols and carboxylic acids in organic phase. The back-extracted fraction and activity yield were varied with the addition of a small amount of various alcohol or carboxylic acid. In the case of OctOH and HexOH, the interface between organic and aqueous phase was very clear with the addition of alcohols, and aggregate of surfactant and protein was not observed. And then, both back-extracted fraction and activity yield were increased with the increase of alcohol concentrations. This result suggests that the back-extraction behaviors can be improved by a small amount of alcohol. On the other hand, carboxylic acids decreased in the back-extracted fraction as well as activity yield with the increase of their concentrations by the additions of OctCOOH and HepCOOH, and it is observed plentifully a precipitate of surfactant and protein aggregate at the interface. This is considered owing to the increase of protein-micellar interaction should by the percolation processes in Fig.1 (b).

Fig.2 Changes of back-extracted fraction (open key) and activity yield (closed key) of protein by the addition of alcohols and carboxylic acids

From these results, therefore, it could be shown that these variations of back-extracted fraction and activity yield are markedly influenced by the alcohols and carboxylic acids, affecting the micellar-micellar or protein-micellar interaction, and they could be explained by the percolation processes of RVMS containing alcohol or carboxylic acid.

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