역상미셀을 이용한 수용액상의 단백질과 금속이온의 분리

박성은, 김형준, 이봉국, 김광, 이성식

동아대학교 화학공학과

Separation of Proteins and Metal ions in Aqueous Solution

Using Reversed Micellar System

Seong-Eun Park, Hyung-Jun Kim, Bong-Kuk Lee, Kwang Kim, Sung-Sik Lee

Department of Chemical Engineering, Dong-A University

INTRODUCTION

A reverse micellar solution is a thermodynamically stable mixture of water, oil and surfactant where the water regions are separated from oil by a monolayer of surfactant. The reverse micelles have a capacity to solubilize a varity of biomolecules and solutes into the monometer-size water pools. Reverse micellar systems have various potentials for separating and concentrating proteins and solutes being scaled up easily.

In the practical application of this separation method, it is important to elucidate the mass transfer mechanism for separation of proteins and metals by reverse micelles. However, there have been only a few papers on the interfacial transport processes of proteins and metal ions between a bulk aqueous and a reverse micellar phase.

In this study, the distribution equilibrium of metal ions and proteins into reversed micellar solution was examined using an anionic surfactant AOT with various pH condition. The solubilization of metal ions or proteins into reverse micelles was carried out by the stepwise injection method. And interactions between micelles were studied by their percolation precesses. Furthermore, forward extraction rate of metals or protein was discussed.

EXPERIMENTAL

1. Materials

AOT(sodium di[2-ethylhexyl] sulfosuccinate) was purchased from Tokyo Kasei Co. and was used without further purification. Bovine serium albumin(BSA, pI=4.9) and C.C.lipase(pI=4.3) were purchased from Sigma Chemical Co.. Cd(No3)2·4H2O, CuCl2·2H2O, CrCl3·6H2O were purchased from Hayashi Pure Chemical Industrial Ltd..

A used buffer solutions were 0.1N HCl - 0.1N Glycine (pH=1.10∼3.50), 0.1N NaOH - 1/100M NaH2C6H5O7 (pH=4.96∼6.69), 0.1N NaOH- 0.1N Glycine (pH=8.53∼12.90).

2. Methods

2-1. Phase transfer method

Fig.1 Stirred cell with a flat liquid           -liquid interface

Forward extraction of metal ions and proteins experimented a stirred cell with a flat liquid-liquid interface described in Fig.1. The cell is a glass cylinder of 8cm inner diameter and 10cm height, and is equipped with four baffle plates. Temperature is kept constant at 25±1℃ with a water jacket.

After the aqueous phase of 200cm3 was supplied to the cell, the reverse micellar phase of 200cm3 was poured slowly on it, and then both phases were stirred at 100rpm and in same direction. BSA and C.C.lipase concentrations in each phase was determined from absorption at 280nm by UV. Cadmium, copper and chromium concentrations were determined by Atomic absorption spectrometer (SHIMADZU-AA6901).

2-2. Stepwise injection method

Fig.2 Solubilization by stepwise injection     method and percolation measurement

The proteins or metals solubilized into AOT/isooctane solution by the stepwise injection method. The experimental method is shown in Fig.2. The electrode was inserted into the reverse micellar solution. Electrical conductivity measurements were performed with dropwise addition of an aqueous solution contaning protein or metal ion to 100mM AOT/isooctane solution until the percolation phenomenon was reached. The conductivity of reverse micellar systems was measured by Metrohm Herissau Switzerland, 644 Conducto-

meter.

RESULTS AND DISCUSSION

1. Separation of proteins and metals by phase transfer method

It is well known that the extraction of protein and metal ion from bulk aqueous solution to reverse micellar solution governed by steric, electroststic, hydrophobic and affinity interactions between the protein(or metal ions) and the micelles.

Solution containing 1mg/ml of protein(or 100ppm metal ions) was contacted with micellar solution at various pH's which controlled by buffer solution. Fig.3 shows the percent of protein transferred from the feed solution to the micellar solution with varying pH, for Bovine serium albumin(BSA, pI=4.9) and C.C.lipase(pI=4.3). As the pH of the aqueous solution is lowered below the isoelectric pH of the protein, there is almost 100% of the protein was extracted. The protein net charge changes from negative to positive as the pH of the aqueous solution is lowered past the protein's pI. Thus, these experiment data indicates that for the case of AOT micellar solutions, it is necessary that the proteins have a positive charge for significant solubilization to occur. AOT is an anionic surfactant, and the reversed micell will therefore have a surface of negative charge surrounding its pore core. Thus it appears that solubilization requires an attractive electrostaic interaction between the micelle and the protein.

In the separation of proteins and metal ions with reverse micellar solution, diffusion resitances through both aqueous and organic boundary film are crucial. And the solubilization kinetics at water-oil interface might become a serious problem. Thus it is nesscary to know the individual mass-transfer rate in the transfer cell. Fig.4 shows the effect of pH on initial phase transfer rate of metal ions. The forward initial phase transfer rate increased with pH value and varied as the first power of pH.

Fig.3 Effect of pH on protein separation

(proteins conc.:1mg/ml,

AOT/isooctane : 100mM, Temp.:25℃)

Fig.4 Effect of pH on initial phase transfer

rate of metal ions

(AOT/isooctane : 100mM, Temp.:25℃)

2. Solubilization of Proteins or metal ions by stepwise injection method and its      effect on Micellar-micellar interaction

The solubilization of proteins or metal ions into AOT/isooctane reverse micellar solution was carried out by the stepwise injection method. The solubilization depends on its aqueous volume fraction as shown Fig.5. With increasing solubilization of protein or metal ions , the conductivities are at first insensitive to the water content. Then, the coductivities are increase very sharply when the water contents exceed respective threshold values indicating

percolation phenomena. The percolation process is clearly reflecting the protein (or metal ions)-micellar interactions. An increase in the concentration BSA (or Cd2+) shifted the percolation threshold(□p) to a higher value of □aq. The percolation threshold depends on the concentration of the proteins (or metal ions) solubilized in the micelles.

Fig.5 Solubilization of cadmium ion and BSA by stepwise injection method and

electrical conductivity (AOT/isooctane : 100mM , Temp. : 25℃ )

CONCLUSION

Separation of protein and metal ion in aqueous solution using AOT/isooctane reverse micellar solution was studied by phase transfer and stepwise injection method.

The partitioning of proteins and metal ions between a bulk aqueous and reverse micellar phase is depend on pH and concentration of proteins and metals. In case of phase transfer method, proteins is well separated lower than isoelectric pH(BSA, pI=4.9 and C.C.lipase, pI=4.3). When highter than pI, it has a poor separation efficiency. And a separation efficiency of metal ions is well overall pH range, especially higher than pH3.

Solubilization of BSA and cadmium ion into reverse micellar solution by stepwise injection method increase with pH and concentration.

REFERENCES

1) Kuboi, R., Hong, D.-P., Komasawa, I., Shiomori, K., Kawano, Y. and Lee, S.-S.,    □Effect of Proteins Solubilized into AOT Reverse Micelles on Their Back-extraction     and Percolation Processes" , Solv. Extr. Res. Dev. Japan, 3, 223 (1996)

2) Yasushi Yamada, Ryoichi Kuboi and Isao Komasawa, "Extraction of Enzymes and     their Activities in AOT Reverse Micellar Systems Modified with Nonionic           Surfactants", Journal of Chem. Eng. of Japan, Vol.27 No.3 (1994)

3) Kelly, B., Wang, D.C. and Hatton, T.A., "Affinity Based Reverse Micellar Protein     Extraction. Ⅱ. Effect of Cosurfactant Tail Length", Biotechnol. Bioeng., 42, 1199     (1993)

4) Hong, D.-P., Kuboi, R., Komasawa, I., "Extraction of Proteins and Polymers Using     Reverse Micelles and Percolation Process", Korean J. of Chem., 14(5), 334-340       (1997)