KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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A Study of the Factors Affecting the Backward Transfer of BSA from AOT Reverse Micellar Solutions

AOT 역미셀 용액으로부터 BSA의 역추출에 영향을 미치는 인자들에 관한 연구

  • 노선균 (전남대학교 공과대학 화학공학과) ;
  • 강춘형 (전남대학교 고분자기술연구소)
  • Published : 2000.12.01
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Abstract

The factors affecting the back-extraction efficiency of Bovine Serum Albumin (BSA, Mw. 65kDa, pl 4.9) solubilized in an AOT reverse micellar solution, prepared by the injection method, to an excess aqueous phase were investigated. In particular, the effects of pH, the types of salts, alcohols added as cosurfactants, and their concentrations in the aqueous phase were examined. Furthermore, by comparing the CD spectra of the back-extracted BSA and the feed BSA, the structural changes of BSA during the extraction process were determined. The addition of 1:1 salt such as KCl or NaCl to the aqueous phase resulted in almost a 100% extraction to the aqueous phase at a pH higher than its isoelectric point pl. This high efficiency of back-extraction might be due to the change in the interactions between the protein and micellar aggregates driven by the added salt. For 1:2 salts like $CaCl_2$ and $MgCl_2$, BSA was back-extracted with lower than 20% extraction efficiency. Maximum extraction efficiencies were attained at about pH=7 and pH=8 for monovalent and divalent salts, respectively. The addition of alcohols as cosurfactants led to an improvement in monovalent and divalent salts, respectively. From the CD spectra of BSA extracted to the aqueous phase, it was observed that denaturation of BSA was not significant. In certain back-extraction conditions, the extracted BSA showed even higher activity than the feed BSA.

주입법을 이용하여 AOT 역미셀 용액에 가용화된 BSA를 과잉 수용액으로 역추출할 때 효율에 영향을 미치는 요인들 에 대하여 조사하였다. 특히 수용액의 pH, 수용액에 가한 염 의 종류와 농도, 알코올의 종류에 따른 영향을 검토하였다. 더하여, 역추출된 BSA의 CD 스펙트럼을 원래의 BSA의 CD 스펙트럼과 비교하여 추출공정 중에 발생하는 구조적 변화를 결정하였다. KCI이나 NaCI과 같은 1: 1염을 수용액에 첨가하 면 BSA의 등전점 이상의 pH에서는 거의 100% 역추출 효율을 보였다. 이것은 첨가된 염에 의하여 유발되는 역미셀과 단백질 간의 전기적 상호작용의 변화에 기인한 것으로 추측 된다 $CaCl_2$ 과 $MgCl_2$, 와 같은 1:2염의 경우에는 20% 이하의 역추출 효율을 보였다. 1가 염과 2가 염의 경우 각각 pH=7 과 pH=8에서 최고의 효율을 보였다 표면활성시약으로써 알 코올을 침가하는 경우에는 역추출 효율이 향상됨을 보였다. 과잉 수용액으로 추출된 BSA의 CD 스펙트럼으로부터 추출 공정 중에는 특이할 만한 구조적 변성은 관찰되지 않았으며, 특정된 추출조건에서는 원래의 단백질 보다 높은 활성을 보이기도 하였다.

Keywords

References

  1. Science v.227 Separation techniques based on the opposition of two counteracting forces to produce a dynamic equilibrium O'Farrell P. H.
  2. Biotechnol. Prog. v.6 no.1 Continuous counteracting chromatographic electrophoresis Ivory C. F.;W. A. Gobie
  3. Chem. Eng. Sci. v.45 no.9 Mass Transfer Rate of Protein Extraction with Reversed Micelles Dekker M.;K. V. Piet;B. H. Bijsterbosch;P. Fijneman;R. Hilhorst
  4. Biotech. Bioeng. v.38 Protein Extraction by Reverse Micelles: A Study of the Factors Affection the Forward and Backward Transfer of alpha-Chymotrypsin and Its Activity Marcozz G.;N. Correa;P. L. Luisi;M. Casell
  5. Chem. Eng. Prog. Liquid-Liquid Extraction for Protein Separations Abbott N. L.;T. A. Hatton
  6. high Selective Separations in Biotechnology Dekker M.;M. E. Leser
  7. Biotech. Bioeng. v.43 The Effect of pH and Ionic Strength on the Partitioning of Four Proteins in Reverse Micelles Systems Andrews B. A.;D. L. Pyle;J. A. Asenjo
  8. Enzyme Microb. Technol. v.8 Reverse micelles as a bioseparation tool Kadam K. L.
  9. J. Ferment. Bioeng. v.86 no.6 Extraction Characteristic of Bovine Serum Albumin Using Sodium Bis(2-ethyl-hexyl) Sulfosuccinate Reverse Micelles Shiomori K.;N. Ebuchi;Y. Kawano;R. Kuboi;I. Komasawa
  10. The 5th International Symp. Effect of addition alcohol on liquid-liquid extraction of BSA using reversed micelles Lee Sung-Sik ;Eun-Ju Lee;Sung-Eun Park;Jong-Hwa KIM;Jong-Gyu Yang
  11. Biotech. Bioeng. v.39 Solubilizing Water Involved in Protein Extraction Using Reversed Micelles Ichikawa S.;M. Imai;M. Shimizu
  12. Biotech. Bioeng. v.47 Extraction of Lysozyne and Ribonucleasea Using Reverse Micelles: Limits to Protein Solubilization Lye G. J.;J. A. Asenjo;D. LPyle
  13. Eur. J. Biochem. v.184 Protein transfer from an aqueous phase into reversed micelles: The effect of protein size and charge distribution Wolbert R. B. G.;R. Hilhorst;G. Voskuilen;H. Nachtegaal;M. Dekker;K. Vantriet;B. H. Bijsterbosch
  14. J. Phys. Chem. v.82 Thermodynamics of the Uptake of Proteins by Reverse Micelles: A First Approximation Model Caselli M.;P. L. Luisi;M. Maestro;R. Roselli
  15. J. Phys. Chem. v.95 Structural Characterization of Chymotrypsin Containing AOT Reversed Micelles Rahaman R. S.;T. A. Hatton
  16. Kor. J. Biotechnol. Bioeng. Spring Symposium Effect of pH and salt type for BSA Solubilization Using Reverse Micelles Rho S. H.;C. H. Kang;D. H. Park
  17. Fluid Phase Equilibria v.122 Generalized water uptake modeling of water in-oil micromulsion. New experimental results for Aersol-ot-water-salts systems Rabie H. R.;J. H. Vera
  18. J. Phys. Chem. v.90 Determination of the Structural Parameters of Reverse Micelles after Uptake of Proteins Zampieri G. G.;H. Jackle;P. L. Luisi
  19. Biotech. Bioeng. v.46 protein Extraction from an Aqueous Phase into a Reversed Micellar Phase : Effect of Water Content and Reversed Micellar Composition Hilhorst R.;M. Sergeerva;D. Heering;P. Rietveld;P. Fijneman;R. B. G. Wolbert;M. Dekker;B. H. Bijsterbosch
  20. J. Chem. Soc., Faraday Trans. I v.82 Interfacial Tension Minima in Oil-Water-Surfactant Systems Aveyard R.;B. P. Binks;S. Clack;J. Mead
  21. Fluid Phase Equilibria v.116 Enzyme recovery from reversed micellar solution through formation of gas hydrates Nagahama K.;H. Noritomi;A. Koyama