KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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A Study on the Solubilization of $\alpha$-Chymotrypsin Using AOT Reverse Micelles; Effects of pH and salts

AOT 역미셀을 이용한 $\alpha$-chymotrypsin의 가용화에 대한 연구;pH와 염의 영향

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

Micellar aggregates are known to be useful for the selective isolation of biologically active materials such as amino acids, proteins, and enzymes from crude mixtures sparsely dispersed in water. In this study, the effects of pH, salt type and its concentration on the solubilization of $\alpha$-chymotrypsin into the organic micellar phase, which consisted of AOT (sodium 야(2-ethylhexy)sulfosuccinate) and iso-octane, were comprehensively examined. It was found that maximum extraction efficiency was attained at a pH below the isoelectric point of $\alpha$-chymotrypsin; at pH=5.0 for NaCl and KCl, and at pH=7.0 for $CaCl_2$and $MgCl_2$. In order to avoid complications stemming from the precipitationof protein at low pH interfaces, the protein concentrations in the organic and aqueous phases were directly measured. The size of the micelle water pool was estimated by measuring the molar ratio of the surfactant to the water, W(sub)o. The resulting values of W(sub)o were nearly constant at 30 and 19 for NaCl and KCl, respectively, and were independent of pH. The addition of 1:2 salts like $MgCl_2$and $CaCl_2$ led to much lower, but a constant value of, W(sub)o than the 1:1 salts.

미셀 집합체들이 수용액에 존재하는 혼합물로부터 아미노산, 단백질, 그리고 효소와 같은 생물학적 물질의 선돼적인 추출에 유용하게 샤용될 수 있음은 널리 알려져 있다. 본 연 구에서는 가용화 과정에 영향을 미치는 변수들을 확인하기 위하여 AOT와 iso-octane의 유기상에서의 $\alpha$-chymotrypsin 가 용화에 대하여 pH의 영향, 염의 종류와 염의 농도의 영향을 연구하였다. NaCI과 KCI에 대하여서는 pH=5.0에서 최대 효 율을 나타내며, $MgCl_2$ 와 $CaCl_2$ 에 대해서는 pH=7.0에서 최대 효율을 나타내었는데 이와 같은 조건은 모두 $\alpha$-chymo$\sigma$ypsin 의 둥전점 이하 영역이다. 낮은 pH에서는 계변에 단백질이 광범위하게 침전되거나 엉킴현상이 나타나기 때문에 유기상 에서의 단백질 농도를 직접 측정하여 실험오차를 줄였다. 미 셀의 water po이 크기는 물에 대한 계면활성제의 몰 비(Wo) 를 측정하여 추산하였다 .. 1: 1염인 NaCI과 KCI에 대하여 w。 값은 각각 30과 19로 거의 일정하고 $MgCl_2$ 와 $CaCl_2$ 의 1:2염 에 대하여서는 1: 1염의 Wo값보다 훨씬 작은 5 이하의 값을 나타내었다

Keywords

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