재조합 β-galactosidase (β-gal) 을 이용하여 transgalactosylation 반응으로 합성된 2-phenoxyethanol galactoside (PE-gal)과 chlorphenesin galactoside (CPN-gal)의 정제를 실시하였다. 먼저 PE와 PE-gal이 포함된 반응물에 ethyl acetate (EA)를 넣고, EA/water 이상계 시스템에서 PE와 PE-gal을 분획하였다. 이 시스템에서 PE는 EA층으로 PE-gal은 water층으로 분획되었다. 그리고, 물층을 모아서 silica gel chromatography를 실시하였다. 최종적으로, silica gel chromatography만 실시하여 PE-gal을 정제한 경우와, EA 처리 후 silica gel chromatography를 실시하여 PE-gal을 정제한 경우의 정제 PE-gal에 대하여 HPLC (High performance liquid chromatography)와 TLC (thin-layer chromatography) 분석을 수행하였다. 그 결과 EA를 처리하여 분획한 후, silica gel chromatography를 수행한 시료에서 잔여 PE가 완전히 제거되는 것을 관찰하였고, silica gel chromatography만 실시하여 PE-gal을 정제한 경우에는 상당량의 잔여 PE가 관찰되었다. 이 때, mole 기준으로 약 21%의 정제 수율을 확인할 수 있었다. 마찬가지로 CPN-gal의 정제에서도 EA 분획 처리 후, silica gel chromatography를 수행하였더니, 잔여 CPN이 거의 없는 순수한 CPN-gal을 얻을 수 있었다.

We investigated the purifications of PE-gal and CPN-gal, synthesized by transgalactosylation reaction using recombinant β-gal. The reaction mixture containing PE and PE-gal was first mixed with EA, and thereafter PE and PE-gal were distributed in two-phase(EA/water) system. In this system, PE and PE-gal was selectively moved into EA and water phase, respectively. Then, the water phase was collected, and silica gel chromatography was carried out using the collected water phase. Finally, we compared two purified PE-gal samples using HPLC and TLC analysis, in which the one was purified only by silica gel chromatography and the other was purified by EA extraction followed by silica gel chromatography. In the latter case, the residual PE was almost completely removed, whereas, in the former case, the residual PE was remained remarkably. Additionally, the purification yield of PE-gal was about 21% on the basis of mole. In the same purification protocol, CPN-gal was able to be purified using EA extraction followed by silica gel chromatography, in which the residual CPN was almost removed when CPN-gal was purified by EA extraction followed by silica gel chromatography.

요 약
 Abstract
 1. 서 론
 2. 실 험
  2.1. 시약
  2.2. β-Gal을 생산하는 재조합 대장균
  2.3. β-Gal 함유 대장균을 이용한 PE-gal과CPN-gal 합성
  2.4. Silica gel chromatography를 이용한PE-gal 정제
  2.5. Extraction of the reaction mixtureusing ethyl acetate
  2.6. TLC 분석
  2.7. HPLC 분석
 3. 결과 및 고찰
  3.1. Silica gel chromatography를 이용한PE-gal 정제
  3.2. PE-gal 정제에 Ethyl acetate 분획 이용
  3.3. Ethyl acetate 분획과 Silica gelchromatography를 이용한 PE-gal 정제
  3.4. Ethyl acetate 분획 및 Silica gelchromatography의 CPN-gal 정제에적용
 4. 결 론
 감사의 글
 References

• 정경환(한국교통대학교 생명공학과, Department of Biotechnology, Korea National University of Transportation) | Kyung-Hwan Jung Corresponding author