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The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Production and Process Monitoring of 5-Aminolevulinic Acid (ALA) by Recombinant E. coli I. Characteristics of ALA Production

유전자 재조합 대장균에 의한 5-Aminolevulinic Acid (ALA)의 생산 및 공정 모니터링 I. ALA의 생산 특성

  • 이종일 (전남대학교 응용화학공학부, 생물공정기술연구실) ;
  • 정상윤 (전남대학교 물질·생물화공과, 생물공정기술연구실) ;
  • 서국화 (전남대학교 물질·생물화공과, 생물공정기술연구실) ;
  • 한경아 (전남대학교 물질·생물화공과, 생물공정기술연구실) ;
  • 조성효 (전남대학교 응용화학공학부) ;
  • 백경환 (전남대학교 응용생물공학부)
  • Published : 2004.02.01
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Abstract

In this study the extracellular production of 5-aminolevulinic aicd (ALA) by recombinant E. coli BL2l (DE3) pLysS harboring the plasmid pFLS45 are investigated. Optimum concentrations of succinic acid and glycine for cell growth and ALA production were found to be 30 mM and 15 mM, respectively. Levulinic acid (LA) as an inhibitor of ALAD was added to the culture medium in the end of exponential cell growth phase and its optimum concentration was 30 mM. Growth of recombinant E. coli BL2l (DE3) pLysS (pFLS45) was largely dependent upon the pH value of culture medium. When the pH of culture medium was in the range of 6.0 and 6.5, high cell mass and ALA production were obtained. IPTG induction for the expression of the fusion gene did not enhance the production of ALA. Recombinant cell grew at 30't faster than at 37$^{\circ}C$, but ALA productivity was lower than at 37$^{\circ}C$. Repeated addition of glycine, succinic acid, and LA increased the production of ALA and the inhibition of intracellular ALA dehydratase activity, with up to 1.3 g/L ALA having been produced in the cultivation.

본 연구에서는 ALA의 생산 공정을 개발하기 위해 재조합 플라스미드, pFLS45를 도입한 재조합 대장균의 성장과 ALA 생산 특성을 조사하였다 30 mM 숙신산과 15 mM 글리신이 첨가된 배양액에서 균체 성장이 원활하고 높은 ALA 생산성을 보였다. 그리고 LA는 균체성장이 거의 다 이루어진 정지기에 첨가될 때 균체 성장에 저해를 일으키지 않으면서 ALA 생산성을 높일 수 있었다. 또한 세포내 효소 ALAD의 활성은 30 mM LA가 첨가되었을 때 가장 효과적으로 저해되었다. 본 연구에서 사용한 재조합 대장균은 pH 조절을 위한 산이나 알칼리 첨가에 민감하여 pH를 조절하지 않은 경우에 균체의 성장이 가장 원활하였고 ALA 생산성도 높았다. 3$0^{\circ}C$에서 배양한 경우 균체의 성장은 원활하였지만 ALA의 생산성이 매우 낮았다. 그리고 일반적인 pET 계열의 재조합 대장균의 발현 양상과 달리 유도 발현하지 않고 LA만을 첨가했을 때 ALA 생산 농도는 800 mg/L 이상으로 매우 높았다. 7L급 생물반응기를 이용하여 MS8 배지에 기질 (포도당, 숙신산, 글리신) 및 LA를 간헐적으로 첨가하여 균체 성장 및 ALA 생산 특성을 살펴보았다. 균체량은 높지 않았지만 기질첨가 이후 ALA 생산량은 꾸준히 증가하여 기존의 ALA 생산량보다 두 배 이상 증가한 1300mg/L 정도 생산되었으며 ALAD의 활성은 다른 실험결과와 비교할 때 30% 정도 낮았다. 한편, LA 및 숙신산의 첨가 이후 낮아진 배양액의 pH는 균체 성장을 저해하였는데 발효 중 기질을 첨가한 후 pH를 잘 제어하면서 유가식 또는 연속식 발효를 수행하면 ALA 생산성을 증대시킬 수 있을 것으로 생각된다. 그러나 고농도의 ALA 생산은 균체 생존에 영향을 주므로(37) 생산된 ALA를 적절히 분리하는 방법이 필요할 것으로 사료된다.

Keywords

References

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