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이승구,노현수,홍승표,이규종,왕지원,태동년,엄기남,방상구,김영준,성문희,Lee, Seung-Goo,Ro, Hyeon-Su,Hong, Seung-Pyo,Lee, Kyu-Jong,Wang, Ji-Won,Tae, Dong-Nyeon,Uhm, Ki-Nam,Bang, Sang-Gu,Kim, Young-Jun,Sung, Moon-Hee 한국미생물 · 생명공학회 1996 한국미생물·생명공학회지 Vol.24 No.1
By using the ${\beta}$-tyrosinase of Citrobacter freundii KCT2006, which was cloned and overexpressed in Escherichia coli, 3,4-dihydroxy phenyl-L-alanine (L-DOPA) was synthesized efficiently from pyrocatechol, sodium pyruvate, and ammonium acetate. Optimal temperature and pH for the reaction were determined to be about 18$^{\circ}C$ and 8.5, respectively. The effects of substrate concentrations were also examined at different concentrations of ammonium acetate, sodium pyruvate, and pyrocatechol. Ammoniumacetate and sodium pyruvate increased the reaction rate until the concentrations reached to 300mM and 50mM, respectively. Although pyrocatechol showed the optimal concentration at 20mM, it was controlled between 20mM and 50mM to avoid the depletion of substrate during the enzymatic synthesis. Meanwhile the synthetic rate was improved about 20% when ethanol was included in the reaction solution. Based on above results, a reaction medium for the productin of L-DOPA was prepared and incubated with 1 unit/ml of ${\beta}$-tyrosinase. Pyrocatechol and sodium pyruvate was added to the reaction solutin intermittently to avoid the substrate depletion during the enzymatic reaction. After 24 hour of reaction, 31.6g/l of L-DOPA was accumulated in the reaction solution as soluble and precipitated ones and the conversion yield was about 85.2%.
제조합 대장균에서 과발현된 Citrobacter freundii KCTC2006 유래의 β-Tyrosinase를 이용한 3,4-Dihydroxyphenyl-L-alanine의 생산
이승구,노현수,홍승표,이규종,왕지원,태동년,엄기남,방상구,김영준,성문희 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.1
재조합 대장균에서 대량발현 시킨 Citrobacter freundii KCTC 2006 유래이 효소 β-tyrosinase를 이용하여 pyrocatechol, sodium pyruvate, ammonium acetate로부터 3,4-dihydroxy phenyl-L-alanine을 생산하기 위한 연구를 수행하였다. 이 효소반응에 적합한 온도 및 pH 조건은 각각 18℃와 8.5로 결정되었고, 반응액 중의 ammonium acetate와 sodium pyruvate의 농도는 각각 300 mM, 50 mM 이상으로 조절하는 것이 적합하였다. Pyrocatechol의 경우는 20 mM에서 가장 높은 반응성을 나타냈으나, 기질을 반복적으로 첨가하며 장시간 동안 효소반응을 수행하는 경우에는 pyrocatechol의 고갈을 피하기 위하여, 20 mM에서 50 mM 사이로 조절하였다. 한편, 반응액 중에 ethanol을 10% 첨가한 경우에는 반응속도가약 20% 증가하였다. 이상과 같은 효소반응특성에 기초하여 조제한 기질용액에 β-tyrosinase를 1 unit/㎖ 농도로 가하고, pyrocatechol과 pyruvate가 고갈되지 않도록 간헐적으로 첨가하면서 효소반응을 수행한 결과, 24시간 만에 85.2%의 수율로 31.6g/ℓ의 3,4-dihydroxyphenyl-L-alanine를 생산할 수 있었다. By using the β-tyrosinase of Citrobacter freundii KCTC2006, which was cloned and overexpressed in Escherichia coli, 3,4-dihydroxy phenyl-L-alanine (L-DOPA) was synthesized efficiently from pyrocatechol, sodium pyruvate, and ammonium acetate. Optimal temperature and pH for the reaction were determined to be about 18℃ and 8.5, respectively. The effects of substrate concentrations were also examined at different concentrations of ammonium acetate, sodium pyruvate, and pyrocatechol. Ammonium acetate and sodium pyruvate increased the reaction rate until the concentrations reached to 300 mM and 50 mM, respectively. Although pyrocatechol showed the optimal concentration at 20 mM, it was controlled between 20 mM and 50 mM to avoid the depletion of substrate during the enzymatic synthesis. Based on above results, a reaction medium for the production of L-DOPA was prepared and incubated with 1 unit/㎖ of β-tyrosinase. Pyrocatechol and sodium pyruvate was added to the reaction solution intermittently to avoid the substrate depletion during the enzymatic reaction. After 24 hour of reaction, 31.6 g/ℓ L-DOPA was accumulated in the reaction solution as soluble and precipitated ones and the conversion yield was about 85.2%.