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3,4-Dihydroxyphenyl-L-alanine의 효소적 생산에 대한 반응첨가물의 영향
이승구,노현수,홍승표,성문희 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.2
재조합 대장균에서 과발현된 Citrobacter freundii KCTC 2006 유래의 tyrosine phenol-lyase(E.C.4.1.99.2) 이용 3,4-dihydroxyphenyl-L-alanine (L-DOPA)의 생산에 대한 반응첨가물의 영향을 조사하였다. 반응액 중 효소 및 조효소 농도의 영향을 조사한 결과, 효소농도는 약 2 units/ml이 적합하였으며, 조효소인 pyridoxal -5-phosphate 는 0.1 mM 이상이 필요하였다. Pyrocatechol과 결합하여 안정한 복합체를 형성하는 sodium borate는 pyrocatechol에 의한 효소의 불활성화를 감소시키는 효과가 있었으나, 효소의 반응성을 현저히 저하시켜 L-DOPA 생산의 관점에서는 불리한 단점도 있었다. 한편, 알콜류 등의 유기용매가 L-DOPA 합성반응에 미치는 영향을 조사한 결과 methanol을 5% 농도로 반응액에 첨가하였을 때, 효소의 반응과 안정성이 크게 증가하여 L-DOPA 합성반응이 지속적으로 수행됨으로써 고농도의 L-DOPA를 효율적으로 생산할 수 있게 되었다. 생산된 L-DOPA의 약 77%가 불용성 상태로 침전되어 쉽게 회수할 수 있었으며, 침전된 L-DOPA를 1N HCI에 용해한 후 재결정화 함으로써 최종적으로 99.96%의 고순도 L-DOPA를 생산할 수 있었다. The enzymatic synthesis of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) was examined for the effects of the reaction additives such as sodium borate, alcohol, and organic solvents. The enzyme used was tyrosine phenol-lyase of Citrobacter freundii KCTC 2006 produced in Escherichia coli. The amounts of tyrosine phenol-lyase and pyridoxal -5-phosphate were optimized to 2.0 units/ml and 0.1 mM, respectively, for the synthetic reaction. Sodium borate, a substance that forms a complex with pyrocatechol, reduced the enzyme deactivation by pyrocatechol although it seriously inhibited the enzyme activity. Among the organic solvents tested, dimethylsulfoxide, dimethylformamide, and alcohol increased the productivity of the L-DOPA synthesis. In a reaction system with 5% methanol, L-DOPA concentration increased up to 210 mM after 24 hours, and 77.1% of which was separated as precipitates. The L-DOPA was purified to 99.96%
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%.
Studies on the Production of Serratiopeptidase from Serratia Culture
Ro, Hyeon Su,Park, Ho Jin,Lee, Byeong Ryong 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.2
세라티아 균주의 배양으로부터 소염제로 사용되는 세라티오펩티다아제의 생산에 관한 연구를 수행하였다. 여러가지 탄소원, 질소원 및 유도제가 효소의 생산에 미치는 영향을 조사하였는데, 탄소원은 효소의 생산이나 세포성장에 좋지 못한 기질이었으며, 특히 citrate의 경우 균체성장량은 포도당과 거의 동일하였으나, 세라티오펩티다제의 생산에 저해효과가 있음이 밝혀졌다. 세라티오펩티다아제는 아미노산인 leucine을 첨가해 주었을 때 그 생산되는 양이 크게 향상되었으며 leucine의 최적 농도는 0.03%였다. 포도당을 기질로 한 유가배양시에 세라티오펩티다아제의 생산이 catabolite repression으로 생각되는 조절기작에 의하여 저해받았으나, yeast extract를 기질로 하였을 때는 저해현상은 나타나지 않았다. An anti-inflammatory agent, serratiopeptidase, was produced from the culture of the Serratia marcescens. The effects of carbon sources, nitrogen sources and inducers on the production were investigated. Citrate was found to be inhibitory in the production of serratiopeptidase. The enzyme was synthesized in the synthetic medium without inducers, albeit low level of synthesis. But the synthesis was increased by the addition of proteinaceous substrate and leucine. Induction of extracellular proteinase by its end-product was discovered. which is not common in the proteinase synthesis in the bacteria. By the glucose fed-batch culture, we found the possible catabolite repression on the production of serratiopeptidase.
Ro, Hyeon-Su,Koh, Byung Hoon,Jung, Sun Ok,Park, Hyun Kyu,Shin, Yong-Beom,Kim, Min-Gon,Chung, Bong Hyun WILEY-VCH 2006 Proteomics Vol. No.
<P>We have developed a surface plasmon resonance (SPR)-based protein microarray to study protein–protein interactions in a high-throughput mode. As a model system, triple protein interactions have been explored with human papillomaviral E6 protein, tumor suppressor p53, and ubiquitin ligase E6AP. Human papillomavirus (HPV) is known to be a causative agent of cervical cancer. Upon infection, the viral E6 protein forms a heterotrimeric protein complex with p53 and E6AP. The formation of the complex eventually results in the degradation of p53. In the present study, a GST-fused E6AP protein was layered onto a glutathione (GSH)-modified gold chip surface. The specific binding of GST-E6AP protein onto the gold chip surface was facilitated through the affinity of GST to its specific ligand GSH. The interacting proteins (E6 and/or p53) were then spotted. Detection of the interaction was performed using a SPR imaging (SPRI) technique. The resulting SPRI intensity data showed that the protein–protein interactions of E6AP, E6, and p53 were detected in a concentration-dependent manner, suggesting that the SPRI-based microarray system can be an effective tool to study protein–protein interactions where multiple proteins are involved.</P>
Effects of Salts on the Conformation and Catalytic Properties of D-Amino Acid Aminotransferase
Ro, Hyeon-Su 생화학분자생물학회 2002 Journal of biochemistry and molecular biology Vol.35 No.3
The effects of salts on the biochemical properties of D-amino acid aminotransferase from Bacillus sp. YM-1 have been studied to elucidate both the inhibitory effects of salts on the activity and the protective effects of salts on the substrate-induced inactivation. The results from UV-visible spectroscopy studies on the reaction of the enzyme with D-serine revealed that salt significantly reduced the rate of the formation of the quinonoid intermediate and its accumulation. The kinetic and spectroscopy studies of the reaction with $\alpha$-[$^2H$]-DL-serine in different concentrations of NaCl provided evidence that the rate-limiting step was changed from the deprotonation of the external aldimine to another step(s), presumably to the hydrolysis of the ketimine. Gel filtration chromatography data in the presence of NaCl showed that the enzyme volume was reduced sharply with the increasing NaCl concentration, up to 100 mM. An additional increase of the NaCl concentration did not affect the elution volume, which suggests that the enzyme has a limited number of salt-binding groups. These results provide detailed mechanistic evidence for the way salts inhibit the catalytic activity of D-amino acid aminotransferase.
Effects of Salts on the Conformation and Catalytic Properties of D - Amino Acid Aminotransferase
(Hyeon Su Ro) 생화학분자생물학회 2002 BMB Reports Vol.35 No.3
The effects of salts on the biochemical properties of Damino acid aminotransferase from Bacillus sp. YM-1 have been studied to elucidate both the inhibitory effects of salts on the activity and the protective effects of salts on the substrate-induced inactivation. The results from UV-visible spectroscopy studies on the reaction of the enzyme with D-serine revealed that salt significantly reduced the rate of the formation of the quinonoid intermediate and its accumulation. The kinetic and spectroscopy studies of the reaction with α-[2H]-DL-serine in different concentrations of NaCl provided evidence that the rate-limiting step was changed from the deprotonation of the external aldimine to another step(s), presumably to the hydrolysis of the ketimine. Gel filtration chromatography data in the presence of NaCl showed that the enzyme volume was reduced sharply with the increasing NaCl concentration, up to 100 mM. An additional increase of the NaCl concentration did not affect the elution volume, which suggests that the enzyme has a limited number of saltbinding groups. These results provide detailed mechanistic evidence for the way salts inhibit the catalytic activity of Damino acid aminotransferase.