• 한국미생물·생명공학회지
• /
• 제24권2호
• /
• pp.222-226
• /
• 1996

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% by solubilizing and recrystallyzing the precipitates.

• 한국미생물·생명공학회지
• /
• 제24권1호
• /
• pp.44-49
• /
• 1996

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%.

• 대한화장품학회지
• /
• 제30권4호
• /
• pp.499-502
• /
• 2004

장미과 식물(에탄을 추출)의 항산화력을 측정하기 위하여 1,1-diphenyl-2-picrylhydrazyl (DPPH) 자유 라디칼 생성 시스템을 이용하여 하였다. 또한 산화 환원 과정을 통하여 피부의 과잉 색소 생성에 관련된 멜라닌 생합성 과정의 조절 가능성을 조사하였다. 장미과 식물중에서 Prunus sargentii, Rubus coreanus, Chaenomeles sinensis, Photinia glabra와 Pyrus pyrifolia은 생합성 과정에서 dopa [3-(3,4-dihydroxyphenyl) alanine]를 도파크롬으로 변환시켜 주는 tyrosinase 저해 효과를 나타내었다. MTT 실험법은 장미과 에탄올 추출물의 사람 섬유아세포에 미치는 독성정도를 실험하기 위해 사용되었다. 장미과 식물중 특히 Prunus sargentii의 껍질, Phorinia glabra의 껍질과 나무 그리고 Chaenomeles sinenis의 잎, 껍질, 나무 모든 부분에서 mushroom tyrosinase에 대해 $100{\;}{\mu}g/mL$에서 50\%$ 이상의 저해 활성을 보였으며, $10{\;}{\mu}g/mL$ 농도에서 강한 라디칼 소거 효과를 나타내었다. 또한 이들 추출물은 사람 섬유아 세포에 대해 높은 생존율을 나타냄으로써 멜라닌 형성 과정을 조절할 수 있을 것으로 기대한다.

• BMB Reports
• /
• 제32권5호
• /
• pp.480-485
• /
• 1999

Tyrosine phenol-lyase of thermophilic Symbiobacterium sp. SC-1, which is obligately and symbiotically dependent on thermophilic Bacillus sp. SK-1, was purified and characterized. The enzyme is composed of four identical subunits and contains approximately 1 mol of pyridoxal 5'-phosphate (PLP) per mol subunit as a cofactor. The enzyme showed absorption maxima at 330 and 420 nm, and lost this absorption profile by treatment with phenylhydrazine. The apparent dissociation constsnt, $K'_D$, for PLP was determined with the apoenzyme to be about $1.2\;{\mu}M$. The isoelectric point was 4.9. The optimal temperature and pH for the $\alpha,\beta$-elimination of L-tyrosine were found to be $80^{\circ}C$ and pH 8.0, respectively. The substrate specificity of the enzyme was very broad: L-amino acids including L-tyrosine, 3,4-dihydroxyphenyl-L-alanine (L-DOPA), L-cysteine, L-serine, S-methyl-L-cysteine, $\beta$-chloro-L-alanine, and S-(o-nitrophenyl)-L-cysteine all served as substrates. D-Tyrosine and D-serine were also decomposed into pyruvic acid and ammonia at rates of 7% and 31% relative to their corresponding L-enantiomers, respectively. D-Alanine, which was inert as a substrate in a, $\beta$-elimination, was the only D-amino acid racemized by the enzyme. The $K_m$ values for L-tyrosine, L-DOPA, S-(o-nitrophenyl)-L-cysteine, $\beta$-chloro-L-alanine, and S-methyl-L-cysteine were 0.19, 9.9, 0.36, 12, and 5.5 mM, respectively.

• KSBB Journal
• /
• 제29권1호
• /
• pp.1-8
• /
• 2014

Tyrosinases catalyze the hydroxylation of monophenolic compounds and the conversion of o-diphenols to oquinones. The enzymes are mainly involved in the modification of tyrosine into L-3,4-dihydroxyphenyl-alanine (L-DOPA) and DOPA/DOPAquinone-drived intermolecular cross-linking, which play the key roles of pigmentation to the cells. It is ubiquitously distributed in microorganisms, plants, and animals all around the nature world. They are classified as copper- containing dioxygen activating enzymes; two copper ions are coordinated with six histidine residues in their active sites and they are distinguished as met-, deoxy-, and oxy-form depending on their oxidative states. Natural extraction and recombinant protein approaches have been tried to obtain practical amounts of the enzymes for industrial application. Tyrosinases have been widely applied to industrial and biomedical usages such as detoxification of waste water containing phenolic compounds, L-DOPA as a drug of Parkinson's disease, biomaterials preparation based on the cross-linking ability and biosensors for the detection of phenolic compounds. Therefore, this review reports the mechanism of tyrosinase, biochemical and structural features and potential applications in industrial field.

• 한국식품과학회지
• /
• 제32권3호
• /
• pp.736-741
• /
• 2000

체내에서 멜라닌 생합성을 촉매하는 tyrosinase에 대한 천연의 지용성 저해제 발굴을 위하여, isooctane/AOT(100 mM)/인산완충용액(20 mM, pH 8.0)으로 구성되고 tyrosinase(105.3 units/mL)와 3,4-dihydroxyphenyl-alanine(0.18 mM)을 함유한 역미셀계에서 식물체 75종의 petroleum ether 추출물이 tyrosinase 반응을 억제하는 효과를 분석하였다. 조사대상 식물체중에서 마늘이 in vitro 멜라닌 합성 반응을 완전히 저해하였고(100%), 모과, 고구마, 양파, 무순, 사과의 저해활성이 60% 이상이었다. 한약재와 향신료 중에서는 로즈마리, 고수, 계피, 산사자, 측백엽, 오매, 두충, 박하, 정향이 60% 이상의 저해능을 보였다. 그리고 각 식물체의 tyrosinase 저해효과와 더불어 추출수율을 함께 고려했을 때, 단위 원재료로부터 tyrosianse 저해제 생산을 많이 할 수 있는 소재를 선정할 수 있었다. 본 연구에서는 역미셀계를 이용함으로서 기존에는 분석이 어려웠던 지용성 물질의 tyrosinase 저해효과를 측정할 수 있었다.

• 대한화학회지
• /
• 제44권6호
• /
• pp.541-546
• /
• 2000

리간드 증감 유발 형광법을 이용하여 Tb(III)-L-dopa (L-3,4-dihydroxyphenyl alanine) 착이온의 방출세기를 측정함으로써 수용액 중의 L-dopa를 정량하는 방법에 대하여 연구하였다. 들뜸파장, pH, 보조 형광증가제의 선택, Tb(III) 이온의 농도, 보조 형광증가제로 사용된 Lu(III) 이온의 농도 및 방출파장의 방출세기에 대한 영향을 조사하였다. 보조 형광증가제로서 Lu(III) 이온을 첨가하였을때 Tb(III) 이온의 방출세기가 현저히 증가함을 관찰하였고, L-dopa의 검출한계를 낮출 수 있었다. 보조 형광증가제를 첨가하지 않았을 경우에 L-dopa 검정곡선의 직선감응범위는 들뜸파장, pH 및 Tb(Ⅲ) 이온의 농도가 각각 300 nm, 8.0 및 $1.0{\times}10^{-4}$ M였을때, $5.0{\times}10^{-7}$ M~$1.0{\times}10^{-4}$ M였다. 이 조건에서의 검출한계는 $4.0{\times}10^{-8}$ M였다. 보조 형광증가제를 첨가하였을 경우에는 들뜸파장, pH, Tb(III) 이온의 농도, 보조 형광증가제로 사용된 Lu(III) 이온의 농도 및 방출파장이 각각 300 nm, 8.5, $1.0{\times}10^{-5}$ M, $1.0{\times}10^{-5}$ M 및 545 nm였을 때, 직선감응범위가 1.0×$10^{-8}$ M~2.0{\times}10^{-4}$ M였고, 이 때의 검출한계는 $1.0{\times}10^{-9}$ M였다.

• 청정기술
• /
• 제22권3호
• /
• pp.175-180
• /
• 2016

본 연구에서는, 저온 및 약산성 조건에서 높은 활성을 보이는 티로시나아제인 tyrosinase-CNK의 반응 안정성을 조사하였다. Tyrosinase-CNK는 지금까지 알려진 중온성(mesophilic) 및 호열성(thermophilic) 환경 유래의 티로시나아제 보다는 열 안정성이 낮았으나 0 ℃에서 효소 안정성이 매우 뛰어났고, 반복적인 동결-해동(freeze-thaw) 과정에서도 효소 활성을 안정적으로 유지하였다. 또한, 물과 ethanol 및 acetonitrile이 혼합된 유기용매 환경에서 초기 상대 활성 값의 변화가 관찰되었으나 유기용매 농도의 증가로 인한 추가적인 활성 저하를 유발하지 않고 활성을 일정하게 유지하였다. 한편, 효소 반응의 염(salt)에 대한 저해는 chaotropic 특성이 많이 나타나는 염일수록 적게 나타났다. 결과적으로, tyrosinase-CNK는 통상의 티로시나아제를 이용하여 반응을 수행하기 어려운 환경에서도 원하지 않는 반응을 억제하고 효소 불활성화를 최소화면서 반응을 촉매할 것으로 기대된다.