• Bulletin of the Korean Chemical Society
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• 제34권9호
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• pp.2731-2736
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• 2013

We report a new tetrameric supramolecular Cu(II) complex ($Cu_4L_4$ = tetrakis(N,N'-bis(salicylidene)-2,2'-ethylenedianiline)Copper(II)) with a Schiff-base ligand ($H_2L$ = N,N'-bis (salicylaldimine)-1,2-ethylenediamine) containing two N,O-bidentate chelate groups. Though the copper sites of $Cu_4L_4$ are non-coupled, the complex exhibits a unsually high catecholase-like activity ($k_{cat}=935h^{-1}$) when the $Cu_4L_4$ solution is treated with 3,5-di-tert-butylcatechol (3,5-DTBC) at basic condition in the presence of air. Combined information obtained from UV-VIS and EPR measurements could lead the suggestion of the reaction pathway in which the substrate may bind to Cu(II) ions by anti-anti didentate bridging mode.

• Bulletin of the Korean Chemical Society
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• 제32권5호
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• pp.1500-1506
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• 2011

Tyrosinase is a widespread enzyme with great promising capabilities. The Lineweaver-Burk plots of the catecholase reactions showed that the kinetics of mushroom tyrosinase (MT), activated by $Co^{2+}$ and $Zn^{2+}$ at different pHs (6, 7, 8 and 9) obeyed the non-essential activation mode. The binding of metal ions to the enzyme increases the maximum velocity of the enzyme due to an increase in the enzyme catalytic constant ($k_{cat}$). From the kinetic analysis, dissociation constants of the activator from the enzyme-metal ion complex ($K_a$) were obtained as $5{\times}10^4M^{-1}$ and $8.33{\times}10^3M^{-1}$ for $Co^{2+}$ and $Zn^{2+}$ at pH 9 and 6 respectively. The structural analysis of MT through circular dichroism (CD) and intensive fluorescence spectra revealed that the conformational stability of the enzyme in these pHs reaches its maximum value in the presence of each of the two metal ions.

• Bulletin of the Korean Chemical Society
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• 제27권5호
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• pp.642-648
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• 2006

Mushroom tyrosinase (MT) structural changes in the presence of $Cu ^{2+}$ and $Ni ^{2+}$ were studied separately. Far-UV CD spectra of the incubated MT with the either of the metal ions indicated reduction of the well-ordered secondary structure of the enzyme. Increasing in the maximum fluorescence emission of anilinonaphthalene-8-sulfonic acid (ANS) was also revealing partial unfolding caused by the conformational changes in the tertiary structure of MT. Thermodynamic studies on the chemical denaturation of MT by dodecyl trimethylammonium bromide (DTAB) showed decrease in the stability of MT in the presence of $Cu ^{2+}$ or $Ni ^{2+}$ using their activation concentrations. Both activities of MT were also assessed in the presence of different concentrations of these ions, separately, with various monophenols and their corresponding diphenols. Kinetic studies revealed that cresolase activity on p-coumaric acid was boosted in the presence of either of the metal ions, but inhibited when phenol, L-tyrosine, or 4-[(4-methylphenyl)azo]-phenol was substrate. Similarly, catecholase activity on caffeic acid was enhanced in the presence of $Cu ^{2+}$ or $Ni ^{2+}$, but inhibited when catechol, L-DOPA, or 4-[(4-methylbenzo)azo]-1,2-benzenediol was substrate. Results of this study suggest that both cations make MT more fragile and less active. However, the effect of the substrate structure on the MT allosteric behavior can not be ignored.

• 한국식품저장유통학회:학술대회논문집
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• 한국식품저장유통학회 1993년도 정기총회 및 제3차 학술발표회
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• pp.19-20
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• 1993

농산물의 수확 후 취급이나 가공중에 일어나는 효소적갈변 현상은 외관의 변색에 의한 소비자의 기호성을 저하시킬 뿐만 아니라 상품의 가치를 하락시키며, 경제적인 손실을 초래함으로서 현재 식품에 종사하는 기술자나 가공업자에게 가장 관심이 있는 분야이다. 효소적갈변의 주역을 담당하고 있는 polyphenol oxidase(PPO)(EC1.14, 18.1)은 대부분의 식품에 존재하고 있으며 monophenol의 hydroxylation에 의한 o-diphenol화합물의 형성반응(cresolase)과 o-diphenol화합물의 o-quinone으로의 산화반응(catecholase)을 촉배한다. 그리고 여기서 일단 생성된 o-quinone은 다시 다른 o-quinone이나 그밖의 단백질, 아미노산, 환원당 등의 여러 성분들과 상호 반응하여 갈변식품의 특유의 색소인 melanine을 형성한다. 효소적갈변에 의한 품질의 저하는 식품가공산업에서 해결되어야 할 하나의 큰 과제이다. 현재까지 식품산업에서 가장 널리 쓰이고 있는 효소적갈변 저해제는 sylfite류 이다. 그들은 새우의melanosis(blackspot)를 억제하며, 감자, 버섯, 사과와 다른 과채류의 갈변을 방지할 뿐 아니라 쥬우스와 포도주의 off flavors를 일으키는 폴리페놀 플리머의 형성을 감소 시킨다. 그러나 최근 sulfite가 사람의 건강에 좋지않는 부작용을 일으키며, 특히 천식을(asthma)을 지니고 있는 사람이 sulfite가 함유된 식품을 먹었을 경우 사망을 초래할 수 있어 식품첨가물로서 그들의 사용이 미국 FDA에 의해 재평가되고 있다. 최근에 FDA는 셀러드바에서의 sulfite사용을 금지 시켰으며 점차 감자가공품에의 사용을 금지하는 방향으로 나아가고 있다. 따라서 현재 실질적이고 기능적인 sulfite의 대체 화합물의 개발이 절실히 필요한 실정이다. 이러한 배경으로 본 강연에서는 효소적갈변 저해제의 개발과 그들의 식품가공에의 적용 현환 및 화장품, 의약품으로의 응용에 대해 설명하고자 한다.

• 생약학회지
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• 제20권2호
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• pp.101-109
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• 1989

Purification of polyphenol oxidase(PPO) from Korean native tobacco variety leaves was carried out through the procedure of acetone preciptation, ammonium sulfate fractionation, and Sephadex G-150 gel filtration, resulting in a 84-fold increase in specific activity. The enzyme was stable in a range of pH 7.5 to 8.0 with an optimum of pH 7.5. The optimum temperature for the enzymic reaction was about $60^{\circ}$. It was thermostable with a half-life equal to 20 min at $70^{\circ}$. Km values for (+)-catechin and pyrogallol were $1.6{\times}10^{-3}$ and $0.5{\times}10^{-3}M$, respectively. It possesses high catecholase activity but little or no cresolase activity. Lineweaver-Burk analysis of inhibition data revealed that the inhibition of (+)-catechin oxidation by potassium cyanide, 4-nitrocatechol, cystein and 2-mercaptoethanol was competitive with Ki values of $1.1{\times}10^{-6}$, $1.8{\times}10^{-6}$, $8.9{\times}10^{-6}$ and $1.3{\times}10^{-5}$, respectively.

• KSBB Journal
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• 제9권5호
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• pp.525-531
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• 1994

본 연구에서는 tyrosinase 효소반응시에 발생하는 반응 비활성화를 막아 조엽 얀정성을 향상시키는 것을 목적으로 연구를 수행하였다. Quinone에 의해 친핵성 공격을 받는 lysine기를 bifunctional rea gent인 glutaraldehyde로 변형시켜 줌으로써 효소 의 비활성화를 줄일 수 있었다. 또한, 고정화된 효소 를 충진층 반응기에 충진시켜 비활생화를 열으키는 생산물을 연속적으로 제거함으로써 효소의 얀정성을 크게 향상시켰다. 고정화 방법으로는 glass bead를 이용한 담체 가교법을 이용하였으며, 이때 glutaral­d dehyde와 효소의 lysine이 반응하게 되어 qumone 에 의한 친핵성 공격을 받지 않게 되어 안정성이 더욱 향상되 었다. 반응매 질로 borate buffer를 이용하 게되면 catechol과 borate의 복합체 형성으로 catechol에서 qumone으로의 전환이 줄어들게 되어 효소 비활성화가 감소하였다.

• 생약학회지
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• 제30권3호
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• pp.306-313
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• 1999

Polyphenol Oxidase(PPO) was purified from an extract of Ginkgo biloba leaves by ammonium sulfate fractionation followed by sephadex G-150 column chromatography, which resulted in a 18-fold increase in specific activity. The enzyme was most active at pH 8.5 and the temperature optimum for the PPO catechol oxidation reaction was $45^{\circ}C$. Heat inactivation studies showed that heating for 7, 9 and 48 min, at 80, 70 and $60^{\circ}C$ respectively caused a 50% loss in enzymatic activity and that the enzyme was completely inactivated after heat treatment at $90^{\circ}C$ for 60 min. Km values of the PPO for catechol, hydroquinone and 4-methylcatechol derived from Lineweaver-Burk plots were $6.06\;{\times}\;10^{-4}M,\;1.02\;{\times}\;10^{-3}M,\;1.41\;{\times}\;10^{-3}M$ respectively. Of the substrates tested, 4-methylcatechol was oxidized most readily and the enzyme did not oxidize monophenols. The enzyme datalyzed browning reaction was completely inhibited in the presence of reducing reagents, namely ascorbic acid, cysteine, glutathione, 2-mercaptoethanol, potassium metabisulfite at 0.5 mM level. Sodium chloride showed very little inhibition effect on Ginkgo biloba leaves PPO. Lineweaver-Burk analysis of inhibition data revealed that the inhibition by cysteine, 2-mercaptoethanol, potassium cyanide was competitive with ki values of $1.1\;{\times}\;10^{-5}M,\;2.4\;{\times}\;10^{-5}M,\;8\;{\times}\;10^{-5}M$, respectively. Among the divalent cations, $Cu^{2+}ion$ was a strong activator on PPO and $Mn^{2+}ion$ was little or no effect on PPO activity $Ni^{2+}ion$ was an inhibitor on PPO.