• 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.

• 한국식품과학회지
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• 제23권4호
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• pp.414-419
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• 1991

돼지감자의 가공과정 중 갈변에 관여하는 polyphenol oxidase를 부분 정제하고 이의 특성을 조사하였다. 인산완충액으로 추출된 조효소가 ammonium sulfate에 의한 침전과 Sephadex G-100에 의한 gel filtration으로 48배 정제되었다 이 효소는 catechol을 기실로 하였을 때 최적 활성 pH가 6.5이었으며 $K_{m}$값은 3mM, 열불활성 화는 1차반응을 보였고, 이때 Z값은 $7.6^{\circ}C$, 활성화에너지($E_{a}$)는 72.6kcal/mole이었다. 이 효소를 인산완충액에 저장하였을 때 $4^{\circ}C$에서는 일주일 이상 안정하였으나 $25^{\circ}C$에서는 활성이 급격히 소실되었다. 이 효소는 monophenol에는 반응하지 않고 diphenol의 기질에만 친화력을 나타내었으며, 그 중 chlorogenic acid와 가장 잘 반응하였다. 그리고 1 mM의 L-cystein(HCl)와 L-hydrosulfite, L-ascorbic acid에 의해서 완전히 저해되었다.

• Applied Biological Chemistry
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• 제30권3호
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• pp.278-284
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• 1987

사과의 건조, 가공 중의 갈변을 방지하기 위한 기초 조사로서 후지 사과로부터 추출한 crude polyphenol oxidase의 특성과 열에 대한 저항성, 갈변 저해제의 저해 효과 등을 조사하였다. Catechol을 기질로 사용하였을 때 polyphenol oxidase의 최적 pH는 5.5, 최적온도는 $20^{\circ}C$, $K_m$ 값은 0.14M이었고, 열불활성화는 유사일차반응을 보였으며 이때 활성화에너지$(E_a)$와 Z값은 각각 23.0cal/kmol, $19.7^{\circ}C$였다. 기질에 따른 친화력은 o-diphenol, 특히 chlorogenic acid에 대하여 높았고, monophenol과 m-diphenol, p-diphenol에 대해서는 나타나지 않았다. Polyphenol oxidase에 의한 갈변은 thiourea와 potassium metabisulfite는 10mM에서, L-cysteine과 ascorbic acid, sodium diethyldithiocarbamate는 1mM에서 현저하게 저해되었다.

• 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.

• 한국환경생물학회:학술대회논문집
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• 한국환경생물학회 2002년도 학술대회
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• pp.123-126
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• 2002

Pollutants that are persistent, bioaccurnulative, and toxic have been linked to numerous adverse effects in human and animals, PBTs include heavy metals, polychlorinated biphenyls (PCBs), dioxins, polycyclic aromatic compounds (PACs) in addition to pesticides. This study focuses on toxic effects of the PBTs except pesticides on insects. Eight PBTs were selected from subgroups: three heavy metals (Pb, Hg, and Cd), two PCB mixtures (Aroclor mixtures 1 and 2), 2,3,7,8-tetrachlorodibenzo-p-dioxin, two monophenols (4-octylphenol and 4-nonylphenol), and tetrabutyltin, Beet armyworm, Spodoptera exigua, was used as test target insect species. Three physiological markers (metamorphosis, immune reaction, and follicle patency) were assessed in each exposure to different doses of the PCBs. Heat-shock proteins as molecular markers were also analyzed in response to the PCBs. All tested PBTs were toxic to metamorphosis from larvae to pupae when they were applied with diet. Two PCB mixtures were the most toxic compounds in this assay by giving significant toxicity at 0.005 ppm, while others had from 10 to 1000 ppm. Dioxin (0.1 ppb), tetrabutyltin (0.1 ppb), Pb (10 ppb), and Hg (0,01 ppb) were potent to inhibit immune reactions analyzed by inducing phenoloxidase activity and blocked phospholipase $A_2$ enzyme, Tetrabutyltin and dioxin significantly induced follicle cell patency, but their effects were lower than that of endogenous juvenile hormone, Dioxin, Pb, Hg, and Cd could induce the expression of heat shock proteins that were detected by immunoblotting against human HSP70 monoclonal antibody. HSP78 and HSP80 were upregulated in response to the PBTs. This expression was detected from the fat body and epidermis at as fast as 4h after injection. All these results clearly suggest that PBTs give significant ecotoxicity to insects that are valuable organisms in our environment.

• Applied Biological Chemistry
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• 제26권1호
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• pp.41-46
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• 1983

송이버섯[Tricholoma matsutake(S. Ito et Imai);Sing.]에서 polyphenol oxidase 조효소액(粗酵素液)을 추출(抽出), 그 성질(性質)을 조사(調査)하여 다음과 같은 결과(結果)를 얻었다. Trihydroxyphenol류(類)와 o-diphenol류(類)에 활성(活性)을 보였고 이들중 pyrogaroll이 최고활성(最高活性)을 보였으며 m-diphenol, p-diphenol 그리고 monophenol류(類)에서는 활성(活性)을 보이지 않았다. 최적(最適) pH는 4.0 부근이었고, 최적온도(最適溫度)는 $45{\sim}55^{\circ}C$였으며, 효소(酵素)의 활성(活性)이 $70^{\circ}C$에서 2분(分) 가열(加熱)로 완전(完全)히 실활(失活)되었다. $Cu^{++}$, $Fe^{++}$ 및 $Mg^{++}$은 $10^{-1}mM$이상(以上) 농도(濃度)에서는 효소력(酵素力)이 증가(增加)되었으나, 그 이상(以上)의 농도(濃度)에서는 오히려 저하(低下)되었다. 저해제(沮害劑)로는 Na-DETC, L-ascorbic acid, L-cysteine 및 NaCl이 양호(良好)하였다. pyrogaroll에 대한 km치(値)는 0.88mM이었다.

• Current Research on Agriculture and Life Sciences
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• 제4권
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• pp.55-64
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• 1986

배과실(果實)의 PPO를 분리(分離) 정제(精製)한 후(後) 그 특성(特性)을 조사(調査)한 결과(結果)는 다음과 같다. 조효소액(粗酵素液)을 전기영동한 결과 2개의 활성분리대(活性分離帶)가 관찰(觀察)되었으며, 이 조효소액(粗酵素液)을 acetone 심몰(沈沒)과 DEAE-cellulose chromatography로 부분정제(部分精製)한 결과 PPO A 분획(分劃)과 PPO B 분획(分劃)을 얻었다. 각 구분을 전기영동(電氣泳動)한 결과 PPO A, B 각각 Rm치가 0.58, 0.68인 단일 활성분리대(活性分離帶)가 관찰(觀察)되었다. PPO A와 PPO B의 최종 정제도(精製度)는 각각 7.8와 8.7배(倍)였다. PPO A의 최적(最適) pH와 온도(溫度)는 pH 7, $33^{\circ}C$인 반면 PPO B의 최적(最適) pH와 온도(溫度)는 pH 4.2, $30^{\circ}C$로 나타났다. PPO A, B의 열(熱) 안정성(安定性)은 $60^{\circ}C$ 이상(以上)의 온도(溫度)에서는 급격히 활성(活性)이 감소했으며 PPO B보다 PPO A가 더 안전(安定)하였다. 배의 두 PPO는 o-diphenol에 강한 활성을 나타냈으며 특히 PPO A는 catechol에 PPO B는 chlorogenic acid에 활성이 강했다. 또한 PPO A는 pyrogallol에도 활성을 나타냈다. 그러나 두 PPO는 m-diphenol, p-diphenol과 monophenol에는 활성(活性)을 나타내지 않았다. 무기염(無機鹽)의 영향(影響)을 본 결과(結果)는 10mM농도(濃度)에서 $Zn^{{+}{+}}$은 PPO A의 활성(活性)을 증가(增加)시켰으나 $Fe^{{+}{+}}$는 저해(沮害)했고 반면 $Fe^{{+}{+}}$, $Zn^{{+}{+}}$는 PPO B는 활성(活性)을 증가(增加)시켰으나 $K^+$, $Mg^{{+}{+}}$, $Ca^{{+}{+}}$, $Hg^{{+}{+}}$는 저해(沮害)했다. $Cu^{{+}{+}}$는 저농도(低濃度)에서는 효소(酵素)를 활성화(活性化)시켰으나 10mM의 고농도(高濃度)에서는 저해작용(沮害作用)을 했다. 저해제(沮害劑)의 작용(作用)을 조사(調査)한 결과(結果), L-ascorbic acid, L-cysteine, thiourea 등이 저해작용(沮害作用)이 강했다. PPO A와 PPO B의 catechol에 대한 Km치는 각각 20mM과 14.3mM이었다.