• Bulletin of the Korean Chemical Society
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• v.27 no.5
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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.

• Korean Journal of Food Science and Technology
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• v.23 no.4
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• pp.414-419
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• 1991

Polyphenol oxidase from Jerusalem artichoke(Helianthus tuberosus L.) tubers was partially purified by precipitation with ammonium sulfate, followed by gel filtration on Sephadex G-100. The enzyme showed maximal activity at pH 6.5 and $4^{\circ}C$. Kinetic studies indicated $K_{m}$ value of 3 mM for catechol and activation energy of 72.6 kcal/mole. As for substrate specificity of polyphenol oxidase the enzyme showed high affinity towards diphenol compounds, but not towards monophenols. The enzamatic browning was completely inhibited at 1 mM concentration of L-ascorbic acid, sodium hydrosulfite and L-cystein(HCl). The activity of polyphenol oxidase in 0.1 M potassium phosphate buffer(pH 6.5) was fairly stable for a week at $4^{\circ}C$, while it decreased remarkably at $25^{\circ}C$.

• Applied Biological Chemistry
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• v.30 no.3
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• pp.278-284
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• 1987

As a basic research for inhibition of enzymatic browning of apples during dehydration or processing, polyphenol oxidase was extracted from Fuji apples to investigate heat inactivation, chemical inhibition and other properties. Polyphenol oxidase showed the highest activity at $20^{\circ}C$ and pH 5.5 with catechol as substrate, and the Michaelis constant of 0.14 M under the same condition of substrate and pH. The thermal inactivation followed pseudo first-order kinetics to have activation energy of 23.0 kcal/mol and z value of $19.7^{\circ}C$. As for substrate specificity the polyphenol oxidase showed high affinity toward the o-diphenolic compounds, particularly chlorogenic acid. Neither the m- and p-dihydroxy phenols nor monophenols were attacked. Browning by polyphenol oxidase was completely inhibited at the concentrations of 10mM for potassiummetasulfite and thiourea and 1mM for L-cysteine, ascorbic acid and sodium diethyldithiocarbamate.

• Korean Journal of Pharmacognosy
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• v.30 no.3
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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.

• Proceedings of the Korea Society of Environmental Biology Conference
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• 2002.11a
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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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• v.26 no.1
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• pp.41-46
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• 1983

The characteristics of crude polyphenol oxidase extracted from mushroom[Tricholoma matsutake(S. Ito et Imai) Sing.] were investigated. The enzyme showed highest affinity to pyrogaroll among trihydroxyphenols. Except for o-diphenols the enzyme was inactive in di-and monophenols. The optimum pH was about 4 and the optimum temperature ranged from 45 to $55^{\circ}C$. The enzyme activity was completely inhibited by $70^{\circ}C$ heat treatment for 2 min. $Cu^{++}$, $Fe^{++}$ and $Mg^{++}$ activated the enzyme at low concentration($10^{-1}mM$), but inhibited at high concentration (1mM). The most potent inhibitors were Na-diethyldithiocarbamate, L-ascorbic acid, L-cysteine and NaCl. The Km value with pyrogaroll was 0.88mM.

• Current Research on Agriculture and Life Sciences
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• v.4
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• pp.55-64
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• 1986

Polyphenol oxidase in japanese pear (Pyrus communis var. mansamkil) was isolated, partially purified and its some properties were investigated. Polyacrylamide disc gel electrophoresis indicated two bands with polyphenol oxidase activity in the extract from acetone dry powder of par flesh. These two polyphenol oxidases (PPO A and PPO B) were purified through acetone precipitation and diethylaminoethyl cellulose column chromatography. PPO A and B were purified 7.8 fold and 8.7 fold by the present procedure, respectively. The Rm values of partially purified PPO A and B were estimated to be 0.58 and 0.68, respectively. The optimum temp, and pH of PPO A activity were $33^{\circ}C$ and pH 7.0, while those of PPO B were $30^{\circ}C$ and pH 4.2, respectively. Two PPO were unstable over the temperature of $60^{\circ}C$. The substrate specificity of pear PPO showed high affinity toward o-diphenolic compounds, especially catechol in PPO A and chlorogenic acid in PPO B, but inactive toward m-diphenol, p-diphenol and monophenols. PPO A showed affinity toward the trihydroxyphenolic compound. $Zn^{{+}{+}}$ activated the PPO A activity but $Fe^{{+}{+}}$ inhibited PPO B activity, while $Fe^{{+}{+}}$ and $Zn^{{+}{+}}$ activated the PPO B activity, while $Fe^{{+}{+}}$ and $Zn^{{+}{+}}$ activated the PPO B activity but $K^+$, $Mg^{{+}{+}}$, $Ca^{{+}{+}}$ and $Hg^{{+}{+}}$ inhibited at 10mM concentration. $Cu^{{+}{+}}$ activated the enzyme action at low concentrations but inhibited at high concentration. Inhibition studies indicated that L-ascorbic acid, L-cysteine and thiourea were most potent. The Km values of PPO A and PPO B for catechol were 20mM and 14.3mM, respectively.