• BMB Reports
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• v.39 no.1
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• pp.76-83
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• 2006

Pyridoxine-5-P oxidase catalyses the terminal step in the biosynthesis of pyridoxal-S-P, the biologically active form of vitamin $B_6$ Which acts as an essential cofactor. Here, a human brain pyridoxine-5-P oxidase gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat-pyridoxine-5-P oxidase fusion protein. Expressed and purified Tat-pyridoxine-5-P oxidase fusion protein transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-pyridoxine-5-P oxidase protein showed catalytic activity and was stable for 48 h. Moreover, the formation of pyridoxal-5-P was increased by adding exogenous Tat-pyridoxine-5-P oxidase to media pre-treated with the vitamin $B_6$ precursor pyridoxine. In addition, the intracellular concentration of pyridoxal-S-P was markedly increased when Tat-pyridoxal kinase was transduced together with Tat-pyridoxine-5-P oxidase into cells. These results suggest that the transduction of Tat-pyridoxine-5-P oxidase fusion protein presents a means of regulating the level of pyridoxal-5-P and of replenishing this enzyme in various neurological disorders related to vitamin $B_6$.

• Microbiology and Biotechnology Letters
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• v.30 no.2
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• pp.142-150
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• 2002

Streptomyces polychromogenes IFO 13072 was used as a strain producing cholesterol oxidase(EC 1.1.3.6). The conditions of cholesterol oxidase production were investigated. The optimum composition of medium for production of the enzyme was 1% dextrin, 0.5% casamino acid, 0.1% $KH_2$PO$_4$, 0.5% $NaNO_3$ and 0.05% $MgSO_4$(pH 7.3). The enzyme was purified specifically by cholesterol affinity column chromatography with a yield of 23.2%. The purified enzyme showed a single polypeptide on SDS-PAGE and the molecular weight was estimated about 52,000 daltons. The optimum pH and temperature of the cholesterol oxidase were pH 7.0 and $37^{\circ}C$, respectively. The enzyme was stable in the range of pH 6.0~7.0 and $25^{\circ}C$. The cholesterol oxidase activity was strongly inhibited by metal ions such as $Hg^{2＋}$ and $Fe^{2＋}$ and inhibitors such as dithiothreitol, mercaptoethanol and isonicotinic acid. The Michaelis constant(Km) for the cholesterol was found to be 25 mM by Lineweaver-Burk plot analysis.

• The Korean Journal of Pesticide Science
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• v.7 no.1
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• pp.45-50
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• 2003

This study was conducted to understand the role of oxidative enzyme cytochrome $P_{450}$ in the bioactivation of benfuracarb and to know metabolites of benfuracarb by cytochrome $P_{450}$. The bimolecular imhibition rate constant $(k_i)$ of benfuracarb on acetylcholinesterase (AChE) was as low as $1.1{\times}10^3\;M^{-1}\;min^{-1}$, suggesting that benfuracarb should be activated for its toxic action. The potency of benfuracarb on AChE in the oxidase system (cytochrome $P_{450}$ + NADPH) in vitro was 10-fold higher than that of control (cytochrome $P_{450}$). Such a similar result was also found in the oxidase + PBO system. In vivo the $I_{50}$ of benfuracarb was 22.7mg $kg^{-1}$, but pie-treatment of piperonyl butoxide (PBO) reduced the $I_{50}$ by >100mg $kg^{-1}$. This result suggests that cytochrome $P_{450}$ was involved in the activation of benfuracarb. Using microsomal oxidase system, metabolites of benfuracarb were elucidated. Fifty-eight percent of benfuracarb was converted to carbofuran, a major toxic metabolite, in the oxidase system, while only less than two percent of benfuracarb was converted to carbofuran in the oxidase + PBO system. These results also suggest that cytochrome $P_{450}$ was involved in the activation of benfuracarb. Overall results indicate that cytochrome $P_{450}$ could be involved in the bioactivation of benfuracarb to carbofuran.

• Journal of the Korean Society of Food Science and Nutrition
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• v.13 no.4
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• pp.397-402
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• 1984

As a basic research for inhibition of enzymatic browning of apple wine, polyphenol oxidase (EC 1.10.3.1) from apple (Jonathan) was extracted, partially purified, and some properties of the enzyme and changes o( active bands by heat treatment were investigated. Optimum conditions for the enzyme reaction were pH6.5 and temperature of $30^{\circ}C$, and o-diphenol was the main substrate for the enzyme. Approximately 35% and 15% of initia lpolyphenol oxidase activity remained after heating at $60^{\circ}C$ and $70^{\circ}C$ for 1 hour, respectively. About 0.5mM of the inhibitor such as sodium metabisulfite, cysteine and ascorbic acid was required for effective inhibition of the enzyme reaction. However, EDTA was found to be a very poor inhibitor. Ethanol did not affect the enzyme activity. The number of active bands of polyphenol oxidase from apple(Jonathan) was found to be four, but two bands and one band were observed after heating at $60^{\circ}C$ and $70^{\circ}C$ for 1 hour, respectively, which showed a significant difference in thermal stability among active bands.

• Korean Journal of Microbiology
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• v.40 no.3
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• pp.248-253
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• 2004

To scrutinize the physiological diversity by BIOLOG microplate, the carbon source utilization patterns of 168 strains of oligotrophic bacteria and 132 strains of psychrotrophic bacteria isolated from Lake Baikal during 2000 and 2002 were investigated. Eighty-six percent (56 strains) of oxidase test positive group (GN-NENT group) and 89 % (92 strains) of oxidase test negative group (GN-ENT group) among oligotrophic bacteria, and 82% (85 strains) of oxidase test negative group among psychrotrophic bacteria were able to utilize $\alpha$-D-glucose as a sole-carbon-source, and 93% (26 strains) of oxidase test positive group among psychrotrophic bacteria were able to utilize bromosuccinic acid as a sole-carbon-source. However, most strains except few oligotrophic bacteria with oxidase test negative group were not able to utilize $\alpha$-D-lactose as a sole-carbon-source. Most dominant genus among 300 strains was Pseudomonas (49 strains). Other dominant genera belonged to Salmonella, Serratia, Buttiauxella, Pantoea, Yersinia, Brevundimonas, Hydrogenophaga, Photorhabdus, Sphingomonas, and Xenorhabdus. Our results by BIOLOG identification system were able to provide basic data to determine community-level carbon source utilization patterns and to accomplish the efficient and reliable identification for microbial community structure in Lake Baikal.

• Applied Biological Chemistry
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• v.39 no.5
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• pp.349-354
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• 1996

The Polyvinyl alcohol(PVA) oxidase involved in PVA degradation by microorganism has been purified to homogeneity from culture broth of Xanthomonas campestris J2Y grown in a minimal medium containing PVA as a sole carbon source. The enzyme was purified by DEAE-cellulose chromatograpy and Sephadex G-150 gel filtration. The purified PVA oxidase was electrophoretically homogeneous both in the absence and presence of SDS. The molecular weight of the enzyme was estimated to be about 55,000 daltons by SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel filtration. The native enzyme existed as a monomer. The optimal pH and temperature was shown to be pH 7 and $37^{\circ}C$ respectively. The activity of enzyme was stable below $55^{\circ}C$ and between pH range of $5{\sim}11$. The enzyme activity was significantly inhibited by metal compounds such as $Ag^{2+},\;Hg^{2+}$. While, metal ions such as $Mn^{2+},\;and\;Cu^{2+}$ stimulated the reaction. Km value of the enzyme for PVA was $7.04{\times}10^{-2}mmol/{\ell}$.

• Korean Journal of Food Science and Technology
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• v.34 no.4
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• pp.552-558
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• 2002

Polyphenol oxidase from Flammulina velutipes was purified and characterized. Purification of polyphenol oxidase was achieved by ammonium sulfate precipitation, Superdex G-200 gel filtration chromatography, Phenyl superose affinity chromatography, Mono-Q anion exchange chromatography and Superdex S-200 gel filtration chromatography on FPLC. After these purification steps specific activity of purified polyphenol oxidase increased to 199.1 units/mg. Polyphenol oxidase from F. velutipes was composed of a single polypeptide with molecular weight of about 40 kDa. Optimum pH and temperature for the enzyme reaction were found to be 6.0 and $25^{\circ}C$, respectively. The activity of the enzyme gradually decreased at acidic pH between 3 and 5, and the enzyme lost its activity at alkaline pH between 8 and 10. This enzyme exhibited high substrate specificity to o-diphenols. Km-values for L-DOPA and caffeic acid were found to be 3.97 mM and 1.78 mM, respectively. 2-mercaptoethanol, L-ascorbic acid, sodium bisulfite, EDTA and $Mg^{2+}$ inhibited the activity of pholyphenol oxidase and $Cu^{2+}$, $Fe^{2+}$, $Zn^{2+}$ and $Ni^{2+}$ increased enzyme activity. The activity of enzyme was well maintained at $-70^{\circ}C$ for over 4 months, and at $-20^{\circ}C$ for 1 months.

• 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 $25^{\circ}C$.

• Journal of Plant Biotechnology
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• v.2 no.2
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• pp.83-87
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• 2000

Carnation petals exhibit autocatalytic ethylene production and wilting during senescence. The autocatalytic ethylene production is induced by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes, whereas the wilting of petals is related to expression of the cysteine proteinase (CP) gene. Until recently, it has been believed that these two phenomena, autocatalytic ethylene production and wilting, are regulated in concert in senescing carnation petals, since the two phenomena occurred closely in parallel. Our studies with petals of a transgenic carnation harboring a sense ACC oxidase transgene and petals of carnation flowers treated with 1,1-dimethyl-4-(phenylsulfonyl) semicarbazide showed that the expression of ACC synthase and ACC oxidase genes and that of CP are regulated differently in carnation psanetals. Interestingly, in the petals of transgenic carnation, the transcript for CP was accumulated but the transcripts for ACC synthase and ACC oxidase were not accumulated in response to exogenous ethylene. Based on these results, we hypothesized that two ethylene signaling pathways, one leading to the expression of ACC synthase and ACC oxidase genes and the other leading to the expression of CP gene, are functioning in senescing carnation petals.

• BMB Reports
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• v.30 no.6
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• pp.397-402
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• 1997

A thiol-specific spin label was attached to cysteine-102 of yeast cytochrome c and electron paramagnetic resonance (EPR) spectra were measured as a function of added cytochrome c oxidase concentration. The intensity decreased due to line broadening as cytochrome c formed a complex with cytochrome c oxidase and reached a minimum when the ratio of cytochrome c to cytochrome c oxidase became one. Replacement of either Lys-72 or Lys-87 of cytochrome c by Glu did not result in a significant change in binding affinity. Interestingly the K72E mutant, unlike K87E, had a much lower rate of electron transfer than the wild type. These results indicate that many positively charged residues as a group participate in complex formation but Lys-72 might be important for cytochrome c to be locked in an orientation for an efficient electron transfer. A stoichiometry of 1 was also confirmed by optical absorption of the cytochrome c-cytochrome c oxidase complex which had been run through a gel chromatography cloumn to remove unbound cytochrome c. The EPR spectrum of this 1:1 complex, however, was a mixture of two components. This explains a biphasic kinetics for a single binding site on cytochrome c oxidase without invoking conformational transition.