• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.1
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• pp.56-60
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• 2001

The pcbC gene encoding (4-chloro-)2,3-dihydroxybiphenyl dioxygenase was cloned from the genomic DNA of Pseudomonas sp. P20 using pKT230 to construct pKK1. A recombinant strain, E. coli KK1, was selected by transforming the pKK1 into E. coli XL1-Blue. Another recombinant strain, Pseudomonas sp. DJP-120, was obtained by transferring the pKK1 of E. coli KK1 into Pseudomonas sp. DJ-12 by conjugation. Both recombinant strains showed a 23.7 to 26.5 fold increase in the degradation activity to 2,3-dihydroxybiphenyl compared with that of the natural isolate, Pseudomonas sp. DJ-12. The DJP-120 strain showed 24.5, 3.5, and 4.8 fold higher degradation activities to 4-chlorobiphenyl, catechol, and 3-methylcatechol than DJ-12 strain, respectively. The pKK1 plasmid of both strains and their ability to degrade 2,3-dihydroxybiphenyl were stable even after about 1,200 generations.

• Korean Journal of Microbiology
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• v.40 no.4
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• pp.275-281
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• 2004

Our previous research has demonstrated that the bacterium, Pseudomonas sp. NFQ-l capable of utilizing quin­oline (2,3-benzopyridine) as the sole source of carbon, nitrogen, and energy was isolated and characterized [Yoon et ai. (2003) Kor. J. Biotechnol. Bioeng. 18(3):174-179]. In this study, we have found that Pseudomonas sp. NFQ-l could degrade quinoline as well as benzoate, and extended this work to characterize the catechol 1,2­dioxygenase (C1,2O) purified from the bacterium cultured in benzoate media. Initially, C1,2O has been purified by ammonium sulfate precipitation, gel permeation chromatography, and Source 15Q. After Source 15Q, puri­fication fold was increased to approximately 14.21 unit/mg. Molecular weight of C1,2O was about 33 kDa. Physicochemical characteristics (e.g., substrate specificity, Km, Vmax, pH, temperature and effect of inhibitors) of purified C1,2O were examined. C1,2O demonstrated the activity for catechol, 4-methylcatechol and 3-meth­ylcatechol as a substrate, respectively. The Km and Vmax value of C1,2O for catechol was 38.54 ${\mu}M$ and $25.10\;{\mu}mol{\cdot}min^{-1}{\cdot}mg^{-1}.$ The optimal temperature of C1,2O was $30^{\circ}C$ and the optimal pH was approximately 8.5. Metal ions such as $Ag^+,\;Hg^+,\;Ca^{2+},\;and\;Cu^{2+}$ show the inhibitory effect on the activity of C1,2O. N-terminal amino sequence of C1,2O was analyzed as ^1TVKISQSASIQKFFEEA^{17}.$ In this work, we found that the amino acid sequence of NFQ-l showed the sequence homology of 82, 71, 59 and $53\%$ compared with C1,2O from Pseudomonas aeruginosa PA0l, Pseudomonas arvilla C-1., P. putida KT2440 and Pseudomonas sp. CA10, respectively.

• Korean Journal of Microbiology
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• v.38 no.2
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• pp.74-80
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• 2002

The purpose of this work was to investigate the characterization and sequence of catechol 1,2-dioxygenase (Cl,2O) purified from Acinetobacter sp. KS-1 which was grown on benzoate as a sole carbon source. Cl,2O demonstrated its enzyme activity to catechol and 4-methylcatechol. The optimum temperature of Cl,2O was $35^{\circ}C$, and the optimal pH was in the range from pH 7.5 to 9.0. $Ag^{+}$, $Hg^{+}$, and $Cu^{2+}$ showed inhibitory effect on the activity of Cl,2O. Molecular weight of the enzyme was determined to approximately 36 kDa by SDS-PAGE and 7-terminal amino acid sequence of Cl,2O was analyzed as $^{1}MNYQQIDALVKQMNVDTAKG^{20}$and exhibited 95% sequence homology with that of Cl,2O from Acinetobacter radioresistens In addition, trypsin digestion and peptide mapping were performed for internal sequencing analysis. Molecular weights of three digested peptide fragments were analyzed as 966.3 Da, 1933.8 Da and 2081.7 Da by MALDI-TOF, which were matched with each internal sequences $^{1}SQSDFNLRR^{9}\, ^{1}HGNRPSHVHYFNSAPGYR^{18}\, ^{1}TIEGPLYVAGAPESVGFAR^{19}$) of. A. radioresistens. PCR product was amplified with the degenerated primers derived from N-terminal and each internal amino acid sequences.

• YAKHAK HOEJI
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• v.45 no.4
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• pp.387-396
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• 2001

The effects of ascorbic acid, thiols such as cysteine, n-acetyl-ι-cyteine, glutathione, thiourea, 2-mercaptoethanol and dithiotreithol and organic acids such as magic acid, citric acid, glycolic acid, taurine and kojic acid on polyphenol oxidate (PPO) activity were studied in order to establish if it reacts with oxidized product and/or directly inhibits the enzyme. To investigate the mechanism, the quantification of t-butylcatechol and 4-methylcatechol (phenolic compounds) as substates, their oxidized product and sulphydryl colorless additional compounds were determined by high performance liquid chromatograph (HPLC) method. Chromatographic results indicate that ascorbic acid, organic acids and lower level of cysteine reduced oxidized product of substrates back to their respective positions uf ο-diphenols. On the other hand, other thiols and high level of cysteine reacted with oxidative product of ο-diphenols and then produced sulphydryl colorless compounds. Cysteine apperars to have two types of mechanism of actions in the formation of oxidative products of substrates depending on its concentration; ascorbic acid-type and other thiols-types. The effect of ascorbic acid with thiols on polyphenol oxidase was determined by same method. Chromatographic results indicate that ascorbic acid was more reactive with oxidized product of substrates than thiols.

• Journal of the Korean Society of Food Science and Nutrition
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• v.33 no.6
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• pp.1013-1016
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• 2004

The inhibitory effect of MRPs (Maillard reaction products) on enzymatic browning of taro was investigated. The MRPs prepared by heating glycine and glucose at 9$0^{\circ}C$ for 7 hr exhibited a strong inhibitory effect on taro polyphenol oxidase (PPO). The maximum inhibitory activity of MRPs against taro PPO was detected toward (＋)-catechin, catechol, 4-methylcatechol followed by L-$\beta$-3,4-dihydroxyphenylalanine (L-DOPA) and pyragallol as a substrate. The MRPs synthesized from fructose and glucose with glycine as a amino acid significantly reduced the taro PPO activity. MRPs prepared by higher glycine or glucose concentration showed stronger inhibition against taro PPO. Increasing reaction time of the glycine and glucose promoted the inhibitory effect of MRPs against the PPO activity of taro, whereas the color formation was gradually increased.

• Microbiology and Biotechnology Letters
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• v.35 no.3
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• pp.245-249
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• 2007

Chloroanilines are widely used in the production of dyes, drugs and herbicides. Chloroanilines, however, are considered potential pollutants due to their toxic and recalcitrant properties to humans and other species. With the increase of necessity of bioremediation, this study was conducted to isolate the chloroanilines-degrading bacteria. A bacterium capable of growth on 3,4-dichloroaniline (DCA) was isolated by the 3,4-DCA-containing enrichment culture. The strain KB35B was identified as Pseudomonas sp. and also able to degrade several chloroanilines. The isolated strain showed high level of catechol 2,3-dioxygenase activity in the presence of 3,4-DCA. The activity of catecho1 2,3-dioxygenase was supposed to be ones of the important factors for 3,4-DCA degradation. The activity toward 4-methykatechol was 60.6% of that of catechol, while the activity toward 3-methylcatechol and 4-chlorocatechol were 27.0 and 13.5%, respectively.

• Journal of Applied Biological Chemistry
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• v.66
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• pp.338-344
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• 2023

Curcuma longa is a plant belonging to the genus Curcuma and is distributed across various Asian regions. This plant is widely known for its rhizomes, which possess a variety of pharmacological properties. However, although the leaves and stems of this plant also contain several health-promoting secondary metabolites, very few studies have characterized these compounds. Therefore, our study sought to quantify the secondary metabolites from the leaves and stems of Curcuma longa L. (LSCL) using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and high-performance liquid chromatography (HPLC). Our LC-ESI-MS analyses detected twenty-one phenolic compounds in the LSCL, among which fifteen compounds were detected via HPLC analysis. Four compounds, namely vanillic acid (0.129 mg/g), p-coumaric acid (0.431 mg/g), 4-methylcatechol (0.199 mg/g), and afzelin (0.074 mg/g) were then quantified. These findings suggest that LSCL is rich in secondary metabolites and holds potential as a valuable resource for the development of functional and nutritional supplements in the future.

• Journal of Microbiology
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• v.35 no.1
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• pp.53-60
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• 1997

Pseudomonas sp. P20 and Pseudomonas sp. DJ-12 isolated from the polluted environment are capable of degrading biphenyl and 4-chlorobiphenyl (4CB) to produce benzoic acid and 4-chlorobenzoic acid (4CBA) respectively, by pcbABCD-encoded enzymes. 4CBA can be further degraded by Pseudomonas sp. DJ-12, but not by Pseudomonas sp P20. However, the meta-cleavage activities of 2, 3-dihydroxybiphenyl (2, 3-DHBP) and 4-chloro-2, 3-DHBP dioxygenases (2, 3-DHBD) encoded by pcbC in Pseudomonas sp. P20 were stronger than Pseudomonas sp. DJ-12. In this study, the pcbC gene encoding 2, 3-DHBD was cloned from the genomic DNA of Pseudomonas sp. P20 by using pKT230. A hybrid plasmid pKK1 was constructed and E. coli KK1 transformant was selected by transforming the pKK1 hybrid plasmid carrying pcbC into E. coli XL1-Blue. By transferring the pKK1 plasmide of E. coli KK1 into Pseudomonas sp. DJ-12 by conjugation, a recombinant strain Pseudomonas sp. P20, Pseudomonas sp. DJ-12, and the recombinant cell assay methods. Pseudomonas sp. DJ12-C readily degraded 4CB and 2, 3-DHBP to produce 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoic acid (HOPDA), and the resulting 4CBA and benzoic acid were continuously catabolized. Pseudomonas sp. DJ12-C degraded 1 mM 4CB completely after incubation for 20 h, but Pseudomonas sp. P20 and Pseudomonas sp. DJ-12 showed only 90% and Pseudomonas sp. DJ-12 had, but its degradation activity to 2, 3-DHBP, 3-methylcatechol, and catechol was improved.

• Proceedings of the Microbiological Society of Korea Conference
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• 2001.11a
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• pp.16-25
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• 2001

A genomic library of biphenyl-degrading strain Comamonas sp. SMN4 was constructed by using the cosmid vector pWE15 and introduced into Escherichia coli. Of 1,000 recombinant clones tested, two clones that expressed 2,3-dihydroxybiphenyl 1,2-dioxygenase activity were found (named pNB 1 and pNB2). From pNB1 clone, subclone pNA210, demonstrated 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, is isolated. 2,3-Dihydroxybiphenyl 1,2-dioxygenase (23DBDO, BphC) is an extradiol-type dioxygenase that involved in third step of biphenyl degradation pathway. The nucleotide sequence of the Comamonas sp. SMN4 gene bphC, which encodes 23DBDO, was cloned into a plasmid pQE30. The His-tagged 23DBDO produced by a recombinant Escherichia coli, SG 13009 (pREP4)(pNPC), and purified with a Ni-nitrilotriacetic acid resin affinity column using the His-bind Qiagen system. The His-tagged 23DBDO construction was active. SDS-PAGE analysis of the purified active 23DBDO gave a single band of 32 kDa; this is in agreement with the size of the bphC coding region. The 23DBDO exhibited maximum activity at pH 9.0. The CD data for the pHs, showed that this enzyme had a typical a-helical folding structures at neutral pHs ranged from pH 4.5 to pH 9.0. This structure maintained up to pH 10.5. However, this high stable folding strucure was converted to unfolded structure in acidic region (pH 2.5) or in high pH (pH 12.0). The result of CD spectra observed with pH effects on 23DBDO activity, suggested that charge transition by pH change have affected change of conformational structure for 23DBDO catalytic reaction. The $K_m$ for 2,3-dihydroxybiphenyl, 3-metylcatechol, 4-methylcatechol and catechol was 11.7 $\mu$M, 24 $\mu$M, 50 mM and 625 $\mu$M.

• Journal of the Korean Society of Tobacco Science
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• v.19 no.2
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• pp.136-144
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• 1997

Cigarette smoking is known to suppress both 1-methy14-phenyl-155,Ltetrahydropy-ridine (MPTP)-induced parkinsonism and idiopathic Parkinson's disease (PD). However, the precise mechanism underlying its protective action against PD is not clearly elucidated yet. In order to find possible clue on the mechanism of protective action of smoking, we investigated the inhibitory effect of cigarette smoke components on rat brain mitochondria1 monoamine oxidase B (MAO-B), responsible enzyme for the activation of MPTP to its toxic metabolitesr and identified the components having an inhibitory potency on this enzyme from cigarette smoke. Total 31 eligible constituents including nicotine were selected from cigarette smoke condensates via solvents partitioning and silica gel chromatographic separation, and inhibitory potencies of 19 components on MAO-B were determined. Hydroquinone and methylcatechol, the phenolic components, showed the strongest inhibitory potencies on MAO-B activity in the components tested. 3,4-Dihydroxybenzylamino, myosmine and indole in basic fracton, eugenol in phenolic fraction, and farnesol in neutral fraction also inhibited the enzyme activity dose-dependently. Among tobacco alkaloids tested only myosmine was effective for the inhibition of this enzyme. These results suggest that the decrease in MAO-B activity by such components derived from cigarette smoke seems to be related to the suppression of MPTP-induced neurotoxicity and to the less incidence of Parkinson's disease in smokers than in nonsmokers.