• Archives of Pharmacal Research
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• v.15 no.4
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• pp.360-364
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• 1992

Methylcatechol 2, 3-dioxygenase encoded in pWWO megaplasmid of Pseudomonas putida mt-2 has been cloned and overexpressed in Escherichia coli. This enzyme gene has been localized inside 2. 3-kb XhoI fragment derived from the pWWO megaplasmid. Analysis of enzyme activity and SDS-PAGE showed that the cloned methylcatechol 2, 3-dioxygenase gene in E. coli was about 100 fold overexpressed compared with the parental gene in P. putida mt-2 (pWWO). The cloned enzyme exhibited higher ring-fission activity to catechol than catechol derivatives including 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.35-42
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• 2000

Reaction characteristics of 4-methylcatechol 2,3-dioxygenase (4MC230) purified from Pseudomonas putida SU10 with a higher activity toward 4-methylcatechol than catechol or 3-cethylcatechol were studied by altering their physical and chemical properties. The enzyme exhibited a maximum activity at pH 7.5 and approximately 40% at pH 6.0 for 4-methylcatechol hydrolysis. The optimum temperature for the enzyme was around $35^{\circ}C$, since the enzyme was unstable at higher temperature. Acetone(10%) stabilized the 4MC230. The effects of solvent and other chemicals (inactivator or reactivator) for the reactivation of the 4MC230 were also investigated. Silver nitrate and hydrogen peroxid severely deactivated the enzyme and the deactivation by hydrogen peroxide severely deactivated the enzyme and the deactivation by hydrogen peroxide was mainly due to the oxidation of ferrous ion to ferric ion. Some solvents acted as an activator and protector for the enzyme from deactivation by hydrogen peroxide. Ascorbate, cysteine, or ferrous ion reactivated the deactivated enzyme by hydrogen peroxide. The addition of ferrous ion together with a reducing agent fully recovered the enzyme activity and increased its activity abut 2 times.

• The Korean Journal of Pain
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• v.21 no.3
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• pp.179-186
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• 2008

Background: The adrenergic nervous system in the spinal cord contributes to the development of neuropathic pain after nerve injury. Brain derived neurotrophic factor may facilitate the sympathetic change in the spinal cord and influence the state of neuropathic pain. We probed the effect of chronic repetitive administration of systemic 4-methylcatechol, which is known to be a neurotrophic factor inducer, in a spinal nerve ligation model. Methods: We made the rat neuropathic pain model by the ligation of the L5 spinal nerve. Intraperitoneal 4-methylcatechol ($10{\mu}g/kg$) or the same volume of saline wasadministrated twice daily just after the operation for 7 days. The tactile allodynia was measured by using von Frey filaments and its change was followed up from 3 days after SNL. The lumbosacral enlargement of the spinal cord was taken out and the mRNA contents of the ${\alpha}_2-adrenoceptor$ subtypes were measured by real time polymerase chain reaction and this was then compared with the control groups. The antiallodynic effect of intrathecal clonidine (3, 10, $30{\mu}g$) was evaluated and compared in the 4-methylcatechol treated rats and the control rats. Results: The expression of the ${\alpha}_{2A}$ and ${\alpha}_{2C}$ adrenoceptor subtypes did not change after 4-methylcatechol treatment. Intrathecal clonidine showed an earlier and better effect at the highest dose ($30{\mu}g$ intrathecal), but not with any other doses. Conclusions: Chronic intraperitoneal administration of 4-methylcatechol may improve the effect of intrathecal clonidine, but we could not prove the increase of ${\alpha}_{2A}$ and ${\alpha}_{2C}$ adrenoceptors in the spinal cord of 4-methylcatechol treated rats.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.249-254
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• 1999

A catechol 2,3-dioxygenase (C23O) was purified to apparent homogeneity from Pseudomonas putida SU10 through several purification steps consisting of ammonium sulfate precipitation and chromatographies on DEAE 5PW, Superdex S-200, and Resource-Q. Gel filtration indicated a molecular mass under nondenaturing conditions of about 130 kDa. The enzyme has a subunit of 34 kDa as was determined by SDS-PAGE. These results suggest that the native enzyme is composed of four identical subunits. The N-terminal amino acid sequence (30 residues) of the enzyme has been determined and exhibits high identity with other extradiol dioxygenases. The reactivity of this enzyme towards catechol and methyl-substituted catechols is somewhat different from that seen for other catechol 2,3-dioxygenases, with 4-methylcatechol cleaved at a higher rate than catechol or 3-methylcatechol. $K_m$ values of the enzyme for these substrates are between 3.5 and 5.7 M.

• Journal of the Korean Wood Science and Technology
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• v.41 no.6
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• pp.583-593
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• 2013

The based-catalyzed depolymerization (BCD) of kraft lignin isolated from black liquor which the chemical pulping of a mixture of various Southeast Asia hardwood chips was carried out in a batch reactor in the presence of different NaOH concentrations with supercritical methanol. The S:G ratio of the kraft lignin determined by pyrolysis-GC/MS analysis turned out roughly 1.4:1 and main products were vanillic acid, syringol and 3-methoxy catechol. The diethyl ether extracts as phenolic monomers from BCD reaction were produced similar yield among different NaOH concentrations. The 21 compounds were identified by GC/MS analysis in all experiments and major products were catechol, 3-methoxycatechol, 4-methylcatechol, syringol and isovanillic acid. However, it had been shown to be different monomer contents depending on the dosage of NaOH. Catechol, 4-methylcatechol and 3-methoxycatechol were shown to be the dominant monomer from BCD reaction using 7.5 and 3.25% of NaOH concentration whereas syringol, isovanillic acid, 3-methoxycatechol and 4-methylcatechol were determined to be the most typical products under the condition of 1.63% NaOH.

• Journal of Microbiology and Biotechnology
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• v.16 no.5
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• pp.778-785
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• 2006

Catechol 1,2-dioxygenase was purified from cells of R. rhodochrous N75 grown at the expense of benzoate and p-toluate as the sole sources of carbon. A single catechol 1,2-dioxygenase was found to be induced with either growth substrate. The enzyme has an estimated $M_r$ of 71,000 consisting of two identical subunits. Catechol 1,2-dioxygenase from R. rhodochrous N75 exhibits some unusual properties including: broad substrate specificity, extradiol cleavage activity with 4-methylcatechol and low $K_m$ values for halocatechols, suggesting that this enzyme is distinct from other known catechol and chlorocatechol 1,2-dioxygenases.

• Korean Journal of Microbiology
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• v.30 no.1
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• pp.8-14
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• 1992

Nucleotide Sequence of phnE Gene Encoding Extradiol Dioxygenase fromPseudomonas sp. Strain DJ77Kim, Young-Chang'.", Myeong-Su Shin1, Kil-Sang Younl, Young-Soon Park1, andUg-Hyeon Kim'.' (Department of Microbiology, C'hungbuk National University.Cheongju 360-763, KOREA. and 'Research Center for Molecular Microbiology,Seoul National University)nal University)

• Journal of Korean Society of Environmental Engineers
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• v.35 no.6
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• pp.389-393
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• 2013

The purpose of this study is to investigate the biodegradation characteristics of the xylene by BTEX-degrading bacteria, Pseudomonas putida BJ10, isolated from oil-contaminated soil and bio-degradation pathway of the xylene. The removal efficiencies of o, m, p-xylene in mineral salts medium (MSM) by P. putida BJ10 were 94, 90 and 98%, respectively for 24 hours. It shows clear difference compared with the control groups which were below 3%. The removal efficiencies of BTEX by P. putida BJ10 in gasoline-contaminated soil were 66% for 9 days. They were clearly distinguished from the control groups (control and sterilized soil) which were 32 and 8%. 3-methylcatechol and o-toluic acid were detected after 6 and 24 hours during the o-xylene biodegradation pathway. Therefore, we confirmed o-toluic acid as the final metabolite. And intermediate-products were somewhat different with previously published studies of the transformation pathway from o-xylene to 3-methylcatechol.

• Korean Journal of Microbiology
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• v.36 no.1
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• pp.26-32
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• 2000

Catechol 2,3-dioxygenase was purified from recombinant strain E. coli CNU312 carrying the tomB gene which was cloned from toluene-degrading Burkholderia cepacia G4. The purification of this enzyme was performed by acetone precipitation, Sephadex G-75 chromatography, electrophoresis and electro-elution. The molecular weight of native enzyme was about 140.4 kDa and its subunit was estimated to be 35 kDa by SDS-PAGE. It means that this enzyme consists of four identical subunits. This enzyme was specifically active to catechol, and$K_(m)$ value and $V_(max)$value of this enzyme were 372.6 $\mu$M and 39.27 U/mg. This enzyme was weakly active to 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol, but rarely active to 2,3-DHBP. The optimal pH and temperature of the enzyme were pH 8.0 and $40^{\circ}C$. The enzyme was inhibited by $Co^(2+)$, $Mn^(2+)$, $Zn^(2+)$, $Fe^(2+)$, $Fe^(3+)$, and $Cu^(2+)$ ions, and was inactivated by adding the reagents such as N-bromosuccinimide, and $\rho$-diazobenzene sulfonic acid. The activity of catechol 2,3-dioxygenase was not stabilized by 10% concentration of organic solvents such as acetone, ethanol, isopropyl alcohol, ethyl acetate, and acetic acid, and by reducing agents such as 2-mercaptoethanol, dithiothreitol, and ascorbic acid. The enzyme was inactivated by the oxidizing agent $H_(2)$$O_(2)$, and by chelators such as EDTA, and ο-phenanthroline.

• Microbiology and Biotechnology Letters
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• v.24 no.3
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• pp.282-289
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• 1996

2,3-DHBP dioxygenase was purified from E. coli CK1092 carrying the pcbC gene, which was cloned from 4-chlorobiphenyl-degrading Pseudomonas sp. P20. Purification of this enzyme was done by acetone precipitation, DEAE- Sephadex A-25 ion exchange chromatography, and preparative gel electrophoresis. The molecular weight of subunit was 34 kDa determined by SDS-PAGE, and that of native enzyme was about 270 kDa. It suggests that this enzyme consist of eight identical subunits. This enzyme was specifically active against only 2,3-DHBP as a substrate with 18 $\mu$M of Km value, but not catechol, 3-methylcatechol, 4-methylcatechol and 4-chlorocatechol. The optimal pH and temperature of 2,3-DHBP dioxygenase were pH 8.0 and 40-60$\circ$C. The enzyme was inhibited by Cu$^{2+}$, Fe$^{2+}$ and Fe$^{3+}$ ions, and was inactivated by H$_{2}$0$_{2}$2 and EDTA. The lower concentrations of some organic solvents such as acetone and ethanol don't stabilize the activity of 2,3-DHBP dioxygenase. The enzyme was completely inactivated by adding the reagents such as N-bromosuccinimide, iodine and p- diazobenzene sulfonic acid.