• 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.

• KSBB Journal
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• v.19 no.1
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• pp.50-56
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• 2004

The aim of this work was to investigate the purification and characterization dixoygenases isolated from Delftia sp. JK-2, which could utilize aniline, benzoate, and p-hydroxybenoate as sole carbon and energy source. Catechol 1,2-dioxygenase (C1, 2O), catechol 2,3-dioxygenase(C2, 3O), and protocatechuate 4,5-dioxygenase(4,5-PCD) were isolated by benzoate, aniline, and p-hydroxybenzoate. In initial experiments, several characteristics of C1 ,2O, C2, 3O, and 4,5-PCD separated with ammonium sulfate precipitation, DEAE-sepharose, and Q-sepharose were investigated. Specific activity of C1 ,2O, C2, 3O, and 4,5-PCD were approximately 3.3 unit/mg, 4.7 unit/mg, and 2.0 unit/mg. C1 ,2O and C2, 3O demonstrated their enzyme activities to other substrates, catechol and 4-methylcatechol. 4,5-PCD showed the specific activity to the only substrate, protocatechuate, but the substrates(e.g., catechol, 3-methylcatechol, 4-methylcatechol, 4-chlorocatechol, 4-nitrocatechol) did not show any specific activities in this work. The optimum temperature of C1, 2O, C2, 3O, and 4,5-PCD were 30$^{\circ}C$, and the optimal pHs were approximately 8, 8, and 7, respectively. Ag$\^$+/, Hg$\^$+/, Cu$\^$2+/ showed inhibitory effect on the activity of C1, 2O and C2, 3O, but Ag$\^$+/, Hg$\^$+/, Cu$\^$2+/, Fe$\^$3+/ showed inhibitory effect on the activity of 4,5-PCD. Molecular weight of the C1, 2O, C2, 3O, and 4,5-PCD were determined to approximately 60 kDa,35 kDa, and 62 kDa by SDS-PAGE.

• Korean Journal of Microbiology
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• v.39 no.1
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• pp.1-7
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• 2003

The aim of this work was to investigate the characterization and sequence of catechol 2,3-dioxygenase isolated from Delfia sp. JK-2, which could utilize aniline as sole carbon, nitrogen and energy source. In initial experiments, several characteristics of C2,3O separated with ammonium sulfate precipitation, DEAE-sepharose were investigated. Specific activity of C2,3O was approximately 4.72 unit/mg. C2,3O demonstrated its enzyme activity to other substrates, catechol and 4-methylcatechol. The optimum temperature of C2,3O was $$Cu^{2+}$^{\circ}C$, and the optimal pH was approximately 8. Metal ions such as $Ag^{+}$, $Hg^{+}$, and $Cu^{2+}$ showed inhibitory effect on the activity of C2,3O. Molecular weight of the enzyme was determined to approximately 35 kDa by SDS-PAGE. N-terminal amino acid sequence of C2,3O was analyzed as $^{1}MGVMRIG-HASLKVMDMDA- AVRHYENV^{26}$, and exhibited high sequence homology with that of C2,30 from Pseudomonas sp. AW-2, Comamonas sp. JS765, Comamonas testosteroni and Burkholderia sp. RPO07. PCR product was amplified with the primers derived from N-terminal amino acid sequence. In this work, we found that the amino acid sequence of Delftia sp. JK-2 showed high sequence homology of C2,3O from Pseudomonas sp. AW-2 (100%) and Comamonas sp. JS765 (97%).

• Biotechnology and Bioprocess Engineering:BBE
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• v.3 no.2
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• pp.112-114
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• 1998

For enhancement of cis,cis-muconate productivity from benzoate, catechol 1,2-dioxygenase (C12O) which catalyzes the rate-limiting step (catechol conversion to cis,cis-muconate) was cloned and expressed in recombinant Pseudomonas putida BCM114. At higher benzoate concentrations (more than 15 mM), cis,cis-muconate productivity gradually decreased and unconverted catechol was accumulated up to 10 mM in the cae of wild-type P. putida BM014, whereas cis,cis-muconate productivity continuously increased and catechol was completely transformed to cis,cis-muconate for P. putida BCM114. Specific C12O activity of P. putida BCM114 was about three times higher than that of P. putida BM014, and productivity was enhanced more than two times.

• BMB Reports
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• v.34 no.2
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• pp.172-175
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• 2001

Catechol 1,2-dioxygenase $I_1$ ($CDI_1$) is the first enzyme of the $\beta$-ketoadipate pathway in Acinetobacter lowffii K24. $CDI_1$ has two cysteines (155, 202) and its enzyme activity is inhibited by the cysteine inhibitor, $AgNO_3$. Two mutants, $CDI_1$ C155V and $CDI_1$ C202V, were obtained by site-directed mutagenesis. The two mutants were overexpressed and the mutated amino acid residues (Cys$\rightarrow$Val) were characterized by peptide mapping and amino acid sequencing. Interestingly, $CDI_1$ C155V was inhibited by $AgNO_3$, whereas $CDI_1$ C202V was not inhibited. This suggests that $Cys^{202}$ is the sole inhibition site by $AgNO_3$ and is close to the active site of the enzyme. However, the results of the biochemical assay of mutated $CDI_1s$ suggest that the two cysteines are not directly involved in the activity of the catechol 1,2-dioxygenase of $CDI_1$.

• Korean Journal of Microbiology
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• v.41 no.1
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• pp.13-17
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• 2005

The aim of this work was to investigate the N-terminal amino acid sequence of catechol 2,3-dioxygenase isolated from Delftia sp. JK-2, which could utilize aniline as sole carbon, nitrogen and energy source. Molecular weight of the enzyme was determined to approximately 35 kDa by SDS-PAGE. N-terminal amino acid sequence of C2,3O from strain JK-2 was $^1MGVMRIGHASLKVMDMDAAVRHYENV^{26}$, and exhibited high sequence similarity with that of C2,3O from Pseudomonas sp., Comamonas sp. JS765, Comamonas test-osteroni, or Burkholderia sp. RP007. Approximately 950-bp C2,3O was obtained through PCR using the primers derived from N-terminal amino acid sequence. Analysis of the DNA sequence revealed that the deduced 296 amino acid sequences were determined, and it showed $100\%$ identity with C2,3O from Pseudomonas sp. AW-2 and $97\%$ similarity with Comamonas sp. JS765.

• Korean Journal of Microbiology
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• v.30 no.4
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• pp.316-321
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• 1992

Catechol 2, 3-dioxygenase (C230) catalyses the oxidative ring cleavage of catechol to 2-hydroxymuconic semialdehyde. This is one of the key reactions in the metabolism of the widespresd pollutant aniline. We have cloned a gene encoding C230 from cells of the aniline degrading bacteria, Pseudomonas acidovorance KCTC2494 strain and expressed in E. coli, A 11.3-kilobase Sau3A partial digested DNA fragment from KCTC2494 was cloned into phagemid vector pBluescript and designated as pLP201. The C230 gene was mapped to a 2.8-kb region, and the derection of transcription was determined. The cloned C230 gene contains its own promoter which can be recognized and employed by E. coli transcriptional apparatus. C230 activities of subclones were identified by enzyme assay and activity staining. The T7 RNA promoter/polymerase system and maxicell analysis showed that a polypeptide with Mw of 35 kDa is the C230 gene product.

• Microbiology and Biotechnology Letters
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• v.35 no.2
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• pp.104-111
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• 2007

In this study, aniline dioxygenase genes responsible for initial catabolism of aniline in Burkholderia sp. HY1 and Delftia sp. HY99 were cloned and the amino acid sequences were comparatively analyzed, which already have been reported as bacteria utilizing aniline as a sole source of carbon and nitrogen, B. sp. HY1 was found to have at least a plasmid, and the plasmld-cured strain, B. sp. HY1-PC obtained using mitomycin C was tested with wild type strain to investigate whether the former maintained the degradability for aniline. This proved that the aniline oxygenase gene from B. sp. HY1 was located in chromosomal DNA, not in plasmid DNA. Aniline dioxygenase small subunits from B. sp. HY1 and D. sp. HY99 were found, based on 146 amino acids, to share 79% similarity. Notably, ado2 genes from B. sp. HY1 and D. sp. HY99 which were found to be terminal dioxygenase of aniline dioxygenase small subunit showed 99% similarity in the deduced amino acid sequences with tdnA2 of Frateuria sp. ANA-18 and danA2 of D. sp. AN3, respectively. Besides, enzyme assay and amino acid sequence analysis of catechol dioxygenase supported the previous report that B. sp. HY1 might occupy ortho-cleavage pathway using catechol 1,2-dioxygenase, while D. sp. HY99 might occupy catechol 2,3-dioxygenase for meta-cleavage pathway.

• Journal of Microbiology
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• v.35 no.4
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• pp.295-299
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• 1997

Pseudomonas sp. S-47 is capable of transforming 4-chlorobenzoate to 4-chlorocatechol which is subsequently oxidized bty meta-cleavage dioxygenase to prodyce 5-chloro-2-hydroxymuconic semialdehyde. Catechol 2,3-dioxygenase (C23O) produced by Pseudomonas sp. S-47 was purified and characterized in this study. The C23O enzyme was maximally produced in the late logarithmic growth phase, and the temperature and pH for maximunm enzyme activity were $30{\sim}35^{\circ}C$ and 7.0, respectively. The enzyme was purified and concentrated 5 fold from the crude cell extracts through Q Sepharose chromatography and Sephadex G-100 gel filtration after acetone precipitation. The enzyme was identified as consisting of 35 kDa subunits when analyzed by SDS-PAGE. The C23O produced by Pseudomonas sp. S-47 was similar to Xy1E of Pseudomonas putida with respect to substrate specificity for several catecholic compounds.

• 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.