• 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.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%).

• Korean Journal of Microbiology
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• v.36 no.2
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• pp.79-83
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• 2000

Activated sludge samples were collected from a municipal sewage treatment plant and used for enrichment of microbial consortia with aniline as the sole carbon and nitrogen source. Threc aniline-degrading bacteria were obtained lrom microbial consortia and an isolate which has excellent aniline degradability was selected for this study. The isolate was Gram-negative, and identified and designated as Delfha sp. JK-2 on the basis of various physiological and biochemical tests. 10 mM aniline was completely degraded within 24 hours after inoculation of the culture. Ammonium ion was liberated in the medium transiently during the incubation and disappeared when aniline was completely degraded. Addition of glucose as a supplementary source to aniline minimal media showed significant decrease in aniline degradat~on rate for the strain Effective degradation of aniline was achieved by the addition of 0.5% nitrate as a nitrogen source, and resulted in approximately 80% higher aniline degradation compared to the absence of nitrate. Phylogenetic analysis based on 16s [DNA sequence revealed that the strain was closely related to De@ia acidovorans, with 96% overall similarity. The 16s [DNA sequence of JK-2 was also found to be closely related to those of six other clonal types, including Acidovoru, Aquaspirillum. Xylophilus, Variovorm, and Rhodofernr.

• 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.