• Journal of Microbiology
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• 제35권1호
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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.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1998년도 한국생물과학협회 학술발표대회
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• pp.324.2-324
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• 1998

No Abstract, See Full Text

• 한국미생물·생명공학회지
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• 제24권3호
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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.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권1호
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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.

• Clinical and Experimental Reproductive Medicine
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• 제28권2호
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• pp.131-140
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• 2001

Objective: To know the effects of xenoestrogen on spermatogenesis, we investigated the expression of cytochrome P450s enzymes (CYPscc, $CYP_{17{\alpha}}$, CYP19) and $3{\beta}$-HSD genes involved in steroidogenesis. Methods: Mouse testicular cells were prepared from 15-day-old ICR mice which had only pre-meiotic germ cells by enzyme digestion using collagenase and trypsin. Testicular cells were cultured in DMEM supplemented with FSH (0.1 IU/ml) and 10% FBS or medium with estrogen ($E_2$), bisphenol-A (BPA), octylphenol (OP; $10^{-9},\;10^{-7},\;10^{-6},\;10^{-5},\;10^{-4}M$, respectively) and aroclor 1254 (A1254) known as PCBs for 48 hours. The gene expression of cytochrome P450 enzymes were examined by semi-quantitive RT-PCR. The production of estrogen and testosterone was examined by RIA. Results: As results, expression of CYPscc mRNA was not significantly decreased, but $3{\beta}$-HSD and $CYP_{17{\alpha}}$. mRNA were significantly dose-dependent decreased. And production of testosterone and estrogen were not different except BPA and OP group ($10^{-5}M$). Conclusion: BPA, OP and A1254 might inhibit steroidogenesis by decreasing CYPscc, $3{\beta}$-HSD and $CYP_{17{\alpha}}$. mRNA expression in the mouse testis. These results suggest that BPA, OP and PCBs like as an endocrine disruptors inhibit the productions of steroidogenic enzymes and decrease the production of T and E by negative feedback mechanism. Therefore, these might disrupt steroidogenesis in Leydig cells of testis and would disturb testicular function and subsequently impair spermatogenesis.

• The Korean Journal of Ecology
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• 제18권1호
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• pp.109-120
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• 1995

As the genetically modified microorganisms (GMMs) and their recombinant plasmid DNAs could be released into natural environments, their stabilities and impacts to indigenous microorganisls have become very importhant research subjects concerning with environmental and ecological aspects. In this study, the genetically modified E. coli CU103 and its recombinant pCU103 plasmid DNA, in which pcbCD genes involving in degradation of biphenyl and 4-chlorobiphenyl were cloned, were studied for their survival and stability in several different waters established under laboratory conditions. E. coli CU103 and its host E. coli XL1-Blue survived longer in sterile distilled water (SDW) and filtered autoclaved river water (FAW) than in filtered river water (FW). A lot of extracellular DNAs were released from E. coli CU103 by lytic action of phages in FW and the released DNAs were degraded by DNase dissolved in the water. Such effects of the factors in FW on stability of the recombinant pCU103 plasmid were also observed in the results of gel electrophoresis, quantitative analysis with bisbenzimide, and transformation assay. Therefore, the recombinant plasmids of pCU103 were found to be readily liberated from the genetically modified E. coli CU103 into waters by normal metabolic processes and lysis of cells. And the plasmid DNAs were quite stable in waters, but their stabilities could be affected by physicoKDICical and biological factors in non-sterile natural waters.