• 한국미생물·생명공학회지
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• 제24권6호
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• pp.657-663
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• 1996

Pseudomonas sp. P20 isolated from the polluted environment is capable of degrading biphenyl and 4-chlorobiphenyl. The pcbABCD genes responsible for degradation of biphenyl and 4-chlorobiphenyl were cloned using pBluescript SK(+) from the chromosomal DNA of Pseudomonas sp. P20 to construct pCK1 and pCK102, harbouring pcbABCD and pcbCD, respectively. The 2, 3-DHBP dioxygenase gene, pcbC, was cloned again from pCK102 by using pKT230 which is known as a shuttle vector and pKK1 hybrid plasmid was constructed. The E. coli KK1 transformant obtained by transforming the pKK1 into E. coli XL1-Blue showed 2, 3-DHBP dioxygenase activity. The specific 2, 3-DHBP dioxygenase activity of E. coli KK1 was similar to that of the E. coli CK102, but much higher than those of the natural isolates, Pseudomonas sp. DJ-12 and Pseudomonas sp. P20.

• 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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• 제9권5호
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• pp.393-399
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• 2004

Useful genes can be Screened from various environments by construction of metagenomic DNA libraries. In this study, water samples were collected from several lakes in mid Korea, and analyzed by T-RFLP to examine diversities of the microbial communities. The crude DNAs r were extracted by the SDS-based freezing-thawing method, and then further purified using an $UltraClean^{TM}$ kit (MoBio, USA). The metagenomic libraries were constructed with the DNAs partially digested with EcoR I, BamH I, and Sac II in Escherichia coli DH 10B using the pBACe3.6 vector. About 44.0 Mb of metagenomic libraries were obtained with average inserts 13-15 kb in size. The bphC genes responsible for degradation of aromatic hydrocarbons via mets-cleavage were identified from the metagenomic libraries by colony hybridization using the bphC specific sequence as a probe. The 2,3-dihydroxybiphenyl (2, 3-DHBP) dioxygenase gene (bphC ), capable of degradation of 2,3-DHBP, was cloned and its nucleotide Sequences analyzed. The genes consisted of 966 and 897 base pairs with an ATG initiation codon and a TGA termination codon. The activity of the 2,3-DHBP dioxygenase was highly expressed to 2,3-DHBP and Showed a broad substrate range to 2,3-DHBP, catechol, 3-methylcatechol and 4-methylcatechol. These results in-dicated that the bphC gene identified from the metagenomes derived from lake water might be useful in the development of a potent strain for degradation of aromatic pollutants.

• 한국미생물·생명공학회지
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• 제21권2호
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• pp.150-156
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• 1993

The 2,3-dihydroxybiphenyl(2,3-DHBP) dioxygenase, the product of pcbC gene, was purified from the biphenyl and 4-chlorobiphenyl degrading Pseudomonas sp. DJ-12 by the methods of acetone precipitation, DEAE-Sephadex A-50 ion exchange chromatography, and Sephadex G-150 gel filtration chromatography. The enzyme was estimated to be about 260 kilodaltons in molecular weight and to be consisted of eight subunits. The Km value of the enzyme was 61 nM to 2,3-DHBP and the highest activity of the enzyme was observed at pH 8 and 30C.

• 한국미생물·생명공학회지
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• 제22권4호
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• pp.353-359
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• 1994

Pseudomonas sp. P20 was a bacterial isolate which has the ability to degrade 4-chlorobi- phenyl(4CB) to 4-chlorobenzoic acid via the process of meta-cleavage. The recombinant plasmid pCK1 was constructed by insetting the 14-kb EcoRI fragment of the chromosomal DNA containing the 4CB-degrading genes into the vector pBluescript SK(+). Subsequently, E. coli XL1-Blue was transformed with the hybrid plasmid producing the recombinant E. coli CK1. The recombinant cells degraded 4CB and 2,3-dihydroxybiphenyl(2,3-DHBP) by the pcbAB and pcbCD gene products, respectively. The pcbC gene was expressed most abundantly at the late exponential phase in E. coli CK1 as well as in Pseudomonas sp. P20, and the level of the pcbC gene product, 2,3-DHBP dioxygenase, expressed in E. coli CK1 was about two-times higher than in Pseudomonas sp. P20. The activities of 2,3-DHBP dioxygenase on catechol and 3-methylcatechol were about 26 to 31% of its activity on 2,3-DHBP, but the enzyme did not reveal any activities on 4-methylcatechol and 4-chlorocatechol.

• 미생물학회지
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• 제32권1호
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• pp.40-46
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• 1994

Polychlorinaed biphenyls(PCBs) 와 biphenyl을 분해하는 Pseudomonas sp. DJ-12에서는 그 초기 분해과정에 pcb ABCD 유전자들이 관여하고 있음이 밝혀졌다. 그 중 pcbCD와 pcdD 유전자를 E. coli XL1-Blue에 클로닝하여 E. coli CU103 과 CU105 균주를 각각 제조하였다. E. coli CU103은 2,3-dehydroxybuphenyl dioxygenase(2,3-DHBP)와 meta-cleavage compound(MCP) hydrolase를 생성하여 2,3-dihydroxybiphenyl을 benzoate로 변환시켜 주었다. E. coli CU1 과 CU103 에서 pcbC 유전자의 산물인 2,3-DHBP dioxygense의 활성도는 Pseudomonas sp. DJ-12에서 보다 약 17배 높았으며, E. coli CU105에서 pcbD의 산물인 MCP hydrolase는 약 3배 더 높게 나타났다.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1995년도 한국생물과학협회 학술발표대회
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• pp.355.2-355
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• 1995

No Abstract, See Full Text

• Journal of Microbiology
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• 제35권2호
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• pp.141-148
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• 1997

Alcaligenes xylosoxydans strain SMN3 capable of utilizing biphenyl grew not only on phenol, and benzoate, but also on salicylate. Catabolisms of biphenyl and salicylate appear to be interrelated since benzoate is a common metabolic intermediate of these compounds. Enzyme levels in the excatechol 2. 3-dioxygenas which is meta-cleavage enzyme of catechol, but did not induce catechol 1, 2-dioxygenase. All the oxidative enzymes of biphenyl and 2, 3,-dihydroxybiphenyl (23DHBP) were induced when the cells were grown on biphenyl and salicylate, respectively. Biphenyl and salicylate could be a good inducer in the oxidation of biphenyl and 2, 3-dihydroxybiphenyl. The two enzymes for the degradation of biphenyl and salicylate were induced after growth on either biphenyl or salicylate, suggesting the presence of a common regulatory element. However, benzoate could not induce the enzymes responsible for the oxidation of these compounds. Biphenyl and salicylate were good inducers for indigo formation due to the activity of biphenyl dioxygenase. These results suggested that indole oxidation is a property of bacterial dioxygenase that form cis-dihydrodiols from aromatic hydrocarbon including biphenyl.

• Journal of Microbiology
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• 제38권3호
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• pp.183-186
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• 2000

Several types of bioluminescent reporter strains have been developed for the detection and monitoring of pollutant aromatics contaminating the environment. In this study, a bioluminescent reporter strain, E. coli SHP3, was constructed by fusing the luc gene of firefly luciferase with the promoter of pcbC responsible for the meta-cleavage of aromatic hydrocarbons. the bioluminescence expressed by the luc gene in the reporter was well triggered by the promoter when it was exposed to 2,3-dihydroxybiphenyI (2,3-DHBP) at 0.5 to 1 mM concentrations. The bioluminescent response was more extensive when the reporter strain was exposed to 5 mM catechol and 2 mM 4-chlorocatechol. These different types of bioluminescent responses by E. coli SHP3 appeared to be characterized by the nature of the aromatics to stress. Since E. coli SHP3 responded to 2,3-DHBP quite sensitively, this reporter strain could be applied for detecting some catecholic pollutants.

• Journal of Microbiology and Biotechnology
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• 제5권2호
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• pp.68-73
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• 1995

Pseudomonas sp. P20 was shown to be capable of degrading biphenyl and 4-chlorobiphenyl (4CB) to produce the corresponding benzoic acids wnich were not further degraded. But the potential of the strain for biodegradation of 4CB was shown to be excellent. The pcbA, B, C and D genes responsible for the aromatic ring-cleavage of biphenyl and 4CB degradation were cloned from the chromosomal DNA of the strain. In this study, the pebC and D genes specifying degradation of 2, 3-dihydroxybiphenyl (2, 3-DHBP) produced from biphenyl by the pebAB-encoded enzymes were cloned by using pBluescript SK(＋) as a vector. From the pCK102 (9.3 kb) containing pebC and D genes, pCK1022 inserted with a EcoRI-HindIII DNA fragment (4.1 kb) carrying pebC and D and a pCK1092 inserted with EcoRI-XbaI fragment (1.95 kb) carrying pebC were constructed. The expression of pcbC and D' in E. coli CK102 and pebC in E. coli CK1092 was examined by gas chromatography and UV-vis spectrophotometry. 2.3-dihydroxybiphenyl was readily degraded to produce meta-cleavage product (MCP) by E. coli CK102 after incubation for 10 min, and then only benzoic acid(BA) was detected in the 24-h old culture. The MCP was detected in E. coli CK1022 containing pebC and 0 genes (by the resting cells assay) for up to 3 h after incubation and then diminished completely in 8 h, whereas the MCP accumulated in the E. coli CK1092 culture even after 6 h of incubation. The 2, 3-DHBP dioxygenases (product of pebC gene) produced by E. coli CK1, CK102, CK1023, and CK1092 strains were measured by native PAGE analysis to be about 250 kDa in molecular weight, which were about same as those of Pseudomonas sp. DJ-12, P. pseudoa1caligenes KF707, and P. putida OU83.