• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.422-427
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• 2003

Streptomyces setonii (ATCC 39116) is a Gram-positive thermophilic soil actinomycetes capable of degrading single aromatic compounds including phenol and benzoate via ortho-cleavage pathway. we isolated approximately 6.3-kb S. setonii DNA fragment containing a thermophilic catechol 1,2-dioxygenase(C12O) gene. Here we further revealed that the 6.3-kb S. setonii DNA fragment was organized into two putative divergently-transcribed clusters with 6 complete and one incomplete open reading frames (ORFs). The first cluster with 3 ORFs showed significant homologies to previously known benA, benB, and benC, implying a part of benzoate catabolic operon. The second cluster revealed an ortho-cleavage catechol catabolic operon with three translationally-coupled ORFs (catR, catB, catA). Each of these individually-cloned ORFs was expressed in E. coli and identified as a distinct protein band with a theoretical molecular weight in SDS-PAGE. The expression of the cloned S. setonii catechol operon was induced in a heterologous S. lividans by specific single aromatic compounds including catechol, phenol, and 4-chlorophenol. The simitar induction pattern was also observed using a luciferase gene-fused reporter system, implying that S. setonii employs an inducer-specific regulatory mechanism for aromatic compound metabolism.

• BMB Reports
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• 제34권2호
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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 $CDI_1$.

• 미생물학회지
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• 제30권4호
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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.

• Bulletin of the Korean Chemical Society
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• 제21권9호
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• pp.923-928
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• 2000

[FeIII(BBA)DBC]ClO4 as a new functional model for catechol dioxygenases has been synthesized, where BBA is a bis(benzimidazolyl-2-methyl)amine and DBC is a 3,5-di-tert-butylcatecholate dianion.The BBA complex has a structuralfeature that iron cent er has a five-coordinate geometry similar to that of catechol dioxygenase-substrate complex.The BBA complex exhibits strong absorptionbands at 560 and 820 nm in CH3CN which are assigned to catecholate to Fe(III) charge transfer transitions. It also exhibits EPR signals at g = 9.3 and 4.3 which are typical values for the high-spin FeIII (S = 5/2) complex with rhombicsymmetry. Interestingly, the BBA complex reacts with O2 within an hour to afford intradiol cleavage (35%) and extradiol cleavage (60%) products. Surprisingly, a green color intermediate is observed during the oxygenation process of the BBA com-plex in CH3CN. This green intermediate shows a broad isotropic EPR signal at g = 2.0. Based on the variable temperature EPR study, this isotropic signalmight be originated from the [Fe(III)-peroxo-catecholate] species havinglow-spin FeIII center, not from the simple organic radical. Consequently,it allows O2 to bind to iron cen-ter forming the Fe(III)-superoxide species that converts to the Fe(III)-peroxide intermediate. These present data can lead us tosuggest that the oxygen activation mechanism take place for the oxidative cleavingcatechols of the five-coordinate model systems for catechol dioxygenases.

• 한국미생물·생명공학회지
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• 제26권4호
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• pp.352-357
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• 1998

방향족 화합물의 기본을 이루고 있는 benzoate와 m-toluate 혼합물 분해를 위한 45일간의 배양 결과 benzoate와 m-toluate 최적 기질 혼합비는 benzoate(75%): m-toluate(25%)일 때 가장 높은 균 생장율과 COD 제거율을 나타내었다. 또한 45일간의 배양 중 혼합기질의 농도가 2,000ppm으로 교체된 30일째의 benzoate와 m-toluate의 기질 분해율은 각각 94%와 79%였고 이때의 COD 제거율은 약 80%였다. 한편 효소 활성측정 결과 초기에 거의 검출되지 않았던 catechol 1,2-dioxygenase의 활성이 검출되어 m-toluate에 의해 본 균주의 효소 대사계가 유도 되었음을 알 수 있었다. 또한 배양 중 기질 농도에 대한 본 균주의 형태변화를 전자현미경으로 관찰한 결과, 기질의 농도가 높을수록 균 형태가 변화된 것으로 볼 때 일정 농도 이상의 방향족 화합물에 대한 내성은 대사에 관련된 효소 활성에 기인할 뿐만 아니라 아니라 세포벽 또는 세포막의 특성에 기인할 수도 있는 것으로 추측된다.

• Applied Biological Chemistry
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• 제31권4호
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• pp.331-338
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• 1988

칡뿌리로 부터 칡뿌리 acetone분말을 제조하고 polyphenol oxidase를 추출, 정제하여 정제효소의 효소학적 특성을 검토하였다. 조효소는 DEAE-Cellulose, DEAE-Sephadex A-50, Sephadex G-100 column chromatography에 의하여 정제되었으며, 정제효소의 비활성은 94배, 정제수율은 45.4%이었다. 정제효소는 catechol 및 pyrogallol에 대하여 감한 친화성을 나타내었으며, km 값은 catechel을 기질로 하였을 때 16.67mM이었다. 작용 최적 pH는 7.5, 최적온도는 $50^{\circ}C$에서 가장 잘 작용하였고 1mM L-ascorbic acid, sodium bisulfite, EDTA, KCN 및 $Fe^{3+}$ 이온에 의하여 심한 저해를 나타내었으며, $Zn^{2+}$ 이온을 약 1.7배 정도의 활성을 증가시켰다. 조효소액을 전기영동하여 catechol로 활성염색 하였을 때 5개 isoenzyme이 확인되었으며 그 중 분자량 35,000의 것이 가장 강한 활성을 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제13권5호
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• pp.700-705
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• 2003

Pseudomonas sp. S-47 is capable of degrading benzoate and 4-chlorobenzoate as well as catechol and 4-chlorocatechol via the meta-cleavage pathway. The three enzymes of 2-oxopenta-4-enoate hydratase (OEH), acetaldehyde dehydrogenase (acylating) (ADA), and 2-oxo-4-hydroxypentonate aldolase (HOA) encoded by xylJQK genes are responsible for the three steps after the meta-cleavage of catechol. The nucleotide sequence of the xylJQK genes located in the chromosomal DNA was cloned and analyzed. GC content of xylJ, xylQ, and xylK was 65% and consisted of 786, 924, and 1,041 nucleotides, respectively. The deduced amino acid sequences of xylJ, xylQ, and xylK genes from Pseudomonas sp. S-47 showed 93%, 99%, and 99% identity, compared with those of nahT, nahH, and nahI in Pseudomonas stutzeri An10. However, there were only about 53% to 85% identity with xylJQK of Pseudomonas putida mt-2, dmpEFG of P. putida CF600, aphEFG of Comamonas testosteroni TA441, and ipbEGF of P. putida RE204. On the other hand, the xylLTEGF genes located upstream of xylJQK in the strain S-47 showed high homology with those of TOL plasmid from Pseudomonas putida mt-2. These findings suggested that the xylLTEGFIJQK of Pseudomonas sp. S-47 responsible for complete degradation of benzoate and then catechol via the meta-pathway were phylogenetically recombinated from the genes of Pseudomonas putida mt-2 and Pseudomonas stutzeri An10.

• BMB Reports
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• 제35권4호
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• pp.432-436
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• 2002

Pseudomonas sp. S-47 is capable of degrading catechol and 4-chlorocatechol via the meta-cleavage pathway. XyITE products catalyze the dioxygenation of the aromatics. The sylT of the strain S-47 is located just upstream of the xylE gene. XylT of the strain S-47 is located just upstream of the xylE gene. XyIT is typical chloroplast-type ferredoxin, which is characterized by 4 cystein residues that are located at positions 41, 46, 49, and 81. The chloroplast-type ferredoxin of Pseudomonas sp. S-47 exhibited a 98% identity with that of P. putida mt-2(TOL plasmid) in the amino acid sequence, but only about a 40 to 60% identity with the corresponding enzymes from other organisms. We constructed two recombinant plasmids (pRES1 containing xylTE and pRES101 containing xylE without xylT) in order to examine the function of XyIT for the reactivation of the catechol 2,3-dioxygenase (XyIE) that is oxidized with hydrogen peroxide was recovered in the catechol 2,3-dioxygenase (C23O) activity about 4 mimutes after incubation, but the pRES101 showed no recovery. That means that the typical chloroplast-type ferredoxin (XyIT) of Pseudomonas sp. S-47 is involved in the reactivation of the oxidized C23O in the dioxygenolytic cleavage of aromatic compounds.

• Korean Chemical Engineering Research
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• 제44권4호
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• pp.387-392
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• 2006

이온 교환법으로 (Fe, Co)/NaY, (Fe, Co)/NaBeta (Fe, Co)/HUSY 등 (Fe,Co)/zeolite 촉매를 제조하였으며, 카테콜을 합성하는 과산화수소에 의한 페놀의 수산화 반응에서 이들의 촉매 성능을 조사하였다. (Fe, Co)/NaBeta 촉매에서는 반응온도가 $70^{\circ}C$, 페놀/과산화수소(몰) 비가 3, 페놀/촉매(무게) 비가 50, 용매(물)/페놀(무게) 비가 6인 조건에서 페놀의 전환율은 22％, 수산화 반응에 대한 선택도는 77％, 카테콜에 대한 선택도는 70％, 카테콜/하이드로퀴논의 생성비는 2.5로 가장 좋은 반응 결과를 얻었다. (Fe, Co)/zeolite 촉매는 재생하여 반복 사용해도 성능이 저하되지 않았다. 반응 전후의 (Fe, Co)/zeolite 촉매를 XRD, UV-VIS DRS, XPS 등으로 조사하여, 이를 근거로 촉매 반응 결과를 해석하였다.

• 대한화학회지
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• 제24권1호
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• pp.25-33
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• 1980

토양에서 분리한 Pseudomonadaceae 속 박테리아로부터 pyrocatechase를 추출, 분리 정제하였으며, 이 효소의 특성을 검토한 결과 pyrocatechase는 catechol에 대하여 기질 특이성을 보여줌을 알았다. 효소 활성도의 최적조건은 pH 7∼10 부근과 온도 $35^{\circ}C$임을 알았으며, catechol에 대한 $K_m$값은 $1.9{\times}10^{-6}M$ 로 얻어졌다. 기질 유도체에 의한 효소 저해 실험결과 벤젠 고리의 ortho 위치에 두개의 수산기는 효소-기질간의 결합반응에 참여될 것이라고 추측했다. 기타 SH-잔기와 작용하는 화합물 또는 중금속 이온등의 첨가에 따른 효소 활성도의 저해 효과를 검토 하였으며, 효소 활성부위에 대하여도 검토해 보았다.