• Journal of Microbiology
• /
• 제34권4호
• /
• pp.341-348
• /
• 1996

The regulation of xylE gene expression was examined by using vector promoter and construction of genetically engineered microorganisms (GEMs) for application in microcosm. When the xylE gene wsa subcloned into pBluscript SK(+) under the control of lac promoter (pTY1) in E. coli, and the expression was induced by IPTG, the enzyme activity of catechol 2, 3-dioxygenase was increased 4.7 times more than that of the crude extracts from transformants harboring pTY1. We suggest that the xylE gene has its own promoter at the upstream portion, because it was able to be expressed even in the absence of IPTG. A recombinant plasmid, pSW3a harboring the xylE gene under the T7 promotor, showed the activity of 14.5 units/mg protein, higher than that of parental strain, E. coli PYT1. The xylE gene in recombinant plasmid pSW3a was used as reporter gene for the application in microcosm ecosystem, since it was used for detection of xylE-positive clones by catechol spray on the agar plates. The pSW3a in E. coli was introduced into Pseudomonas patida to construct GEM strain, and examined for the exxpression and functional stability in microcosms.

• Journal of Microbiology and Biotechnology
• /
• 제13권4호
• /
• pp.487-494
• /
• 2003

2,3-Dihydroxybiphenyl 1,2-dioxygenase (23DBDO), an enzyme of the biphenyl biodegradation pathway encoded by the bphC gene of Comnmonas sp. SMN4, was expressed and purified using column chromatographies. SDS-PAGE of purified 23DBDO showed a single band with a molecular mass of 32 kDa, which was consistent with the data from the gel filtration chromatography (GFC). The purified enzyme exhibited a maximum 23DBDO activity at pH 9.0 and was stable at pH 8.0. The enzyme showed maximum activity at $40^{\circ}C$ and maintained activity at $30^{\circ}C$ for 24 h. Kinetic parameters represented by Michaelis-Menten constants such as $K_m\;and\;V_{max}$ values for various substrates were determined by Lineweaver-Burk plots: The purified enzyme 23DBDO from Comamonas sp. SMN4 had the highest catalytic activity for 2,3-dihydroxybiphenyl and 3-methylcatechol, and had very poor activity with catechol and 4-methylcatechol.

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

• Journal of Microbiology
• /
• 제40권1호
• /
• pp.86-89
• /
• 2002

Rhodococcus sp. strain T104, which is able to grow on either biphenyl or limonene, was found to utilize phenol as sole carbon and energy sources. Furthermore, T104 was positively identified to possess three separate pathways for the degradation of limonene, phenol, and biphenyl. The fact that biphenyl and limonene induced almost the same amount of catechol 1,2-dioxygenase activity indicates that limonene can induce both upper and lower pathways for biphenyl degradation by T104.

• 한국환경보건학회지
• /
• 제18권2호
• /
• pp.92-101
• /
• 1992

Halogen substituted-phenol and analog phenol degrading strains were identified as Aeromonas, Moraxella, and Flavobacterium genus. Optimal degrading condition was generally 50~100 $\mu$M substituted-phenol as carbon source, $NH_4NO_3$ as nitrogen source, 30$\circ$C , and initial pH 7.2. $\rho$-Chlorophenol degrading strain of Aeromonas sp. C4 had biodegradability to the various substituted-phenols. Flavobacterium sp. M9 had substrate specificity to methyl substituted-function. Catechol was cleavaged by catechol 1, 2-dioxygenase in Aeromonas sp. C4, Moraxella sp. N7, and Flavobacterium sp. M9.

• Journal of Microbiology and Biotechnology
• /
• 제10권1호
• /
• pp.111-114
• /
• 2000

Streptomyces are widespread in nature and play a very important role in the biosynthesis as well as biodegradation of natural and unnatural aromatic compounds. Both qualitatively and quantitatively through TLC and UV spectrophotometric assays, it was observed that the thermophilic soil bacteria S. setonii (ATCC 39116), which can utilize a benzoate as a sole carbon and energy source in a minimal liquid culture, was not very sensitive to the benzoate concentation and to the culture conditions such as the pH and temperature. The in vitro conversion of a catechol to a cis, cis-muconic acid by a crude S. setonii lysate implies that the aromatic ring cleavage by S. setonii is initiated by a thermostable catechol-1,2-dioxygenase, the key enzyme in the ortho-cleavage pathway of aromatic compound biodegradation. Unlike non-degrading S. lividans, S.setonii was also highly resistant to other similar hazardous aromatic compounds, exhibiting almost no adverse effect on its growth in a complex liquid culture.

• Journal of Microbiology and Biotechnology
• /
• 제19권4호
• /
• pp.339-345
• /
• 2009

We evaluated the activity and abundance of the crude-oil-degrading bacterium Nocardia sp. H17-1 during bioremediation of oil-contaminated soil, using real-time PCR. The total petroleum hydrocarbon(TPH) degradation rate constants(k) of the soils treated with and without H17-1 were $0.103\;d^{-1}$ and $0.028\;d^{-1}$ respectively. The degradation rate constant was 3.6 times higher in the soil with H17-1 than in the soil without H17-1. In order to detect and quantify the Nocardia sp. H17-1 in soil samples, we quantified the genes encoding 16S ribosomal RNA(16S rRNA), alkane monooxygenase(alkB4), and catechol 2,3-dioxygenase(23CAT) with real-time PCR using SYBR green. The amounts of H17-1 16S rRNA and alkB4 detected increased rapidly up to 1,000-folds for the first 10 days, and then continued to increase only slightly or leveled off. However, the abundance of the 23CAT gene detected in H17-1-treated soil, where H17-1 had neither the 23CAT gene for the degradation of aromatic hydrocarbons nor the catechol 2,3-dioxygenase activity, did not differ significantly from that of the untreated soil($\alpha$=0.05,p>0.22). These results indicated that H17-1 is a potential candidate for the bioaugmentation of alkane-contaminated soil. Overall, we evaluated the abundance and metabolic activity of the bioremediation strain H17-1 using real-time PCR, independent of cultivation.

• 한국환경과학회:학술대회논문집
• /
• 한국환경과학회 2001년도 정기총회 및 봄 학술발표회 초록집
• /
• pp.164-165
• /
• 2001

본 연구는 방향족 화합물질 중 페놀폐수에 대한 생물학적 처리를 위해 본 실험실에서 분리한 페놀분해능이 우수한 Rhodococcus sp. EL-GT의 catechol 분해시 1,2-dioxygenase 분해활성이 높은 것으로 보아 분해경로가 ortho-pathway임을 알 수 있었다. 향후 Rhodococcus sp. EL-GT의 페놀분해 균의 유전학적 연구를 통하여 방향족 화합물의 분해에 보다 우수한 균으로 개발시켜 효율적인 처리에 이용가능성을 예측할 수 있었다.

• 대한화학회지
• /
• 제24권1호
• /
• pp.25-33
• /
• 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.

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