순천만 갈대근권 토양으로부터 얻은 PAH 분해세균의 특성 분석

김성현,강성미,오계현,김승일,윤병준,강형일,Kim Sung-Hyun,Kang Sung-Mi,Oh Kye-Heon,Kim Seung-Il,Yoon Byoung-Jun,Kahng Hyung-Yeel 한국미생물학회 2005 미생물학회지 Vol.41 No.3

This study was accomplished in order to collect fundamental data on microbial roles in recycling process of reed rhizosphere. Sunchon bay, which is considered as one of the marsh and mud environments severely affected by human activities such agriculture and fisheries, was selected as a model place. In our initial efforts, two bacterial consortia were obtained by enrichment culture using PAH mixtures containing anthracene, naphthalene, phenanthrene and pyrene as the sources of carbon and energy, and four pure bacteria capable of rapid degradation of PAH were isolated from them. Four strains designated as SCB1, SCB2, SCB6, and SCB7 revealed by morphological, physiological and molecular analyses were identified as Burkholderia anthina, Alcaligenes sp., Achromobacter xylosoxidans., and Pseudomonas putida, respectively with over $99{\%}$ confidence. Notably, Burkholderia anthina SCB1 and Alcaligenes sp. SCB2 were found to utilize anthracene and pyrene more quickly than naphthalene and phenanthrene, whereas Achromobacter xylosoxidans SCB6 and Pseudomonas putida SCB7 exhibited similar growth and degradation patterns except for pyrene. These facts suggest that the rhizosphere microorganisms capable of PAH degradation might be used to clean up the contamination sites with polycyclic aromatic hydrocarbons. 본 연구는 농업과 어업, 그리고 생태체험과 같은 인간들의 활동으로 인하여 상당히 영향을 받는 갯벌환경 중의 하나인 순천만을 모델장소로 갈대의 환경정화 기능에 있어 근권에 분포하는 미생물의 역할에 대한 기초 자료를 얻고자 수행하였다. 우선, 순천만의 갈대근권 토양을 시료로하고 anthracene, naphthalene, phenanthrene, pyrene 등이 첨가된 다환성 방향족 화합물(polycyclic aromatic hydrocarbons; PAH)을 탄소원 및 에너지원으로 하는 농화 배양을 통하여 두 개의 consortium을 획득하였다. 두 consortium으로부터 순수 분리된 우수한 PAH분해능을 갖는 4개의 균주(SCB1, SCB2, SCB6,그리고 SCB7)를 형태 및 생리학적 특성과 16S rRNA유전자서열을 기초로 분석한 결과 각 균주는 $99{\%}$ 이상의 신뢰도로 Burkholderia sp., Aicaligenes sp., Achromobacter sp., and Pseudomonas sp.로 동정되었다. 주목할 만한 점은 Burkholderia sp. SCB1과 Alcaligenes sp. SCB2는 naphthalene이나 phenanthrene보다 훨씬 안정되어 있는 구조의 anthracene이나 pyrene에서 더 빠른 성장률과 기질 분해율을 나타내는 것으로 밝혀졌다. 반면,Achromobacter sp. SCB6와 Pseudomonas sp. SCB7은 pyrene을 제외한 다른 시험기질에 대하여 유사한 성장 및 분해패턴을 나타내었다. 이러한 결과는 주요한 염습지 식물중의 하나인 갈대의 근권에서 살아가는 이들 PAH 분해 균주들이 PAH와 같은 물질로 오염된 근권 환경의 정화작용에 중요한 역할을 할 수 있음을 제시해 주었다.

Stenotrophomonas sp. OK-5에서 분리한 NAD(P)H-Nitroreductase의 생리학적 및 분자생학적 특성 연구

호은미,강형일,오계현,Ho Eun-Mi,Kahng Hyung-Yeel,Oh Kye-Heon 한국미생물학회 2004 미생물학회지 Vol.40 No.3

TNT 분해 세균 Stenotrophomonas sp. OK-5는 세 개의 다른 NAD(P)H-nitroreductase의 활성 fractions (NTR fractions I, II, III)을 갖고 있는 것으로 확인된 바 있다. 본 연구에서는 NTR fractions I, II, III에 대한 생리학적 특성과 분자생물학적 특성을 규명하고자 하였다. TNT에 대한 균주 OK-5의 NTR fractions I, II, 그리고 III의 활성은 억제 물질인 $\beta$-mercaptoethanol의 첨가 시에 효소의 활성 이 모두 억제되는 것으로 확인되었다. TNT와 그 유사 기질을 이용하여 균주 OK-5에서 분리된 NTR의 기질 특이성을 조사한 결과, nitrobenzene, 그리고 RDX에 대 해서는 비교적 활성 이 높게 나타났으나 2,6-DNT와 2,4-DNT에서는 낮은 활성을 나타내는 것으로 확인되었다. 균주 OK-5에서 정제된 NTR fraction I의 N-말단 아미노산 서 열은 $^1MSDLLNADAVVQLFRTARDS^20$로 분석되었고, Xanthomonas campestris의 NTR과 X. axonopodis의 NTR에서 각각 70%와 65%로 비교적 높은 유사성을 가지는 것으로 나타났다. 균주 OK-5의 NTR fraction I의 효소를 암호화하는 SmOK5nrI 유전자의 염기서열을 확인하고 분석된 유전자로부터 유추되는 아미노산 서열을 각각 비교한 결과 X. campestris의 NTR과 81%, X. axonopodis의 NTR과 75%,그리고 Streptomyces avermitilis의 NTR과 30%의 유사성이 있는 것으로 조사되었으나, Pseudomonas putida KT2440의 NTR (pnrB)과는 16%로 낮은 유사성이 있는 것으로 확인되었다. Stenotrophomonas sp. OK-5 capable of degrading TNT has been found to have three nitroreductase fractions designated as NTR fractions I, II, and III. NTR in a previous study. This study was attempted to reveal physiological and molecular characteristics of NTR fractions I, II, and III in strain OK-5. Several chemicals (e.g., EDTA, NaCl, dithiothreitol, $\beta$-mercaptoethanol) were tested for their effect on enzyme activity of NTRs, demonstrating that enzyme activities of NTR fractions I, II, and III from OK-5 were inhibited in the presence of $\beta$-mercaptoethanol. Substrate specificity test showed that NTR fractions I, II, and III all have over 70% enzyme activities for nitrobenzene or RDX as a substrate. N-terminal amino acid sequence of NTR fraction I from Stenotrophomonas sp. OK-5 was $^1MSDLLNADAVVQLFRTARDS^20$ and exhibited 70% sequence homology with that of NTR from Xanthomonas campestris. NTR I gene from Stenotrophomonas sp. OK-5 (SmOK5nrI) shared extensive sequence homology in deduced amino acid sequence of PCR product with NTRs from Xanthomonas campestris (81 %), X. axonopodis (75%), Streptomyces avermitilis(30%), whereas they had low homology with that from P. putida KT2440 (pnrB) (16%).

유류누출 지역에서 유래한 토착세균, Pseudoalteromonas sp. HK-3 배양에서 생물계면활성제의 최적 생산

조수희(Su-Hee Cho),마채우(Chae-Woo Ma),오계현(Kye-Heon Oh) 한국생물공학회 2011 KSBB Journal Vol.26 No.1

The principal objective of this study was to determine the optimal conditions for the production of biosurfactant by the indigenous bacterium, Pseudoalteromonas sp. HK-3, originating from oil-spilled areas. The relationship between total biosurfactant production and the factors affecting biosurfactant production were evaluated by statistical analysis using SPSS software. The effects of various supplemental carbon sources (e.g., glucose, dextrose, mannitol, citrate, acetate) on the maximal production of biosurfactant by the test culture of Pseudoalteromonas sp. HK-3 was then evaluated. As a result, mannitol was found in this study to be the best supplemental carbon source for the production of biosurfactant. A spot inoculation of crude cultural liquid containing the HK-3 cells generated the largest clear zone, whereas only small clear zones appeared around the spots inoculated with either supernatant only or cell pellets following centrifugation. Our results demonstrated that the HK-3 test culture supplemented with 2% mannitol at an initial pH of 6 generated the maximal amount of biosurfactant within 72 h of incubation.

방향족 화합물인 Aniline, Benzoate, p-Hydroxybenzoate를 분해하는 Delftia sp. JK-2에서 분리된 Dioxygenases의 특성연구

황선영(Seon-Young Hwang),천재우(Jae-Woo Chun),강형일(Hyung-Yeel Kahng),오계현(Kye-Heon Oh) 한국생물공학회 2004 KSBB Journal Vol.19 No.1

폐광지역에서 분리한 quinoline 분해 세균인 Pseudomonas sp. NFQ-1의 특성연구

윤경하(Kyung-Ha Yoon),황선영(Seon-Young Hwang),권오성(O-Sung Kwon),오계현(Kye-Heon Oh) 한국생물공학회 2003 KSBB Journal Vol.18 No.3

오염된 토양으로부터 분리된 세균에 이한 2,4-Dinitrotoluene과 2,6-Dinitrotoluene의 생물학적 제거

오계현,조윤석,이명석 순천향대학교 기초과학연구소 1999 순천향자연과학연구 논문집 Vol.5 No.2

Dinitrotoluenes (e.g., 2.,4-Dinitrotoluene and 2,6-Dinitrotoluene) are the major impurity resulting from the manufacture of 2,4,6-trinitrotoluene(TNT) and are primary starting materials for the synthesis of toluenediisocyanate which are used in the production of polyurethane foam. Both 2,4-DNT(2,4-dinitro- toluene) and 2,6-DNT(2,6-dinitrotoluene) are recalcitrant and have been shown to be toxic to aquatic and terrestrial organisms in the environment. Because little information is available for the biological degradation of DNTs, they have has not been evaluated for the possible treatment processes of industrial waste streams where DNTs may represent a serious disposal problem. In the present work, we explored the feasibility using a bacterium for DNTs degradation, with the ultimate aim of application for the industrial treatment. A DNTs-degrading bacterium was isolated from soil samples collected from an explosive contaminated area. Complete degradation of 2,4-DNT at the initial concentration of 50 ㎎ 2,4-DNT per liter was achieved within 10 days of incubation in bench-scale bioreactors, whereas 2,6-DNT degraded completely within 14 days under the same conditions, respectively. The bacterium was able to utilize DNTs as the sole nitrogen source. Co-substrate was essential for the bacterial growth and DNTs degradation in the medium. Neither bacterial growth nor DNTs degradation were observed in the absence of co-substrate. The detection of the parent substrates and their intermediates were based on HPLC and GC-MS methodology.

TNT 분해세균 Pseudomonas sp. HK-6에서 정제한 NAD(P)H-nitroreductase의 특성연구

호은미,오계현 순천향대학교 기초과학연구소 2003 순천향자연과학연구 논문집 Vol.9 No.2

The aim of this work was to characterize NAD(P)H-nitroreductase purified from Pseudomonas sp. HK-6, which could utilize 2,4,6-trinitrotoluene. In initial experiments, NAD(P)H-nitroreductase from actively growing HK-6 cells was separated by ammonium sulfate precipitation, DEAE-sepharose, and Q-sepharose. Specific activity of NAD(P)H-nitroreductase was approximately 4.46 unit/㎎. Optimum temperature and pH of the enzyme were approximately 30℃ and 7.5, respectively. Specific activity of NAD(P)H-nitroreductase on the effect of several metal ions was monitored. Fe^(3+) showed stimulatory effect on the activity of NAD(P)H-nitroreductase, whereas the specific activity of the enzyme was determined to approximately 27 kDa by SDS-PAGE.

토양에서 분리된 Acrylamide 분해 세균 JK-7의 분리 및 특성

천재우,오계현 순천향대학교 기초과학연구소 2003 순천향자연과학연구 논문집 Vol.9 No.2

The feasibility of using pure culture for acrylamide degradation, with the ultimate aim of application for biological treatment process, was explored. The present study reports on the test cultures which were developed to grow aerobically with acrylamide as the sole source carbon and nitrogen. A bacterial isolate, strain JK-7 was isolated from paddy soil samples, Strain JK-7 could degrade 50 mM acrylamide completely within 72 hours of incubation. Major intermediates resulting from acrylamide degradation were not detected with the HPLC methodology except acrylic acid which appeared to accumulate transiently in the growth medium. Initial pH 7.2 of the cultures became to increase 8.4 at the end of incubation. When JK-7 cells were grown at over 100 mM acrylamide, there was a pause of cell growth, resulting in a reduction in the rates of acrylamide degradation. Survival test revealed that cells exposed to low concentrations of acrylamide enable to the strain JK-7 to survive at a lethal concentration of 100 mM acrylamide. The relationships between the acrylamide degradation by JK-7 and several relevant physicochemical environmental parameters were examained. The effect of supplemented carbons(e.g., glucose, fructose, citrate, succinate) on the acrylamide degradation by the test culture of JK-7 was evaluared. The results indicated that the addition of carbons accelerated the bacterial growth and acrylamide degradation compared to in the absence of supplemented carbons. The effect of supplemented nitrogens on the degradation was monitored. Increasing concentrations of yeast extract resulted in higher growth yield, based on the turbidity measurement, and complete degradation of acrylamide. However, acrylamide degradation was essentially uninfluenced by the addition of (NH_(4))_(2)So_(4),NH_(4)Cl, or urea. The bacterium was identified as belonging to the genus Pseudomonas in the basis of use BIOLOG test, and designated as Pseudomonas sp. JK-7

활성 슬러지에서 분리한 세균 Delftia sp. JK-2에 의한 산업 유기용매의 제거

황선영,유미옥,오계현 순천향대학교 기초과학연구소 2004 순천향자연과학연구 논문집 Vol.9 No.2

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청국장 발효세균인 Bacillus sp. SK-6의 혈전용해활성 및 특성

송유진,정윤정,정민재,오계현 순천향대학교 기초과학연구소 2008 순천향자연과학연구 논문집 Vol.14 No.2

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