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

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.

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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Sodium chloride에 노출된 Aeromonas sp. MH-8의 세포반응 조사

석지원,김동민,오계현 순천향대학교 기초과학연구소 2017 순천향자연과학연구 논문집 Vol.23 No.2

The purpose of this study is to investigate the cellular responses of Aeromonas sp. MH-8 exposed to sodium chloride (NaCl). Strain MH-8 was isolated from stale fish. Survival of the MH-8 strain with time in the presence of different concentrations of NaCl under sublethal conditions was monitored. Analysis of SDS-PAGE and Western blot using anti-DnaK and anti-GroEL revealed that two stress shock proteins, 70-kDa DnaK and 60-kDa GroEL were found to decrease in proportion to the NaCl concentrations in exponentially growing cultures. Scanning electron microscopic analysis demonstrated the presence of wrinkled surface containing perforations and irregular rod-shaped forms after treatment with NaCl. These results provide clues for understanding the mechanism of NaCl-induced stress and cytotoxicity on Aeromonas sp. MH-8

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%).

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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도시폐수처리장의 활성슬러지에서 분리한 Aniline 분해세균 Delftia sp. JK-2의 특성연구

조윤석,강형일,장효원,오계현 한국미생물학회 2000 미생물학회지 Vol.36 No.2

도시폐수처리장의 활성슬러지 표본으로부터 유일 탄소원 및 질소원으로 aniline을 이용할수 있는 미생물 컨소시엄을 농화배양하였다. 농화배양으로부터 분리된 3개의 분해세균 가운데 분해능이 탁원한 세균을 본 연구에 사용되었다. 분리세균운 그람 음성세균으로서 여러 가지 생리.생화학적 시험을 통하여 Delftia acidovorans로 동정되었으며 이를 Delftia sp. JK-2로 명명하였다. 10mM의 aniline이 포함된 액체배지에 Delftia sp. JK-2를 접종한 결과 24시간 이내에 aniline이 완전히 분해되엇다. 이 가간동안 배양액 내에 $NH_4^+$이온은 일시적으로 생성되었다가 aniline이 완전히 분해됨에 따라 완전히 사라졌다. Aniline을 포함하여 무기배지에 부가탄소로서 glucose를 첨가하였을 때 분해능은 크게 감소하였다. 질소원으로서 0.5% nitratef,f 첨가하였을 때 첨가하지 않은 대조군에 비해 anline의 분해가 80% 이상 향상되었다. Aniline 분해세균의 16S rDNA 염기서열을 이용하여 phylogenetic 분석을 실시한 결과 이 세균은 Delftia acidovorans와 96%의 유사성을 나타내었으며, Acidovorax, Aquaspirllum, Xylophilus, Variovorax, Rhodoferax 등의 세균과도 상당한 유사성을 나타내었다. Activated sludge samples were collected from a municipal sewage treatment plant and used for enrichment of microbial consortia with aniline as the sole carbon and nitrogen source. Threc aniline-degrading bacteria were obtained lrom microbial consortia and an isolate which has excellent aniline degradability was selected for this study. The isolate was Gram-negative, and identified and designated as Delfha sp. JK-2 on the basis of various physiological and biochemical tests. 10 mM aniline was completely degraded within 24 hours after inoculation of the culture. Ammonium ion was liberated in the medium transiently during the incubation and disappeared when aniline was completely degraded. Addition of glucose as a supplementary source to aniline minimal media showed significant decrease in aniline degradat~on rate for the strain Effective degradation of aniline was achieved by the addition of 0.5% nitrate as a nitrogen source, and resulted in approximately 80% higher aniline degradation compared to the absence of nitrate. Phylogenetic analysis based on 16s [DNA sequence revealed that the strain was closely related to De@ia acidovorans, with 96% overall similarity. The 16s [DNA sequence of JK-2 was also found to be closely related to those of six other clonal types, including Acidovoru, Aquaspirillum. Xylophilus, Variovorm, and Rhodofernr.

느타리버섯에서 분리한 Leuconostoc mesenteroides의 동정, 특성조사, 버섯추출물 첨가에 따른 생장변화

석지원,이현호,박상국,오계현 순천향대학교 기초과학연구소 2018 순천향자연과학연구 논문집 Vol.24 No.2

The aim of this study was to identify and characterize Leuconostoc mesenteroides isolated from oyster mushrooms, and to assess the enhanced production of organic acids by the addition of mushroom extracts. The isolate from mushroom was obtained via the enrichment culture technique. Both the BIOLOG system and phylogenetic analysis using 16S rRNA sequencing were utilized for identification, and the strain was designated as Leuconostoc mesenteroides. Phylogenetic tree of Leuconostoc mesenteroides was plotted based on 16S rRNA sequence comparisons. During an incubation period of 72 hours, the changes of bacterial growth, production of organic acids (e.g., lactic acid and acetic acid) as metabolites, and pH were monitored. HPLC methodology was used to analyze the production of organic acids in the test cultures. The isolate produced both lactic acid and acetic acid in the presence (10%) or absence of mushroom extracts, which were responsible for the pH decrease during growth. Compared to the cultures in the absence of mushroom extracts, the production of organic acids was increased in the presence of mushroom extracts. In the addition of mushroom extracts, maximum concentrations of lactic acid and acetic acid reached approximately 308.8 mM and 85.2 mM for this isolate.