Purification and Characterization of theNAD(P)H-nitroreductase for the Catabolismof 2,4,6-Trinitrotoluene (TNT) in Pseudomonas sp. HK-6

오계헌,강형일,이병욱,조윤석 한국생물공학회 2007 Biotechnology and Bioprocess Engineering Vol.12 No.4

The NAD(P)H-nitroreductase of the Pseudomonas sp. HK-6, which is capable of catabolizing 2,4,6-trinitrotoluene (TNT), was purified and biochemically characterized. The specific activity of the purified TNT nitroreductase was approximately 1.47 units/mg, and was concentrated to 10.1-fold compared to the crude extract. The optimal temperature and pH of the highest nitroreductase activity was 30℃ and 7.5, respectively. The substrate specificity test revealed that the nitroreductase exhibited the highest enzyme activity for the TNT substrate of the nitroaromatic compounds tested in this study. Moreover, the molecular weight of the TNT nitroreductase was approximately 27 kDa on the SDS-PAGE. The N-terminal amino acid sequence of the purified protein was 5'-MDTVSLAKRRYTTKAYDASR, which is identical to pnrB of Pseudomonas putida JLR11, and is capable of TNT reduction. The molecular analysis of the approximately 650-bp PCR product, originating from the HK-6, revealed that the oxygen-insensitive NAD(P)H-nitroreductase gene, which transforms TNT in strain HK-6 with five unique amino acid sequences and diverges from the nitroreductases identified so far in Pseudomonas, Burkholderia, and Ralstonia, is frequently found amidst the powerful degraders of aromatic compounds.

TNT에 대한 세균의 반응기작: 생존율, 스트레스 유도단백질의 SDS-PAGE 및 2-D 전기영동 분석

오계헌,장효원,강형일,김승일 한국미생물학회 2002 미생물학회지 Vol.38 No.2

폭약 2,4,6-trinitrotoluene (TNT)스트레스 조건하에서 토양세균 Pseudomonas sp. HK-6의 세포반응에 대하여 조사하였다. 다양한 농도의 TNT에 노출됨으로서 약 70-kDa DnaK와 60-kDa GroEL의 스트레스 충격단백질 (stress shock proteins, SSPs)이 단떠질이 유도되었다. 이들 SSPs의 존재는 SDS-PAGE과 anti-DnaK와 anti-GroEL monoclonal antibodies를 이용한 Western bolt을 통하여 확인되었다. SSPs은 0.5 mM TNT로 6-12 시간 처리된 세포에서 나타났으며, TNT에 노출 후8시간대 에서 최대의 단백질 유도가 관찰되었다. $30^{\circ}C$에서 $42^{\circ}C$로 열변환충격을 주었을 때의 SSPs는 TNT노출에서와 유사한 유도양상을 보여주었다. TNT에 노출된 Pseudomonas sp. HK-6세포에서 유도된 SSPs의 존재는 배양된 세포의 수용성 단백질 분획에 대하여 2-D PAGE를 통하여 확인되었다. Coomassie brilliant blue R25O로 염색된 젤로부터 pH 3-10 범위에서 약 450 개의 spots이 탐침되었으며, 이들 가운데 12 개의 spots이 TNT 스트레스에 대하여 현저하게 유도되었다. Gel상에서 가장 짙게 나타난 대표적 인 spot에 대한 N-말단 아미노산 서열을 분석한 결과, $^1XXAKDVKFGDSARKKML^17$로서, Pseudomonas putida의 GroEL의 N-말단 아미노산 염기서열인 $^1XXAKDVKFGDSARKKML^17$과 동일한 것으로 분석되었다. The cellular responses of soil-borne bacterium, Pseudomonas sp. HK-6 to explosive 2,4,6-trinitrotoluene (TNT) were examined. Two stress shock proteins (SSPs), approximately 70-kDa DnaK and a 60-kDa GroEL were found in HK-6 cells in response to TNT. Analyses of SDS-PAGE and Western blot using anti-DnaK and GroEL revealed that SSPs were induced in HK-6 cells exposed to 0.5 M of TNT far 6-12 hrs. The maximum induction of proteins was achieved at 8-hr incubation point after HK-6 cells'exposure to TNT. Similar SSPs were found to be induced in HK-6 cells by heat shock (shift of temperature, from $30^{\circ}C$ to $42^{\circ}C$) or cold shock (shift of temperature,$30^{\circ}C$ to $4^{\circ}C$).2D-PAGE of soluble protein tractions from the culture of Pseudomonas sp. HX-6 exposed to TNT demonstrated that approximately 450 spots were observed on the silver stained gels ranging from pH 3 to pH 10. Among them, 12 spots significantly induced and expressed in response to TNT were selected and analyzed. Approximately 60-kDa protein, which was assumed highly expressed on the gel, was used for amino acid sequencing. N-terminal microsequencing with in-gel digestion showed that N-terminal sequence of the TNT-induced protein, <$^1XXAKDVKFGDSARKKML^17$, shared extensive similarity with $^1XXAKDVKFGDSARKKML^17$, N-terminal sequence of (P48216) GroEL of Pseudomonas putida.

TNT-분해세균에 의한 s-Triazine계 제초제인 Atrazine과 Simazine의 미생물학적 분해

오계헌,이명석,장효원,소재성 한국미생물학회 2000 미생물학회지 Vol.36 No.3

이연구는 물리화학적 환경요인에 따른 TNT 분해 세균에 의한 s-triazine 계열의 atrazine과 simazine의 분해를 조사하기 위하여 실시되었다. TNT 분해세균은 atrazine과 simazine에 대해서도 높은 분해능을 나타내었다. Atrazine(<50 mg/l)과 simazine(<15 mg/l) 은 각각 30시간과 4일의 배양기간중에 완전히 분해되었다. 배양내에서 atrazine과 simazine 의 농도가 증가함에 따라 이들의 분해는 지연되었다. 부가 탄소원은 atrazine과 simazine의 분해를 위하여 필수적이었으며 부가 탄소원이 없는 상태에서는 분해되지 않았다. Atrazine 과 simazine의 분해에서 부가 질소원의 효과에 대하여 조사하였다. Atrazine이나 simazine을 포함하는 생장배지에 부가 질소원이 첨가된 뱅야에서 배양기간동안 이들 제초제는 일부만 문해되었다. 그러나 부가 질소원이 첨가되지 않은 배양에서 atrazine과 simazine은 완전히 분해되지 않았다. 이 연구에서 효모추출물의 첨가는 atrazine과 simazine의 분해를 억제하였 다. TNT 분해세균은 작은 그람 음성의 간균이었다. BIOLOG system을 이용한 생리학적 분 석으로 이 균주는 Stenotrophomonas maltophilia로 동정되었다. The purpose of U7is work was to iilvestigate the degradation of s-h~azine hel-hicidcs, ahilzine and simazine by TNT-degrader under several relevaut physicochemical environ~nental parameters. TNT-degrader showed effective degradability of atrazine and snnazine as well. Both atrazme (GO 1i1~11) and simazine ( 4 5 rng//) were completely degraded within 30 hrs and 4 days of incubation, respectively. As d ~ e concentrations of atrazine and sunazine increased in the media, the degradation ofthose compounds were delayed. Additional caubans were essential to degrade atrazine and simazule, and no degradation was achieved in the absence of additional carbons. The effect of supplemented nitrogens on the degradation of atrazine and sunazine was evalualed. Addition of a suppleinented nitrogen in he growth medium containing ah-azine or siinazine showed partial degr-adation olihose herbicides duriug the incubation period. However, complete degradation of atrazine and simazu~e was examined ul the absence or any supplemented nitrogens. Addltion of yeast extract in this study was inhibilory to atrazine aud siinazine degradations, respectively. TNT-degrader was a small Gram-negative cocco-bacillus. Physiological analysis using BIOLOG sysleln revealed that this strain was Ste~~ol~~opl~orno~~ns rrialtophilia.

Biological Removal of Explosive 2,4,6-Trinitrotoluene by Stenotrophomonas sp. OK-5 in Bench-scale Bioreactors

오계헌,소재성,Myung-Seok Lee,Hyo-Won Chang,Hyung-Yeel Kahng 한국생물공학회 2002 Biotechnology and Bioprocess Engineering Vol.7 No.2

The biological removal of 2,4,6-trinitrotoluene (TNT) was studied in a bench-scale bioreactor using a bacterial culture of strain OK-5 originally isolated from soil samples contaminated with TNT. The TNT was completely removed within 4 days of incubation in a 2.5 L bench-scale bioreactor containing a newly developed medium. The TNT was catabolized in the presence of different supplemented carbons. Only minimal growth was observed in the killed controls and cultures that only received TNT during the incubation period. This catabolism was affected by the concentration ratio of the substrate to the biomass. The addition of various nitrogen sources produced a delayed effect for the TNT degradation. Tween 80 enhanced the degradation of TNT under these conditions. Two metabolic intermediates were detected and identified as 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene based on HPLC and GC-MS analyses, respectively. Strain OK-5 was characterized using the BIOLOG system and fatty acid profile produced by a microbial identification system equipped with a Hewlett Packard HP 5890 II gas chromatograph. As such, the bacterium was identified as a Stenotrophomonas species and designated as Stenotrophomonas sp. OK-5.

Benzoate 분해세균 Acinetobacter sp. kS-1에서 분리된 catechol 1,2-dioxygenase의 특성 및 N 말단 아미노산 서열 분석

오계헌,송승열,김승일,윤경하 한국미생물학회 2002 미생물학회지 Vol.38 No.2

단일 탄소원 및 에너지원으로 benzoate를 이용하는 Acinetobacter sp. KS-1에서 분리 정제한 catechol 1,2-dioxygenase (Cl,2O)의 특성과 아미노산 서 열을 분석하였다. Cl,2O는 catechol과 4-methylcatechol에 대해서 효소활성을 나타내었으며, 활성 최적온도는 $35^{\circ}C$이고, 활성 최적 pH는 7.5-9.0의 범위 내에 있었다. 효소활성 저해제로서 은, 수은, 그리고 구리는 Acinetobacter sp. KS-1의 Cl,2O 활성을 억제하였다. SDS-PAGE에 의해 측정된 Cl,2O의 분자량은 약 36 kDa 였으며, N-말단 아미노산 서 열을 분석한 결과, $^{1}MNYQQIDALVKQMNVDTAKG^{20}$로 Acinetobacter radioresistens의 Cl,2O와 95%의 유사성을 보여주었다. In-gel 아미노산 서열 분석을 위하여 trypsin 처리와 peptide mapping을 실시하였다. MALDI-TOF를 이용하여 trypsin으로 처리된 세 개의 peptide flagmen써 분자량을 분석한 결과 966.3 Da, 2081.7 Da, 그리고 1933.8 Da으로 각각 나타났는데, 이는 A. radioresistens의 Cl,2O와 내부 서 열$^{1}SQSDFNLRR^{9}\, ^{1}HGNRPSHVHYFNSAPGYR^{18}\, ^{1}TIEGPLYVAGAPESVGFAR^{19}$ 이 일치하는 것으로 분석되었다. N-말단 서열과 내부 서열을 바탕으로 primer를 제작하여 polymerase chain reaction을 실시하였다. The purpose of this work was to investigate the characterization and sequence of catechol 1,2-dioxygenase (Cl,2O) purified from Acinetobacter sp. KS-1 which was grown on benzoate as a sole carbon source. Cl,2O demonstrated its enzyme activity to catechol and 4-methylcatechol. The optimum temperature of Cl,2O was $35^{\circ}C$, and the optimal pH was in the range from pH 7.5 to 9.0. $Ag^{+}$, $Hg^{+}$, and $Cu^{2+}$ showed inhibitory effect on the activity of Cl,2O. Molecular weight of the enzyme was determined to approximately 36 kDa by SDS-PAGE and 7-terminal amino acid sequence of Cl,2O was analyzed as $^{1}MNYQQIDALVKQMNVDTAKG^{20}$and exhibited 95% sequence homology with that of Cl,2O from Acinetobacter radioresistens In addition, trypsin digestion and peptide mapping were performed for internal sequencing analysis. Molecular weights of three digested peptide fragments were analyzed as 966.3 Da, 1933.8 Da and 2081.7 Da by MALDI-TOF, which were matched with each internal sequences $^{1}SQSDFNLRR^{9}\, ^{1}HGNRPSHVHYFNSAPGYR^{18}\, ^{1}TIEGPLYVAGAPESVGFAR^{19}$) of. A. radioresistens. PCR product was amplified with the degenerated primers derived from N-terminal and each internal amino acid sequences.

플라스크와 발효조에서 세균혼합배양에 의한 페녹시계 제초제 MCPP의 분해

오계헌 순천향대학교 1992 논문집 Vol.15 No.1

제초제 생산공장 주변으로부터 MCPP[2-(2-methyl-4-chlorophenoxy) propionic acid]를 이용하는 세균배양을 얻었다. UV-흡광법을 이용하여 플라스크와 발효조에서, 배양액의 잔존 MCPP 농도를 분석하였다. 잔존 MCPP 농도와 무기염소의 형성에 근거한 mass blance가 만들어졌다. 배양액에 유리되는 무기염소의양은 MCPP농도의 변화로부터의 기대치와 일치하였다. MCPP는 본 실험조건하에서 완전 분해되지 않았다. 세포단백질은 실험기간동안 배양액에 유리되는 무기염소의 농도에 비례하였다. MCPP분해효율과 분해율은 발효조에서 약 96%와 560 μmol/l·day 그리고 플라스크에서 약 50%와 140 μmol/l·day였다. Soil samples were collected from a fertilizer manufacturing plant site and used for enrichment of bacterial cultures with 2-(2-methyl-4-chlorophenoxy) propionic acid(MCPP) as the sole carbon and energy source. UV-spectrometry was used to analyze the residual MCPP concentration in the test cultures growing in shake flasks and a stirred tank reactor. A mass balance was established based upon the residual concentration of MCPP and formation of inorganic chloride in the test cultures. The amount of inorganic chloride released into solution was in agreement with the expected value calculated from the changes in MCPP concentration. MCPP was not completely degraded under the experimental condition. Cell protein paralleled a similar time profile to that of the inorganic chloride during the experiment. The efficiency and rate of MCPP degradation were approximately 96% and 560 μmol/l·day in the stirred tank reactor, and approximately 50% and 140 μmol/l·day in shake flasks.

토양농화배양에 의한 페녹시 제초제 4-Chloro-2-Methylphenoxyacetic Acid의 생분해에 영향을 미치는 물리화학적 요인

임상원,김영진,오계헌 순천향대학교 기초과학연구소 1997 순천향자연과학연구 논문집 Vol.3 No.1

The relationships between the phenoxyherbicide 4-chloro-2-methylphenoxyacetic acid(MCPA) degradation by soil enrichment culture and several relevant physicochemical environmental parameters were examined. As concentrations of MCPA were increased, the degradation became inhibited. Biodegradation activity was higher at neutral pH or slightly acidic pH, but the activity was showed only partial degradation of MCPA at basic pH. The effect of inoculum size on the MCPA degradation was monitored. An increase in the inoculum reduced the lag period in the growth curve and accelerated the MCPA degradation. Increased concentrations of nitrogen and phosphorus as supplemental nutrients were inhibitory to the degradation of MCPA, respectively. Addition of yeast extract accelerated MCPA degradation, whereas the supplemented glucose inhibited the degradation of MCPA.

미생물 컨소시엄에 의한 페놀수지 Resole의 분해

최원식,차민석,오계헌 순천향대학교 기초과학연구소 1997 순천향자연과학연구 논문집 Vol.3 No.1

Microbial consortia were screened for their ability to degrade resole at the sole carbon source. Five microbial consortia were derived from a plant site where a biological treatment of liqid waste stream of a manufacturing facility was located. Among the consortia, the test consortium, designated as MS2, displayed approximately 70% degradation of the substrate, 0.1 g of resole per liter, within the first twelve days of incubation but the degradation did not proceed to completion. pH was decreased and resole degradation was inhibited under the conditions. UV-spectroscopy was used quantitatively to monitor the residual concentrations of resole in the present work. UV-scans of spent culture showed that wavelength of maximum absorption was 216nm for resole.

미생물 컨소시엄에 의한 페놀수지 Resole의 분해

오계헌(Kye-Heon Oh),최원식(Won-Sik Choi) 한국생물공학회 1998 KSBB Journal Vol.13 No.2

TNT-분해세균인 Stenotrophomonas maltophilia에 의한 s-Triazine계 제초제인 Atrazine의 미생물학적 분해

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

The agricultural use and relative persistence of s-triazine herbicide such as atrazine have led to increasing concern about environmental contamination. Stenotrophomonas maltophilia capable of utilizing TNT as sole nitrogen source has been isolated from contaminated soils and the bacterium showed excellent degradability for several-triazine herbicides including atrazine. Complete depletion of atrazine was achieved within 30 hours of incubation. Atrazine was catabolized in the presence of different supplemented carbons. Among the co-substrates used in this experiment (e.g., glucose, fructose, starch, succinate, acetate), fructose was the best co-substrate. However, no atrazine degradation was monitored without the supplemented co-substrates in the cultures. The relationships between atrazine degradation by S. maltophilia and several relevant physicochemical parameters (e.g., N-sources, yeast extract) were examined. High performance liquid chromatographic methodology was used to measure this substrate and it also resolved unknown intermediates.