• 한국환경농학회지
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• 제41권2호
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• pp.115-124
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• 2022

BACKGROUND: Methane is a major greenhouse gas attributed to global warming partly contributed by agricultural activities from ruminant fermentation and rice paddy fields. Methanotrophs are microorganisms that utilize methane. Their unique metabolic lifestyle is enabled by enzymes known as methane monooxygenases (MMOs) catalyzing the oxidation of methane to methanol. Rice absorbs, transports, and releases methane directly from soil water to its stems and the micropores and stomata of the plant epidermis. Methylobacterium species associated with rice are dependent on their host for metabolic substrates including methane. METHODS AND RESULTS: Methylobacterium spp. isolated from rice were evaluated for methane oxidation activities and screened for the presence of sMMO mmoC genes. Qualitatively, the soluble methane monooxygenase (sMMO) activities of the selected strains of Methylobacterium spp. were confirmed by the naphthalene oxidation assay. Quantitatively, the sMMO activity ranged from 41.3 to 159.4 nmol min^-1 mg of protein^-1. PCR-based amplification and sequencing confirmed the presence and identity of 314 bp size fragment of the mmoC gene showing over 97% similarity to the CBMB27 mmoC gene indicating that Methylobacterium strains belong to a similar group. CONCLUSION(S): Selected Methylobacterium spp. contained the sMMO mmoC gene and possessed methane oxidation activity. As the putative methane oxidizing strains were isolated from rice and have PGP properties, they could be used to simultaneously reduce paddy field methane emission and promote rice growth.

• KSBB Journal
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• 제13권5호
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• pp.483-490
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• 1998

Two unidentified methanotrophic strains (MM-white and MM-red) secreting soluble methane monooxygenase (sMMO) involved in thrichloroethylene biodegradation have been isolated from mixed methanotrophic consortium (MM) around Taejon area. Subsequently four methanotrophic strains were isolated from MM and named according to their color; white (MS-white), yellow (MS-yellow), pink (MS-pink) and reddish brown (MS-rbrown). All strains except MS-yellow which can take glucose as well as methane, metabolized methane as a sole carbon source. They all showed symbiotic behavior when methane was used as the sole carbon source. Optimum conditions of cell growth for MM were pH of 6.8 - 7.2, temperature of 29 - 32$^{\circ}C$, and gas flow rate of 6 (for methane), 40 (for air), and 4 ml/min (for carbon dioxide). The sMMO activity was expressed as naphthalene oxidation rate (${\mu}$mol/ mg protein/ hr). The sMMO activity for MM grown in flask culture with 1 ${\mu}$M of CuSO4 was 36, while it was 61 without copper. The activity for MM grown in the fermentor without CuSO4 was 1077, but is was 197 after reaction with 5 ppm of TCE. The methanotrophs showed significantly high sMMO activity despite the presence of 1 ${\mu}$M of CuSO4, although most of other strains already known could not express sMMO activity under this condition.

• Journal of Microbiology and Biotechnology
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• 제32권3호
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• pp.287-293
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• 2022

The hydroxylation of methane (CH₄) is crucial to the field of environmental microbiology, owing to the heat capacity of methane, which is much higher than that of carbon dioxide (CO₂). Soluble methane monooxygenase (sMMO), a member of the bacterial multicomponent monooxygenase (BMM) superfamily, is essential for the hydroxylation of specific substrates, including hydroxylase (MMOH), regulatory component (MMOB), and reductase (MMOR). The diiron active site positioned in the MMOH α-subunit is reduced through the interaction of MMOR in the catalytic cycle. The electron transfer pathway, however, is not yet fully understood due to the absence of complex structures with reductases. A type II methanotroph, Methylosinus sporium 5, successfully expressed sMMO and hydroxylase, which were purified for the study of the mechanisms. Studies on the MMOH-MMOB interaction have demonstrated that Tyr76 and Trp78 induce hydrophobic interactions through π-π stacking. Structural analysis and sequencing of the ferredoxin domain in MMOR (MMOR-Fd) suggested that Tyr93 and Tyr95 could be key residues for electron transfer. Mutational studies of these residues have shown that the concentrations of flavin adenine dinucleotide (FAD) and iron ions are changed. The measurements of dissociation constants (K_ds) between hydroxylase and mutated reductases confirmed that the binding affinities were not significantly changed, although the specific enzyme activities were significantly reduced by MMOR-Y93A. This result shows that Tyr93 could be a crucial residue for the electron transfer route at the interface between hydroxylase and reductase.

• KSBB Journal
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• 제13권4호
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• pp.391-398
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• 1998

The effect of culture medium copper availability on the specific growth rate(${\mu}$) and carbon conversion efficiency (CCE) was sutided for an obligatory methanotroph Methylosinus trichosporium OB3b under various combinations of carbon and nitrogen sources. Methane or methanol was used as a carbon source, and nitrate or ammonium was used as a nitrogen source. Medium copper availability determined the intracellular location or kind of methane monooxygenase (MMO), cell-membrane (particulate or pMMO) when copper was present and cytoplasm (soluble or sMMO) when copper was deficient. When methane was used as a carbon source, copper-containing medium exhibited higher ${\mu}$ and CCE than copper-free medium regardless of the kind of nitrogen source. When methanol was used as a carbon source, however, the effect of copper disappeared. Ammonium gave the higher ${\mu}$ and CCE than nitrate for both methane and methanol. Those observation suggest that there exist an important difference in energy utilization efficiency for methane assimilation between sMMO and pMMO.

• 대한환경공학회지
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• 제22권6호
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• pp.991-1000
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• 2000

메탄올 주요 탄소원으로 사용하며 가용성 메탄산화효소 (soluble methane monooxygenase, sMMO)를 분비하는 혼합 메탄차화균을 celite R-635에 고정화시켜 TCE를 함유한 폐수를 연속적으로 처리하였다. 2 ppm의 TCE를 공급했을 때 각각 6. 20시간의 체류시간에서 약 80.4, 84.5%의 처리 효율을 얻었으며, 체류시간이 증가함에 따라서 제거율도 서서히 증가하였다. 5 ppm의 TCE를 공급하고 10시간 동안 체류시켰을 때, '초기에는 TCE의 제거능이 낮았으나 점차 81%까지 증가하였다. 또한 산소를 공급하면서 메탄을 주기적으로 공급할 때 5 ppm의 TCE가 체류시간 10. 15시간에서 각각 88.5, 96.5%까지 제거되었다. 반응기 내에 산소가 고갈된 상태에서 메탈을 고농도로 공급하면 MMO에 흡착된 메탄의 산화반응이 쉽게 진행되지 않아 TCE 분해능이 떨어졌다. 파일롯트 플랜트 규모의 생물막 반응기에서의 TCE 분해 실험 결과, 실제 크기 규모의 공장에도 충분히 적용 가능할 것으로 사료되었다.

• KSBB Journal
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• 제8권4호
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• pp.341-350
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• 1993

본 연구에서는 sMMO를 갖는 메탄 자화균인 M. triclwsporium OB3b를 이용하여 메탄올 생산을 위한 기초실험을 수행하였다. 중요한 결과를 요약하면 다음과 같다(Table 2). 1. 세포 내 NADH의 재생을 위해 개미산을 첨가 할 때 whole-cell의 sMMO 활성은 pH 7.0 및 $30^{\circ}C$ 에서 최대값을 보이며 propylene을 기질로 할 경우 약 130nmol/mg cell min 정도이다. 2. 인산은 MMO와 MDH 활성을 모두 저해하나 M MDH에 대한 저해 정도가 훨씬 크므로 메탄올 합성 에 사용이 가능하다. Noncompetitive mode를 가정 할 때 저해상수는 각각 185mM(MMO) 및 42mM ( (MDH)이었다. 3. 메탄올은 MMO 활성을 저해하며 noncompeti­t tive mode를 가정할 때 propylene기질의 경우 2 21mM 이었다. 4. 균체 내 sMMO 활성은 성장이 멈춰진 상태에 셔 비교적 때}른 속도로 감소하며 고농도 인산용액에 서 그 속도가 더 빨라진다. 5. 인산농도 91mM에서 메탄은 메탄올로 산화되 어 축적되며 4.5시간 동안 에탄올의 생성속도는 평 균 79nmol/mg min이었다.

• 한국동물학회:학술대회논문집
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• 한국동물학회 2001년도 한국생물과학협회 학술발표대회
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• pp.244.2-244
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• 2001

No Abstract, See Full Text

• KSBB Journal
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• 제11권6호
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• pp.642-648
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• 1996

본 연구에서는 에탄 자화균인 Methylosinus trichosporim OB3b를 이용하여 메탄으로부터 에탄올 생성에 관한 실험을 수행하였다. 에탄으로부터 에탄올을 생성하기 위해서는 메탄 산화과정 중 두번 째 효소인 methanol dehydrogenase 효소의 활성을 부분 저해해야 하므로 이를 위해 EDTA를 사용한 결과 EDTA가 methanol dehydrogenase의 저해제 임을 확인하였고 배지에 6mM EDTA를 첨가하였을 때 전혀 첨가하지 않았을 때와 비교하여 메탄올 생 성이 약 5배 정도 증가되어 lOmmole/L의 에탄율을 얻을 수 있었다. 또한 Cu의 존재유무가 에단올 생성 에 미치는 영향을 실험한 결과 ImM Cu 존재시 $5\mu\textrm{M}$ Cu 존재하에 비해 메탄올 생성이 약 2.5배 증가되어 약 11mmole/L의 메탄올을 얻을 수 있었는데 이는 Cu 존재가 입자상(particulate) MMO의 생성을 촉 진시키며 생성된 이 세포 단위중량당 MMO 활성이 높은 pMMO가 에탄으로부터 에탄올의 생성을 촉진 시키는 것으로 생각된다. 그리고 온도가 에탄올 생 성에 미치는 영향을 실험한 결과 온도가 3TC에서 $30^{\circ}C , 25^{\circ}C$ 로 낮아점에 따라 생성 메단올 농도가 증 가하여 15.5mmole/L에 이르렀고 메탄 소비속도도 증가됨을 알 수 있였다. 또한 메단과 산소의 구생성 분비가 에탄올 생성에 미치는 영향을 실험한 결과 산소대비 에탄 농도가 증가할수록 생성 에탄올의 농 도 및 세포농도가 증가됨을 알 수 였다. 그리하여 50% 메탄, 50% 산소 존재하에 비해 70% 에탄, 30% 산소 경우에는 약 50% 증가된 15.3 mmole/L 농도의 머l단올을 얻을 수가 있였으며 세포농도도 많이 증가됨을 알 수 있다.

• 대한환경공학회지
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• 제22권5호
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• pp.971-980
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• 2000

가용성 메탄산화효소를 분비하는 혼합 메탄자화균총을 celite R-635에 고정화시켜 TCE 분해를 위한 새로운 형식의 가압 산기식 혼합 메탄자화균총 고정층 생물막 반응기를 설계하였다. Celite R-635에서 용출되는 용액의 pH는 약 4시간 후부터 안정화되어서 중성 영역에 도달하므로 더 이상 중화할 필요가 없었다. 혼합 메탄자화균 생물막을 완전히 형성하기 위해서는 130일이 걸렸으며, 처음에 흰색을 띠고 있었던 celite는 점차 붉게 변해 갔었다. 생물막이 형성된 후에는 메탄과 산소를 각각 2.5~4, 8~10 ppm씩 공급할 때 하루 동안 체류한 후 0.5~1, 1~2 ppm 정도로 농도가 낮아졌다. 초기에 2 ppm의 TCE를 메탄자화균 고정층 생물막 반응기에서 10시간 동안 체류시켰을 때 79.9%의 분해 효율을 보였다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권5호
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• pp.309-312
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• 2003

The transformation capacity (T$\_$c/) of Methylosinus trichosporium OB3b in the degradation of ethylene chlorides was determined by measuring the decrease of soluble methane monooxygenase (sMMO) activity of resting cells in batch experiments. All measurements of sMMO activity were taken in the presence of 20 mM formate to avoid the deficiency of reducing power, and within 2 hrs to avoid the effect of natural inactivation from instability of the resting cells. The constant T$\_$c/ values of 0.58 ${\pm}$ 0.132 and 0.80 ${\pm}$ 0.17 ${\mu}$mol/mg cell were obtained for trichloroethylene (TCE) and 1,2-dichloroethylene (cis and trans-1,2-DCE), respectively, regardless of their concentrations. The transformation capacity measured by this method can be used to predict the amount of cells that should be stimulated in in-situ bioremediation.