• 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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• 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.

• 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.

• 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.

• 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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• 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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• 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.

• 한국수처리학회지
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• 제26권6호
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• pp.27-42
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• 2018

This study analyzed the utility of ammonium chloride ($NH_4Cl$) as a nitrogen source for methanotroph communities. When cultured in nitrate mineral salt (NMS) medium, the methanotroph community we identified four families, seven genera, and 16 type I and type II species of methanotrophs. Among species in the Methylobacter genus, Methylobacter marinus could be actively cultured in NMS medium without NaCl addition. Following the addition of 25 mM $NH_4Cl$, the numbers of the type I genera Methylomonas, Methylococcus, and Methylobacter were increased, whereas the numbers of the type II genera Methylocystis and Methylosinus were decreased after 5 days. In methanotroph communities, certain concentrations of $NH_4Cl$ affected methane consumption and growth of methanotrophs at the community level. $NH_4Cl$ caused a considerable decrease in the methane consumption rate and the expression of soluble methane monooxygenases (sMMOs) but did not inhibit the growth of Methylomonas methanica expressing sMMO. These results could be attributed to competitive antagonism of MMOs due to their direct involvement in ammonia oxidation.

• 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.