• Journal of Microbiology and Biotechnology
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• v.32 no.3
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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.

• Prospectives of Industrial Chemistry
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• v.19 no.2
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• pp.28-35
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• 2016

천연가스, 셰일가스 및 바이오가스의 주성분인 메탄은 지구온난화 가스로, 감축대상인 동시에 차세대 탄소 자원으로 주목을 받고 있다. 기존의 화학적 메탄전환방법은 대규모 설비투자가 요구되는 규모의 경제가 적용되어 소규모 한계 가스전에는 활용이 어렵다. 이러한 문제점을 극복하기 위하여 최근에 생물학적 전환법이 대안으로 고려되고 있다. 메탄자화균은 메탄산화효소(methane monooxygenase)를 이용하여 상온 상압에서 메탄을 탄소원으로 사용하여 생장할 수 있다. 따라서 메탄자화균의 메탄 대사경로를 기반으로 대사공학을 활용하면 메탄으로부터의 다양한 종류의 고부가가치 산물 생산이 가능하다. 본고에서는 메탄자화균을 이용한 메탄의 바이오전환 기술의 현황 및 전망에 대하여 논의하였다.

• KSBB Journal
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• v.13 no.5
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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.

• Korean Journal of Environmental Agriculture
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• v.41 no.2
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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.

• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1597-1605
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• 2014

Methane is considered as a next-generation carbon feedstock owing to the vast reserves of natural and shale gas. Methane can be converted to methanol by various methods, which in turn can be used as a starting chemical for the production of value-added chemicals using existing chemical conversion processes. Methane monooxygenase is the key enzyme that catalyzes the addition of oxygen to methane. Methanotrophic bacteria can transform methane to methanol by inhibiting methanol dehydrogenase. In this paper, we review the recent progress made on the biocatalytic conversion of methane to methanol as a key step for methane-based refinery systems and discuss future prospects for this technology.

• Applied Biological Chemistry
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• v.34 no.4
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• pp.386-392
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• 1991

Whole cells of Methylosinus trichosporium OB3b, the obligate methylotroph, were used to produce propylene oxide from propane. This strain has methane monooxygenase, which catalyzes the conversion methane to methanol and can catalyze also the conversion propane to propylene oxide. Optimal condition for the production of propylene oxide was investigated in resting-whole cell system. The optimal pH and temperature was 7.0 and $35^{\circ}C$ respectively. The end product, propylene oxide, didn't inhibit the production of propylene oxide and was not further metabolized in reaction mixture. The addition of methane metabolites (methanol, formaldehde and formic acid) to the reaction mixture stimulated formation of propylene oxide by $3{\sim}4$ times, and methanol was the most effective especially. Under the optimal conditions, the 14.2 mM of propylene oxide was produced after incubation of 60 min. and the conversion ratio of propane to propylene oxide was approximately 8%.

• Proceedings of the Zoological Society Korea Conference
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• 2001.10a
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• pp.244.2-244
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• 2001

No Abstract, See Full Text

• Journal of Korean Society of Environmental Engineers
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• v.22 no.6
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• pp.991-1000
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• 2000

Methanotrophic consortium utilizing methane as the primary carbon source and secreting soluble methane monooxygenase (sMMO) was immobilized on celite R-635 to continuously treat a wastewater containing trichloroethylene (TCE). With influent 2 ppm of TCE. 80.4 and 84.5% of TCE was degraded in 6 and 20 hour of hydraulic retention time (HRT). respectively. and the removal efficiency of TCE was increased with an increase in HRT in methanotrophic consortium biofilm reactor (MCBR). With influent 5 ppm of TCE and 10 hour of HRT. average efficiency of TCE removal was decreased in initial stage. but gradually increased to 81%. TCE was degraded to 88.5 and 96.5% with 10 and 15 hour of HRT. respectively. when methane was supplied alternately with continuous oxygen supply at influent 5 ppm of TCE. The efficiency of TCE degradation was decreased probably because oxidation reaction of methane was proceeded slowly on MMO. when high concentration of methane was supplied with depletion of oxygen. As results of the pilot-scale study. biodegradation of TCE by MCBR system might be feasible at full-scale operation.

• KSBB Journal
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• v.13 no.4
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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.

• Journal of Korean Society of Environmental Engineers
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• v.22 no.5
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• pp.971-980
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• 2000

Mixed methanotrophs (MM) secreting soluble methane monooxygenase(sMMO) were immobilized on celite R-635 to degrade trichloroethylene(TCE) in methanotrophic consortium biofilm reactor(MCBR) system. Further neutralization of celite R-635 was not needed for immobilization because effluent pH was stabilized at neutral after 4 hour washing. It took 130 days to develop biofilm on celite R-635 and the color of the celite changed gradually from white to red. After biofilm developed, influent methane and oxygen were decreased from 2.5~4 and 8~10 ppm to 0.5~1 and 1~2 ppm, respectively, With influent 2 ppm of TCE and 10 hours of retention time, 79.9% of TCE was degraded in the MCBR system.