Purification and Biochemical Characterization of Soluble Methane Monooxygenase Hydroxylase from<i>Methylosinus trichosporium</i>IMV 3011

Shi, Hua; Li, Shuben; Jia-yin, Xin; Niu, Jianzhong; Xia, Chungu; Tan, Haidong; Tang, Wei

doi:10.1271/bbb.60402

Cited by 5 publications

(7 citation statements)

References 16 publications

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“…On the contrary, M. silvestris BL2 yielded a fluorescence signal both when cultured with and without copper because of its copper-independent expression of sMMO. Moreover, sMMO of M. trichosporium cells showed higher specific activity than that of M. capsulatus, which agrees with a previous report (Shaofeng et al, 2007). Therefore, the GESSlet successfully quantified different levels of sMMO activity from various methanotrophs, demonstrating its potential applications for methanotroph screening.…”

Section: Functional Phenotyping Of Methanotrophs With Smmo Activitysupporting

confidence: 90%

Sensitive and Rapid Phenotyping of Microbes With Soluble Methane Monooxygenase Using a Droplet-Based Assay

Lee

Baek

Kim

et al. 2020

Front. Bioeng. Biotechnol.

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Methanotrophs with soluble methane monooxygenase (sMMO) show high potential for various ecological and biotechnological applications. Here, we developed a high throughput method to identify sMMO-producing microbes by integrating droplet microfluidics and a genetic circuit-based biosensor system. sMMO-producers and sensor cells were encapsulated in monodispersed droplets with benzene as the substrate and incubated for 5 h. The sensor cells were analyzed as the reporter for phenol-sensitive transcription activation of fluorescence. Various combinations of methanotrophs and biosensor cells were investigated to optimize the performance of our droplet-integrated transcriptional factor biosensor system. As a result, the conditions to ensure sMMO activity to convert the starting material, benzene, into phenol, were determined. The biosensor signals were sensitive and quantitative under optimal conditions, showing that phenol is metabolically stable within both cell species and accumulates in picoliter-sized droplets, and the biosensor cells are healthy enough to respond quantitatively to the phenol produced. These results show that our system would be useful for rapid evaluation of phenotypes of methanotrophs showing sMMO activity, while minimizing the necessity of time-consuming cultivation and enzyme preparation, which are required for conventional analysis of sMMO activity.

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Section: Functional Phenotyping Of Methanotrophs With Smmo Activitysupporting

confidence: 90%

Sensitive and Rapid Phenotyping of Microbes With Soluble Methane Monooxygenase Using a Droplet-Based Assay

Lee

Baek

Kim

et al. 2020

Front. Bioeng. Biotechnol.

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“…Characterization of MMOH from M. trichosporium IMV3011, a type II methanotroph, showed that oxidized MMOH exhibits optimal intensity at 281 nm and weak absorption is detected at approximately 395-420 nm due to the oxo-bridged iron The UV-visible spectrum of purified MMOH is shown in Figure 3C, which exhibited optimal absorption at 280 nm with a calculated extinction coefficient of 561,220 cm −1 M −1 , and a weak charge transfer band was observed at approximately 390-430 nm. Characterization of MMOH from M. trichosporium IMV3011, a type II methanotroph, showed that oxidized MMOH exhibits optimal intensity at 281 nm and weak absorption is detected at approximately 395-420 nm due to the oxo-bridged iron clusters [38]. The yellow color of MMOH arises from the charge transfer band around the 300-350 nm region, and spectral studies have evinced that this phenomenon is usually observed for the (µ-oxo)diiron(III) centers of the synthetic analogues.…”

Section: Expression and Purification Of Hydroxylase From M Sporiummentioning

confidence: 99%

“…The yellow color of MMOH arises from the charge transfer band around the 300-350 nm region, and spectral studies have evinced that this phenomenon is usually observed for the (µ-oxo)diiron(III) centers of the synthetic analogues. Preliminary reports have shown that the weak absorption spectrum from 400 to 500 nm by hydroxylase represents tyrosine radical formation and ligation, and M. sporium 5 showed these weak spectra, but M. trichosporium OB3b did not [17,[38][39][40].…”

Section: Expression and Purification Of Hydroxylase From M Sporiummentioning

confidence: 99%

“…These results imply that MMOH has reasonable solubility in bacterial cells, but MMOB has higher solubility compared to MMOH. clusters [38]. The yellow color of MMOH arises from the charge transfer band around the 300-350 nm region, and spectral studies have evinced that this phenomenon is usually observed for the (μoxo)diiron(III) centers of the synthetic analogues.…”

Section: Expression and Purification Of Hydroxylase From M Sporiummentioning

confidence: 99%

“…Our results demonstrated SEA of 28 mU/mg in the absence of MMOB ( Figure 5B); reports have also proposed that MMOB is essential for the activity because it can improve activity by more than 20-fold. Another type II methanotroph, M. trichosporium IMV 3011, exhibits SEA of 603.6 mU/mg, and the optimal activity is obtained at pH 7.2 in 25 mM Tris-HCl buffer [38]. Highly purified enzymes are required for sMMO studies, possibly because of other components that are co-expressed in methanotrophs.…”

Section: Activities Of Essential Enzymes From M Sporiummentioning

confidence: 99%

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Biocatalytic Oxidations of Substrates through Soluble Methane Monooxygenase from Methylosinus sporium 5

et al. 2018

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Methane, an important greenhouse gas, has a 20-fold higher heat capacity than carbon dioxide. Earlier, through advanced spectroscopy and structural studies, the mechanisms underlying the extremely stable C–H activation of soluble methane monooxygenase (sMMO) have been elucidated in Methylosinus trichosporium OB3b and Methylococcus capsulatus Bath. Here, sMMO components—including hydroxylase (MMOH), regulatory (MMOB), and reductase (MMOR)—were expressed and purified from a type II methanotroph, Methylosinus sporium strain 5 (M. sporium 5), to characterize its hydroxylation mechanism. Two molar equivalents of MMOB are necessary to achieve catalytic activities and oxidized a broad range of substrates including alkanes, alkenes, halogens, and aromatics. Optimal activities were observed at pH 7.5 for most substrates possibly because of the electron transfer environment in MMOR. Substitution of MMOB or MMOR from another type II methanotroph, Methylocystis species M, retained specific enzyme activities, demonstrating the successful cross-reactivity of M. sporium 5. These results will provide fundamental information for further enzymatic studies to elucidate sMMO mechanisms.

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Epoxypropane biosynthesis by whole cell suspension of methanol-growth Methylosinus trichosporium IMV 3011

Xin

Zhang

Dong

et al. 2009

World J Microbiol Biotechnol

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Methanotrophs containing methane monooxygenase (MMO) can catalyze the epoxidation of propene to epoxypropane. Methane cannot support dense biomass growth due to its low aqueous solubility. Low growth rate is important limiting factor for the application of methanotrophs. Methanol can act as growth substrate, but direct addition of methanol is toxic to most methanotrophs. The MMO activity during growth on methanol is also uncertain. In this paper, methanol-adapted Methylosinus trichosporium IMV 3011 was successfully cultivated at high cell densities using methanol as sole carbon source. A biomass density of 1.68 g dry weight cell l -1 was achieved and cells contained almost 80% of the MMO activity measured for cells grown with methane. It has been found that methanol can also act as the electron-donating substrate to regenerate the NADH and drive epoxypropane synthesis. The effect of methanol supply on the epoxidation capacity of Methylosinus trichosporium IMV3011 was studied in batch reactor. 0.016% methanol concentration was found to give the highest propene epoxidation capacity.

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Purification and Biochemical Characterization of Soluble Methane Monooxygenase Hydroxylase fromMethylosinus trichosporiumIMV 3011

Cited by 5 publications

References 16 publications

Sensitive and Rapid Phenotyping of Microbes With Soluble Methane Monooxygenase Using a Droplet-Based Assay

Sensitive and Rapid Phenotyping of Microbes With Soluble Methane Monooxygenase Using a Droplet-Based Assay

Biocatalytic Oxidations of Substrates through Soluble Methane Monooxygenase from Methylosinus sporium 5

Epoxypropane biosynthesis by whole cell suspension of methanol-growth Methylosinus trichosporium IMV 3011

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