Masayuki Takeguchi

Semicontinuous methanol biosynthesis by Methylosinus trichosporium OB3b

Abstract Methylosinus trichosporium OB3b is a methanotrophic bacterium containing methane monooxygenase, catalyzing hydroxylation of methane to methanol. When methane is oxidized, the product is subsequently oxidized by methanol dehydrogenase contained in the same bacterium. To prevent further oxidation of methanol, the cell suspension was treated by cyclopropanol, an irreversible inhibitor for methanol dehydrogenase, leading to extracellular methanol accumulation. However, the batch type of methanol synthesis by M. trichosporium OB3b was terminated at ca. 6 mM of methanol, for MMO is inhibited by increasing methanol concentration. For prolonged methanol accumulation, a semicontinuous process was carried out. In this process, the reaction was repeated several times and the produced methanol was 36.1 μmol for 6 h.

Optimization of methanol biosynthesis byMethylosinus trichosporium OB3b: An approach to improve methanol accumulation

Methylosinus trichosporium OB3b is a methanotrophic bacterium containing methane mono-oxygenase, catalyzing hydroxylation of methane to methanol. When methane is oxidized, the product is subsequently oxidized by methanol dehydrogenase contained in the same bacterium. To prevent further oxidation of methanol, the cell suspension was treated by cyclopropanol, an irreversible inhibitor for methanol dehydrogenase, leading to extracellular methanol accumulation. However, the reaction was terminated at approx 3 h with a final methanol concentration below 2.96 mmol/g dry cell. The methanol production efficiency (the ratio of the produced methanol per methane consumption) was 2.90%. By selecting the culture conditions and the reaction conditions, the reaction continued for 100 h, resulting in a methanol concentration of 152 mmol/g dry cell. This level was 51 times higher than that of the conventional reaction, and the methanol production efficiency was 61%.

Role of iron and copper in particulate methane monooxygenase of Methylosinus trichosporium OB3b

The role of iron and copper in particulate methane monooxygenase (pMMO) of Methylosinus trichosporium OB3b is described, and an overview of the enzymes properties is presented. The pMMO from M. trichosporium OB3b was solubilized in the detergent n-dodecyl-β-D-maltoside and purified by chromatographic techniques. The enzyme consists of 0.9 iron atoms and 12.8 copper atoms per molecule. The iron site in pMMO may be mononuclear non-heme iron. Copper exists as either copper ion coupled to four nitrogen atoms and/or trinuclear copper cluster wherein copper ions are ferromagnetically coupled.

The role of copper in particulate methane monooxygenase from Methylosinus trichosporium OB3b

Abstract The effect of metal ions on particulate methane monooxygenase (pMMO) was studied. The pMMO activity in the membranes was partially inhibited by ethylenediaminetetraacetic acid (EDTA), but remained more than 70% of the as-isolated membranes. The activity of the EDTA-treated membranes was strongly influenced by the addition of metal ions. Among the metal ions, copper ion stimulated the activity, indicating that copper was needed for the activity. When duroquinol and dioxygen were introduced to the EDTA-treated membranes, the electron spin resonance signal of copper did not change, suggesting that the copper cluster did not play as an electron transport and may have another function, such as active site of pMMO or regulator of the activity. On the other hand, the iron signal (g=5.98) decreased by the addition of duroquinol, dioxygen and acetylene, showing an iron atom is contained in the active site of pMMO.

PROPERTIES OF THE MEMBRANES CONTAINING THE PARTICULATE METHANE MONOOXYGENASE FROM METHYLOSINUS TRICHOSPORIUM OB3B

The particulate methane monooxygenase (pMMO) from Methylosinus trichosporium OB3b was partiallypurified and characterized by measuring the effects of reducing agents and additives, and the stability ofpMMO was studied. Duroquinol was a suitable reducing agent, and pMMO was stabilized by bovine serumalbumin (BSA). Among the additivies, the copper (II) ion stimulated pMMO at low concentration andinhibited at high concentration. The optimum conditions for pMMO activity were as follows: 45 ° C, pH 6.5and 55 mM 3-morpholinopropanesulfonic acid (MOPS) buffer, and the rate of propene epoxide formationwas 13.6 nmol min mg protein. ESR spectra indicate that the copper cluster in the membrane fraction isreduced by duroquinol and oxidized by dioxygen. The result suggests that the copper cluster is containedin the active site of pMMO.

Purification and properties of bilirubin oxidase from Penicillium janthinellum

Purification of bilirubin oxidase (BOX) from Penicillium janthinellum was carried out and some properties of the BOX were clarified. The BOX contained copper, zinc, and iron ions. The effect of metal ions on BOX activity was abnormal and, in the presence of mercury ions, a remarkable increase of the activity was observed. The product of bilirubin oxidation with the BOX was biliverdin only and biliverdin oxidation did not occur. Thus, the BOX is a new type of BOXs and its reaction mechanism is discussed.

Biocatalytic methanol production from methane with Methylosinus trichosporium OB3b: An approach to improve methanol accumulation

INTRODUCTION Methylosinus trichosporium OB3b is a methanotrophic bacterium containing methane monooxygenase (MMO), catalyzing hydroxylation of methane to methanol. When methane is oxidized by the intact cell, produced methanol is subsequently oxidized by methanol dehydrogenase containing in the same bacterium (Anthony, 1982; Shimoda and Okura, 1991). To prevent further oxidation, the cell suspension was treated with cyclopropanol which was a selective and irreversible inhibitor for methanol dehydrogenase (Shimoda and Okura, 1991), and methanol production has been established. However, the reaction was terminated at ca. 3 h. Methanol produced was at most 0.41 mmol per g-wet wt.-cell as shown by the circles in Fig. 1. Since OB3b grows using methane as a sole carbon source, the methanol production efficiency, the ratio of the produced methanol per methane consumption, was 4.1%. In this study, we tried to improve methanol yield, the efficiency, by selecting the reaction conditions.

ROLE OF IRON IN PARTICULATE METHANE MONOOXYGENASE FROM METHYLOSINUS TRICHOSPORIUM OB3B

The effect of iron ions on particulate methane monooxygenase was studied by using the EDTA-treated membranes from Methylosinus trichosporium OB3b. When the membrane was treated with EDTA the activity remained 82% of the as-isolated membranes, and the activity of the EDTA-treated membranes was strongly influenced by the addition of metal ions. Among the metal ions, ferric, ferrous and cupric ions stimulated the activity, indicating those ions were needed for the activity. When propargylamine was added, pMMO activity decreased and also the iron ESR signal decreased. As the ESR signal involves the ferrous nitrosyl complex in EDTA-treated membranes, the active site of pMMO may contain a mononuclear non-heme iron.

REDOX BEHAVIOR OF COPPER IN PARTICULATE METHANE MONOOXYGENASE FROM METHYLOSINUS TRICHOSPORIUM OB3B

The redox properties of the copper in particulate methane monooxygenase from Methylosinus trichosporium OB3b were investigated. The ESR spectrum of the pMMO-containing membranes from M. trichosporium OB3b indicated a typical type II copper (II) signal (g∥ = 2.24, A∥ = 18.4 mT, g⊥ = 2.06, α2= 0.84). By anaerobic addition of excess amounts of duroquinol, an optimum reductant of pMMO, the ESR spectra indicated that the copper cluster in membranes was reduced and successively oxidized by dioxygen, a substrate of pMMO. The result suggests that the copper is the active site of pMMO or an electron carrier. During the titration, the intensity of the type II copper signal decreased with decreasing potential and the multiple hyperfine structure at g = 2.06 appeared clearly. Although the copper signal did not change by treatment of the EDTA-treated membranes with duroquinol and dioxygen, the copper signal intensity decreased with decreasing potential in the redox titration. These results suggest that some redox mediators play a role as an electron carrier between the active site and a reductant, and the presence of at least two types of copper sites in pMMO- containing membranes. On the basis of the ESR spectra of the EDTA-treated membranes and the as-isolated membranes, it is concluded that one type of the copper sites functions as the active site of pMMO (A-site), and the other type of copper sites plays a role as an electron carrier (E-site)

On the molecular weight of bilirubin oxidase from Penicillium janthinellum

Abstract When bilirubin oxidase from Penicillium janthinellum was treated with bilirubin BOX was dissociated to the compound with the molecular weight between 0.5 kDa and 5 kDa. When BOX was treated with EDTA, a remarkable increase of the reaction rate was observed. The reaction mechanism of these effects is discussed.