KR20190049573A - Method for Methanol Production Using methanotroph without External Reducing Power - Google Patents

Abstract

The present invention relates to a methane-using methanol production method using methanotrophs that do not require an external reducing power. According to the method, methanol production can be increased by partially inhibiting MDH during a methanol production process and keeping the amounts of catalyst and reducing power constant, which can be effectively used in the methanol production process.

Description

Technical Field [0001] The present invention relates to a methanotrophic methanol production method using methanotrophic bacteria which does not require external reducing power,

More particularly, the present invention relates to a method for producing methane-using methanol using a methane-magnetizing microorganism that does not require external reducing power, and more particularly, to a method for producing methane using a methane- / RTI >

Currently reported methane - using methanol production method using methane magnetism is a method in which methanol produced from methane is not metabolized through blocking of methanol dehydrogenase and is accumulated in a culture solution.

Although methane conversion is possible under mild conditions, there are two reasons why methanol production by biological methods is difficult. First, methane monooxygenase (MMO) requires a reducing power to convert methane. Second, the produced methanol is decomposed by methanol dehydrogenase (MDH).

In methanotroph cells, the reducing power required for methane conversion is recovered from methanol oxidation, and since the formaldehyde produced in this process is used for growth through cell metabolism, induction of natural methanol accumulation is very difficult. Therefore, studies on methanol production by methane magnetism to date have shown that MDH inhibitors are used to prevent degradation of methanol produced. However, when the activity of methanol dehydrogenase is inhibited artificially, methane oxidation is not performed due to lack of reducing power.

Therefore, in order to solve this problem, conventional methods have been proposed to inhibit methanol dehydrogenase and provide formic acid, which is more expensive than methanol, as an external reducing power.

Therefore, the present inventors confirmed that when the methanol dehydrogenase (MDH) was partially inhibited, the methanol production process could be performed without supplying the external reducing power.

Accordingly, an object of the present invention is to provide a process for the preparation of a compound of formula (I) or a salt thereof, wherein Methylomonas, Methylobacterium, Methylomicrobium, Methylobacter, Methylococcus, But are not limited to, Methylosphaera, Methylocaldum, Methyloglobus, Methylosarcina, Methyloprofundus, Methylolucumthus, Methylothalobus, Methylohalobius, Methylogaea, Methylomarinum, Methylovulum, Methylomarinovum, Methylorabicin, Methylothiophene, The compounds of the present invention may be used in combination with one or more compounds selected from the group consisting of Methylorubrum, Methyloparacoccus, Methylosinus, Methylocystis, Methylocella, Methylocapsa, Methylofurula, Methyl acid ethylphilum and methylacidimicrobium, and ethylenediaminetetraacetic acid (EDTA). The present invention also provides a method for producing methanol, comprising the steps of:

It is another object of the present invention to provide a method for the treatment and / or prophylaxis of a disease or disorder which comprises administering to a mammal an effective amount of at least one compound selected from the group consisting of methylolmonas, methyllobaterium, methylolubium, methylrobacter, methylloccus, methylrosperes, There may be mentioned methylcyclohexane, methylcyclohexane, methylcyclohexane, methylcyclohexane, methylcyclohexane, methylcyclohexane, methylcyclohexane, methylcyclohexane, And a mixture of methyl ascorbic acid and methyl ascorbic acid, and a mixture of methyl ascorbic acid, methyl ascorbic acid, methyl ascorbic acid, methyl ascorbic acid, methyl ascorbic acid, methyl ascorbic acid, methyl ascorbic acid, And a conversion step of inducing a conversion reaction in a culture medium containing EDTA from at least one strain selected from the group consisting of EDTA.

The present invention relates to a method for producing methane-using methanol using a methanotrophic bacterium which does not require an external reducing power, and the method according to the present invention uses methane- Of production.

The present inventors increased the production of methanol without supplying external reducing power by producing methanol using methane magnetizing bacteria under the condition of containing a certain amount of EDTA.

In order to realize the present invention, 1) a cell culture apparatus which continuously produces cells and 2) a methanol production apparatus which produces methanol, and the cells produced in the culture apparatus are supplied to the methanol production process, To increase methanol production.

Hereinafter, the present invention will be described in more detail.

One embodiment of the present invention is a method for producing a compound of formula (I) or a salt thereof, wherein the compound is selected from the group consisting of Methylomonas, Methylobacterium, Methylomicrobium, Methylobacter, Methylococcus, But are not limited to, Methylosphaera, Methylocaldum, Methyloglobus, Methylosarcina, Methyloprofundus, Methylothermus, ), Methylohalobius, Methylogaea, Methylomarinum, Methylovulum, Methylomarinovum, Methylolabrum, Methyloglucarnide, But are not limited to, Methylorubum, Methyloparacoccus, Methylosinus, Methylocystis, Methylocella, Methylocapsa, Methylofurula, Methylacid at least one microorganism belonging to the genus Escherichia, and at least one microorganism belonging to the genus Escherichia, and at least one microorganism belonging to the genus Escherichia.

The strain may be, but is not limited to, Methylomonas DH-1 deposited with Accession No. KCTC 13004BP.

Wherein the composition comprises EDTA in an amount of 0.01 to 5.00 mM, 0.01 to 2.00 mM, 0.01 to 1.00 mM, 0.01 to 0.80 mM, 0.10 to 5.00 mM, 0.10 to 2.00 mM, 0.10 to 1.00 mM, 0.10 to 0.80 mM, For example, at a concentration of 0.20 to 2.00 mM or 0.20 to 1.00 mM, for example, 0.20 to 0.80 mM, but the present invention is not limited thereto.

Another aspect of the present invention is a method for producing a compound of formula (I) or a pharmaceutically acceptable salt thereof, which comprises administering to a mammal in need thereof an effective amount of at least one compound selected from the group consisting of methylolmonas, methyllobaterium, methylolmobium, methylolbacter, methylolcocuses, methylrosperes, There may be mentioned methylcyclohexane, methylcyclohexane, methylcyclohexane, methylcyclohexane, methylcyclohexane, methylcyclohexane, methylcyclohexane, methylcyclohexane, And a mixture of methyl ascorbic acid and methyl ascorbic acid, and a mixture of methyl ascorbic acid, methyl ascorbic acid, methyl ascorbic acid, methyl ascorbic acid, methyl ascorbic acid, methyl ascorbic acid, methyl ascorbic acid, A step of inducing a conversion reaction in a culture medium containing EDTA from at least one strain selected from the group consisting of:

As used herein, the term " conversion " refers to the process by which a strain produces a specific substance using a substance in a culture fluid.

The strain may be, but is not limited to, Methylomonas DH-1 deposited with Accession No. KCTC 13004BP.

Wherein the composition comprises EDTA in an amount of 0.01 to 5.00 mM, 0.01 to 2.00 mM, 0.01 to 1.00 mM, 0.01 to 0.80 mM, 0.10 to 5.00 mM, 0.10 to 2.00 mM, 0.10 to 1.00 mM, 0.10 to 0.80 mM, For example, at a concentration of 0.20 to 2.00 mM or 0.20 to 1.00 mM, for example, 0.20 to 0.80 mM, but the present invention is not limited thereto.

The conversion step may be carried out while maintaining the ratio of NADH / NAD + in the cell to 0.5 or more, but is not limited thereto.

The conversion step may be performed at a concentration of 10-50% v / v, 10-45% v / v, 10-40% v / v, 10-35% v / v / v), 20 to 45% (v / v), 20 to 40% (v / v), 20 to 35% (v / v), 25 to 50% / v) or 25 to 40% (v / v), for example 25 to 35% (v / v) methane.

The conversion step may be performed by exhausting the gas for 10 to 900 minutes, but is not limited thereto. The optimum time for exhausting the gas can be appropriately adjusted according to the variation of the conditions including the volume of the whole culture liquid, the execution time of the process, the type of the used strain and the composition of the medium.

The conversion step may be performed at a temperature of 25 to 35 DEG C, 27 to 35 DEG C, 29 to 35 DEG C, 25 to 32 DEG C, or 27 to 32 DEG C, for example, 29 to 32 DEG C, It is not.

The present invention relates to a method for producing methane-using methanol using a methanotrophic bacterium which does not require an external reducing power, and in which the methanol production is partially inhibited during the methanol production process to maintain the amount of the catalyst and the reducing power constant, So that it can be effectively used in the methanol production process.

FIG. 1 is a schematic diagram showing the methanol production route of the present invention. FIG.
2 is a graph showing the effect of ethylenediaminetetraacetic acid (EDTA) on the activity of methanol dehydrogenase (MDH) of DH-1 strain.
FIG. 3 is a graph showing MDH activity of DH-1 strain cells according to EDTA concentration. FIG.
FIG. 4A is a graph comparing methane consumption and methanol production of DH-1 strain under the condition of no EDTA-added medium. FIG.
FIG. 4B is a graph comparing methane consumption and methanol production of DH-1 strain under a medium containing 0.50 mM EDTA.
FIG. 4C is a graph comparing methane consumption and methanol production of the DH-1 strain under a medium condition in which EDTA was added at 10.00 mM. FIG.
FIG. 5A is a graph showing the ratio of NADH / NAD ^{+ in} DH-1 strain cells under the condition of no EDTA-added medium. FIG.
FIG. 5B is a graph showing the ratio of NADH / NAD ^{+ in the} DH-1 strain cells under the condition of addition of EDTA at 0.50 mM.
FIG. 5C is a graph showing the ratio of NADH / NAD ^{+ in} DH-1 strain cells under a medium condition in which EDTA was added at 10.00 mM. FIG.
FIG. 6 is a graph showing the relationship between the absorbance and the culture conditions in which the concentration of EDTA was varied in the conversion reaction of DH-1 strain.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Determination of optimum concentration of EDTA

Two electrons are required for the conversion of one molecule of methane, and four electrons can be produced through the oxidation of methanol, so theoretically, up to 1/2 mole of methanol can be obtained from 1 mole of methane through proper control of MDH enzyme activity .

Ethylenediaminetetraacetic acid (EDTA) was used for the selective production of methanol through the control of methanol dehydrogenase (MDH) activity. EDTA is a metal chelator that can interfere with the electron transfer of MDH by interfering with the initial binding through the immobilization of lysyl or arginyl residues in MDH and has been widely used in previous methanol production studies.

Inoculate 150 mL of DH-1 stock (KCTC 13004BP) into a 500 mL baffled flask containing 50 mL of NMS medium, periodically inject methane / air and measure the absorbance (OD 600) of the culture at 600 nm. Was cultured at 30 DEG C and 230 rpm in a shaking incubator until the concentration reached 5 to 6, and the reaction was induced by the same route as in Fig. A cap with a butyl rubber septum was used in the flask to prevent the inflow of outside air and the leakage of methane or propane into the flask during the culture. The composition of the NMS medium is shown in Table 1 below.

NMS composition Amount for 1L MgSO _{4 ·} 7H ₂ O 1 g KNO3 1 g CaCl ₂ .2H ₂ O 0.228 g Fe-EDTA 0.0038 g Na ₂ MoO ₄ 0.0006 g FeSO ₄ .7H ₂ O 0.5 mg ZnSO ₄ .7H ₂ O 0.4 mg MnCl ₂ .7H ₂ O 0.02 mg CoCl ₂ .6H ₂ O 0.05 mg NiCl ₂ .6H ₂ O 0.01 mg H ₃ BO ₃ 0.015 mg EDTA 0.25 mg KH ₂ PO ₄ 0.26 g Na ₂ HPO ₄ .7 (H ₂ O) 0.62 g Biotin 0.02 mg Folic acid 0.02 mg Thiamine HCl 0.05 mg Ca pantothenate 0.05 mg Vitamin B12 0.001 mg Riboflavin 0.05 mg Nicotiamide 0.05 mg CuSO _{4 ·} 5H ₂ O 2.5 mg

For the methanol conversion experiment, the cultured medium was centrifuged, the supernatant was removed, and then washed with PBS buffer twice. Then, the EDTA concentration was set to 0.00, 0.01, 0.10, 0.50, 1.00, 5.00 and 10.00 mM in a PBS buffer of pH 8.5, and the washed methanotrophic cells were added to the OD 30. Methane: air = 3: 7 by volume gas was evacuated for 30 minutes and then stirred at 30 rpm at 230 rpm in a shaking incubator.

The effect of EDTA on the MDH activity of Methylomonas sp. DH-1 was observed at various EDTA concentrations. The activity of MDH was determined by measuring the rate of reduction of DCPIP (2,6-dichlorophenolindophenol) by electrons that occurs when MDH oxidizes methanol (References: Kim Hee-Gon, Lee Sik Ei, Kim Sik Wook, Biosynthesis of Methanol Using Methylobacterium sp. HG-1 ", Journal of Engineering Technology, 1 (1), 33-37 (2008))

As can be seen in FIG. 2, the inhibitory effect of EDTA did not appear until 0.01 mM, and then rapidly increased until the concentration of 1.00 mM. When 0.50 mM EDTA was added, the specific activity of MDH was 2.095 nmol / mg protein / min, which was 31% of the original activity. In the case of EDTA above 1.00 mM, the activity was slowly inhibited. Finally, when the EDTA concentration reached 10.00 mM, the activity of MDH was completely inhibited. Considering the electrons required to convert methane to methanol and the electrons obtained by oxidation of methanol, it is expected that most of methanol can be accumulated when the activity of MDH is 1/3 to 1/2, so that the optimum concentration of EDTA is 0.50 mM Respectively.

Example 2: Confirmation of MDH inhibitory effect by EDTA

In order to confirm that the inhibition of MDH enzyme by EDTA is also achieved in vivo, the initial methanol concentration in the reaction buffer was 45 mM, and Methylomonas sp. DH-1 cells were injected and methanol degradation was observed. The amount of methanol consumed per hour of DH-1 resting cells was analyzed at 0.00 mM, 0.50 mM, and 10.00 mM EDTA concentration derived from enzyme activity experiments.

As shown in FIG. 3, when EDTA was not included, DH-1 cells consumed methanol in 4 hours. However, in the condition that EDTA containing 10.00 mM EDTA containing MDH activity was mostly inhibited, it was confirmed that methanol decomposition was hardly achieved by consuming only 1.80 mM of methanol even after 4 hours. On the other hand, DH-1 cells consumed 13.9 mM methanol, which is 31% of the input methanol, for 4 hours under the condition of containing 0.50 mM of EDTA, which is a partial inhibition condition of MDH.

Example 3: Determination of conversion and accumulation of methanol

Actual methane conversion experiments were performed using the enzymatic activity tests performed in Examples 1 and 2 and the MDH partial inhibitory effect obtained from the dormant cell experiments. Methylomonas sp. DH-1 cells were injected and the consumption of methane was observed.

As can be seen from FIG. 4A, the methanol consumption was 36.28 mM and 37.10 mM at 4 hours and 8 hours, respectively, as a result of methanol conversion experiment under the condition of no EDTA without MDH inhibition. This is the most abundant methane consumption in the cell because it is not inhibited by the metabolic process.

However, in the absence of MDH inhibition, methanol was continuously oxidized and methanol accumulation was scarcely occurred. Maximum methanol production was 0.273 mM at 4 hours and methanol yield was 0.8%. At 8 hours after the reaction, methanol was further decreased to 0.224 mM and methanol yield was 0.6% compared to methane consumption.

As can be seen in FIG. 4B, methanol was successfully accumulated in the condition of MDH activity reduced to 1/3 level (EDTA 0.50 mM). Methane consumption was 29.41 mM and 35.24 mM at 4 and 8 hours, respectively, but the methanol concentration was rapidly increased to 21.52 mM (0.69 g / l) at 4 hours after the reaction. Methanol production increased to 27.85 mM (0.892 g / l) at the end of time. Methanol production yields of methane were 73.1% and 79.0% at 4 and 8 hours, respectively.

As can be seen from FIG. 4C, under the condition of EDTA 10.00 mM in which complete inhibition of MDH occurs, no reducing power was supplied through methanol oxidation, so no methane conversion occurred and almost no methane consumption and methanol accumulation occurred. At the 4th hour of the reaction, the amount of methane consumed was 3.62 mM and the methanol concentration was 0.06 mM.

In conclusion, the MDH partial inhibition was able to produce up to 0.892 g / l of methanol without external reducing power.

Example 4: Measurement of intracellular reducing power by NADH / NAD + ratio

Methane conversion and methanogen production using methanogenic bacteria play a very important role. Methylomonas sp. To determine the level of intracellular reducing power in methanol conversion using DH-1 dormant cells, the NADH / NAD ⁺ ratio was determined.

As can be seen in FIG. 5A, it was confirmed that the NADH / NAD ⁺ ratio remained in the initial state during the conversion time in the absence of EDTA without MDH inhibition. Methylomonas sp. This means that methanol produced from methane in DH-1 is all re-used to supply the reducing power needed for methane production.

As can be seen in FIG. 5B, the NADH / NAD ⁺ ratio in the condition containing 0.50 mM EDTA partially inhibited MDH was partially reduced at the early stage during the conversion time, but it remained to some extent. The methanol produced is partially supplied as a reducing power required for methane conversion depending on the activity of the partially inhibited MDH, and some of it is considered to be accumulated in the reaction buffer. However, the overall NADH / NAD + ratio is low, suggesting that methane conversion rate is slower than without EDTA.

As shown in FIG. 5C, the ratio of NADH / NAD ⁺ in the condition containing 10.00 mM of EDTA completely inhibiting MDH activity was continuously decreased. It can be interpreted that only a trace amount of methane conversion is observed since the reductive power required for methane conversion and methanol oxidation are not smoothly achieved. At the EDTA 10.00 mM, the NADH / NAD ⁺ ratio was significantly lowered from 0.766 at the beginning of the reaction to 0.254 at the 8th hour of the reaction.

Example 5: Confirmation of change in cell concentration during conversion reaction

The optical density (OD) value during the conversion reaction was measured.

As can be seen from FIG. 6, when the EDTA was 0.00 mM, the cell OD value was 31 at the initial stage but decreased by 2.8 after 4 hours. It was confirmed that no significant inhibition of cell maintenance was observed in the absence of EDTA during the entire reaction.

However, when EDTA was added, the cell concentration was steadily decreased, and the degree of cell concentration was increased with concentration. When 0.50 mM EDTA containing MDH partial inhibitor was added, the cell OD values were decreased by 6.5 and 10.8 after 4 and 8 hours, respectively. The slower production rate of methanol after 4 hours was considered to be caused by the decrease of these biocatalysts .

In addition, it was confirmed that the cells contained EDTA 10.00 mM completely inhibited MDH more rapidly than the previous condition. After 4 hours and 8 hours after the reaction, the cell OD values decreased by 24.5 and 27.55, respectively, and most of the cells were killed.

From these results, it was found that EDTA concentration affects not only MDH activity but also decomposition of dormant cells, and the maintenance of continuous cell activity and intracellular reducing power should be prepared for commercial methanol production process.

Claims (10)

Methylomonas, Methylobacterium, Methylomicrobium, Methylobacter, Methylococcus, Methylosphaera, Methylcyclohexanone, and the like. The compounds of formula (I) are preferably selected from the group consisting of Methylocaldum, Methyloglobus, Methylosarcina, Methyloprofundus, Methylothermus, The compounds of the present invention can be used in the form of Methylohalobius, Methylogaea, Methylomarinum, Methylovulm, Methylomarinovum, Methylorubrum, Methyloparcoccus, Methylosinus, Methylocystis, Methylocella, Methylocapsa, Methylofurula, Methylcyclohexyl, Methylcyclohexyl, Methylcyclohexyl, Methylacidiphilum and Methyl Acid At least one strain selected from the group consisting of Methylacidimicrobium and ethylenediaminetetraacetic acid (EDTA). The composition for producing methanol according to claim 1, wherein the strain is Methylomonas DH-1 deposited with Accession No. KCTC 13004BP. The composition for producing methanol according to claim 1, wherein the composition comprises EDTA at a concentration of 0.01 to 5.00 mM. Methylomonas, Methylobacterium, Methylomicrobium, Methylobacter, Methylococcus, Methylosphaera, Methylcyclohexanone, and the like. The compounds of formula (I) are preferably selected from the group consisting of Methylocaldum, Methyloglobus, Methylosarcina, Methyloprofundus, Methylothermus, The compounds of the present invention can be used in the form of Methylohalobius, Methylogaea, Methylomarinum, Methylovulm, Methylomarinovum, Methylorubrum, Methyloparcoccus, Methylosinus, Methylocystis, Methylocella, Methylocapsa, Methylofurula, Methylcyclohexyl, Methylcyclohexyl, Methylcyclohexyl, Methylacidiphilum and Methyl Acid And a conversion step of inducing a conversion reaction in a culture solution containing ethylenediaminetetraacetic acid (EDTA) from at least one strain selected from the group consisting of Methylacidimicrobium. 5. The method of producing methanol according to claim 4, wherein the strain is Methylomonas genus DH-1. 5. The method according to claim 4, wherein the culture medium contains EDTA at a concentration of 0.01 to 5.00 mM. 5. The method according to claim 4, wherein the step of converting is carried out while maintaining the ratio of NADH / NAD + in the cell of the strain to 0.5 or more. 5. The process according to claim 4, wherein the conversion is carried out in the presence of a gas comprising 10 to 50% (v / v) methane. 9. The method according to claim 8, wherein the conversion is carried out by evacuating the gas for 10 to 900 minutes. 5. The process according to claim 4, wherein the conversion step is carried out at a temperature of 25 to 35 占 폚.

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