KR20190049575A - Composition for the production of propanol containing methanotroph and propanol production method - Google Patents

Abstract

The present invention relates to a method for producing a high concentration of propanol by supplying methanol and cultivating methanotrophs in the presence of propane-containing gas. The high concentration of propanol can be stably produced according to the method, which can be effectively used to produce high-concentration propanol.

Description

TECHNICAL FIELD The present invention relates to a composition for producing propanol containing methane magnetism and a method for producing propanol,

TECHNICAL FIELD The present invention relates to a composition for producing propanol containing a methanogenic bacterium and a method for producing propanol, and more particularly, to a method for producing a high concentration of propanol by culturing methanotrophic bacteria in the presence of methanol and propane-containing gas.

Methane monooxygenase (MMO) possessed by methanogenic bacteria has a low substrate specificity and propanol production from propane is possible. However, since the produced propanol is further converted to acetone or the like by methanol dehydrogenase (MDH), it is impossible to produce propanol at a high concentration.

The propanol production process is a concept in which the reducing power for oxidizing propane is obtained from inexpensive methane or methanol and propanol is selectively recovered. Methanol hydrolase, which is possessed by methanogenic bacteria, decomposes the produced alcohol to produce reducing power, which is helpful in terms of reducing power supply. However, appropriate production strategies and strains are needed because propanol produced can also be decomposed.

Therefore, the inventors of the present invention have found that the production efficiency of propanol is excellent when methanol is supplied and the methanogenic bacteria are cultured in the presence of propane-containing gas.

Accordingly, an object of the present invention is to provide a process for producing a compound of formula (I), which is a compound of formula (I), wherein Methylomonas, Methylomicrobium, Methylobacter, Methylococcus, Methylosphaera, But are not limited to, Methylocaldum, Methyloglobus, Methylosarcina, Methyloprofundus, Methylothermus, Methylollobuides, Methylmorpholinamide, Methylohalobius, Methylogaea, Methylomarinum, Methylovulum, Methylomarinovum, Methylorubrum, Methylorubarin, But are not limited to, Methyloparacoxus, Methylosinus, Methylocystis, Methylocella, Methylocapsa, Methylofurula, Methylocyadine, Methylacidiphilum and Methyl Acid Micro Propanol containing culture medium of the strain or the strains at least one selected from the group consisting of Titanium in (Methylacidimicrobium) to provide a composition for production.

It is another object of the present invention to provide a method for the treatment and / or prophylaxis of a disease or condition in a mammal, such as a mammomonas genus, a methylorubicus genus, a methylorubic genus, a methylorbacter genus, a methylrococcus genus, a methylrosperace genus, There may be mentioned methylolpropanol, methylolfull, methylolfull, methylrogeran, methyllumarnol, methylolulomon, methylromarinol, methyloluburum, methylol, Wherein the microorganism belonging to the genus Escherichia is a species selected from the group consisting of the genus Escherichia, the genus Escherichia, the genus Escherichia, the genus Escherichia, the genus Escherichia, the genus Escherichia, the genus Escherichia, And a conversion step of inducing a conversion reaction in the above-mentioned strain or a culture medium of the strain.

The present invention relates to a composition for producing propanol containing a methanogenic bacterium and a method for producing propanol, and the method according to the present invention can be utilized for producing a high concentration of propanol.

The present inventors cultured Methylomonas genus DH-1 or Methylomicrobium alcaliphilum 20Z using methanol and propane-containing gas, and methanol dehydrogenase preferentially uses methanol due to competitive substrate inhibition It was confirmed that propanol was accumulated in the culture medium, and it was found that propanol can be stably produced with high added value because the activity of the bio catalyst is continuously maintained through methanol metabolism.

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, Methylomicrobium, Methylobacter, Methylococcus, Methylosphaera, But are not limited to, Methylocaldum, Methyloglobus, Methylosarcina, Methyloprofundus, Methylothermus, Methyloligobius, Methylohalobius, Methylogaea, Methylomarinum, Methylovulm, Methylomarinovum, Methylorubrum, Methyloraparacicus, But are not limited to, Methyloparacoxus, Methylosinus, Methylocystis, Methylocella, Methylocapsa, Methylofurula, Methylacidiphilum and methyl acedimicrobi The propanol for production of a composition comprising in a first culture medium of the strains or isolates or more selected from the group consisting of (Methylacidimicrobium).

The strain may be, but is not limited to, Methylomonas genus DH-1 deposited with accession number KCTC 13004BP or Methylomicrobium alcaliphilum 20Z deposited with accession number DSM 19304.

The composition may be formulated to contain, for example, from 0.5 to 4.0 g / L, from 0.5 to 3.0 g / L, from 0.5 to 2.0 g / L, from 1.0 to 4.0 g / L or from 1.0 to 3.0 g / L, But the present invention is not limited thereto.

V / v), 5 to 20% (v / v), 10 to 30% (v / v), 10 to 25% (v / (v / v), such as 10-20% (v / v), 15-30% (v / v) or 15-25% (v / v), such as 15-20% But it is not limited thereto.

The gas may comprise at least one gas selected from the group consisting of 5 to 30% (v / v), 5 to 25% (v / v), 5 to 20% (v / v), 10 to 30% (v / v), such as 10-20% (v / v), 15-30% (v / v) or 15-25% But is not limited thereto.

Another aspect of the present invention is the use of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for the treatment or prophylaxis of a disease or condition selected from the group consisting of methylromonas, methylromycibium, methylrobacter, methylrococcus, methylrosperacea, There may be mentioned methylolpropanol, methylolfull, methylolfull, methylrogeran, methyllumarnol, methylolulomon, methylromarinol, methyloluburum, methylol, Wherein the microorganism belonging to the genus Escherichia is a species selected from the group consisting of the genus Escherichia, the genus Escherichia, the genus Escherichia, the genus Escherichia, the genus Escherichia, the genus Escherichia, the genus Escherichia, And a conversion step of inducing a conversion reaction in the above-mentioned strain or a culture medium of said strain.

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 Methylomonas DH-1 deposited with the deposit number KCTC 13004BP or methylmicrobium alkali filament 20Z deposited with the deposit number DSM 19304, but is not limited thereto.

The conversion step may be carried out in a culture medium at a concentration of 0.5 to 4.0 g / L, 0.5 to 3.0 g / L, 0.5 to 2.0 g / L, 1.0 to 4.0 g / L or 1.0 to 3.0 g / L, L of methanol is added, but the present invention is not limited thereto.

The conversion step may be performed by additionally adding 0.5 to 4.0 g / L of methanol to the culture medium when the methanol in the culture liquid is depleted.

(V / v), 5 to 20% (v / v), 10 to 30% (v / v), 10 to 25% (v / v) / v), 10 to 20% (v / v), 15 to 30% (v / v) or 15 to 25% (v / v), such as 15 to 20% (v / v) But the present invention is not limited thereto.

The gas may comprise at least one gas selected from the group consisting of 5 to 30% (v / v), 5 to 25% (v / v), 5 to 20% (v / v), 10 to 30% (v / v), such as 10-20% (v / v), 15-30% (v / v) or 15-25% But is not limited thereto.

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 propanol at a high concentration by culturing a methanogenic bacterium in the presence of methanol and propane-containing gas. Since it is possible to stably produce propanol at a high concentration by the above method, it can effectively produce a high concentration of propanol .

Fig. 1 is a schematic diagram showing a production route of propanol using methane magnetism.
Fig. 2 shows the results of the measurement of Methylomonas sp. ≪ RTI ID = 0.0 > 1, < / RTI > 1 is a graph showing the inhibition effect of propanol from DH-1 strain.
Figure 3 is a methane and air volume ratio of 3: Methylomonas under the conditions supplied to 7 and add supplied to the methanol per liter 0.5 g, 1 g, 2 g (0.5 g / L, 1 g / L, 2 g / L) sp . FIG. 2 is a graph showing the effect of inhibition of growth of methanol from DH-1 strain. FIG.
4 is a graph showing the growth curves of DH-1 strain when 2 g / L of methanol and 30% of propane were used as a substrate.
5 is a graph showing the amount of metabolites when 2 g / L of methanol and 30% of propane are used as a substrate.
FIG. 6 is a graph showing the growth curve of DH-1 strain when experiments in which methane and air were supplied at a volume ratio of 3: 7 and methanol was used as a single carbon source at various concentrations.
FIG. 7 is a graph showing the concentration of propanol, acetone, and methanol measured every 6 hours by adding 2 g / L of methanol to a culture solution of DH-1 strain having an initial OD of 10 and supplying 30% of propane.
FIG. 8 is a graph showing the concentration of propanol, acetone, and methanol measured every 3 hours by adding 2 g / L of methanol to a culture solution of DH-1 strain having an initial OD of 4 and supplying 30% of propane.
FIG. 9 is a graph showing the concentration of propanol, acetone, and methanol measured every 2 hours by adding 1 g / L of methanol to a culture solution of DH-1 strain and supplying 30% of propane.
10 is a graph showing the concentration of propanol, acetone, and methanol in a cumulative pattern every 2 hours while feeding 1 g / L of methanol to a culture solution of DH-1 strain and supplying 30% of propane.
11 is a graph showing the production of propanol by supplying 1 g / L of methanol after the methanol is depleted in the culture solution of DH-1 strain.
12 is a graph showing the production pattern of propanol production in an accumulation pattern by supplying 1 g / L of methanol after the methanol is depleted in the culture solution of DH-1 strain.
FIG. 13 is a graph showing the production of propanol by supplying 0.5 g / L of methanol at a rate of 1.5 g / L after the methanol is depleted in the culture solution of the DH-1 strain.
FIG. 14 is a graph showing the production pattern of propanol production in an accumulation pattern by supplying 0.5 g / L of methanol with 1.5 g / L of methanol after the methanol is depleted in the culture solution of the DH-1 strain.
FIG. 15 is a graph showing production of propanol by feeding methanol at a rate of 0.3 g / L of methanol 1.5 g / L after the methanol is depleted in the culture solution of the DH-1 strain.
FIG. 16 is a graph showing the production pattern of propanol produced by supplying 0.3 g / L of methanol at a rate of 1.5 g / L after the methanol is depleted in the culture solution of DH-1 strain.
17 is a graph showing the production amount of propanol by simultaneously supplying methane and propane to the culture solution of DH-1 strain.
Figure 18 is a graph alcaliphilum < / RTI > 20Z strain by simultaneous supply of methane and propane.

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 Preparation of Strain and Experimental Method

1-1. Methylomonas sp. DH-1

1 mL of DH-1 stak (stock, KCTC 13004BP) was inoculated into a 500 mL baffled flask containing 50 mL NMS medium, periodically methane / air was injected and incubated at 30 ° C , And cultured at 230 rpm. 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 KNO ₃ 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

When the initial OD was 10 or more, the OD 4 medium was concentrated by centrifugation, and the cell concentration was adjusted by adding NMS medium.

Propanol production experiments were conducted by feeding a certain amount of methanol as a reducing power to the above-mentioned DH-1 culture broth, evacuating propane: air = 3: 7 gas for 30 minutes, and then stirring at 230 rpm at 30 DEG C in a shaking incubator.

In the case of providing methane as a reducing power, the gas of methane: propane: air = 1.5: 1.5: 7 was evacuated for 30 minutes without adding methanol, and then stirred at 230 rpm at 30 DEG C in a shaking incubator.

1-2. Methylomicrobium alcaliphilum 20Z

The preparation was carried out under the same conditions as the above-mentioned 1-1, and the composition of the medium was as shown in Table 2 below.

Medium composition Amount for 1L NaCl 30 g MgSO ₄ .7H ₂ O 0.2 KNO ₃ 1 g CaCl ₂ .2H ₂ O 0.02 g EDTA 5 mg CuCl ₂ .5H ₂ O 0.1 mg FeSO ₄ .7H ₂ O 2 mg Na ₂ MoO ₄ 0.03 mg ZnSO ₄ .7H ₂ O 0.1 mg MnCl ₂ .4H ₂ O 0.03 mg CoCl ₂ .6H ₂ O 0.2 mg NiCl ₂ .6H ₂ O 0.02 mg H ₃ BO ₃ 0.03 mg KH ₂ PO ₄ 0.28 g Na ₂ HPO ₄ .12 (H ₂ O) 0.6 g 1M NaHCO ₃ 50 mL 1M Na ₂ CO ₃ 5 mL

Example 2 Analysis of Propanol Metabolism from Strain

1, Methylomonas sp. The DH-1 strain is a methane metabolic strain and has the property of converting methane to methanol using MMO. The strain was identified and the production of propanol was analyzed when a propane or methane-propane mixed gas or methanol-propane was fed to the strain.

Prior to the production of propanol, methane and air were fed at a volume ratio of 3: 7 to determine whether the strain could metabolize propane and inhibit the growth of the strain by the produced propanol, and 1 g (1 g / L) of propanol was further fed, and the growth curve of the microorganism was confirmed.

As can be seen in FIG. 2, slight growth inhibition occurred, but it was judged that the degree of growth was not so large, and the subsequent experiments were conducted.

Methane and air were supplied at an initial OD 2 of 3: 7 by volume in a volume ratio of 3: 7 to determine the optimal methanol feed concentration. Methanol (0.5 g / l, 0.5 g / L, 1 g / And the growth curve of the bacteria was confirmed.

As can be seen from FIG. 3, it was judged that there was almost no inhibition effect by methanol even at a methanol concentration of 2 g / L as shown in the figure below.

Example 3 Analysis of Production of Propanol from Strain

2 g / L of methanol and 30% of propane were given as a substrate to confirm whether propanol and methanol were actually supplied to the strain with an initial OD of 10.

As can be seen in FIG. 4, cell concentration decreased over time.

As shown in FIG. 5, 116 mg / L of propanol was produced. However, after 6 hours of incubation, the metabolism was almost unchanged. Thereafter, additional experiments on growth and metabolism inhibitory factors were carried out. As a result, the production of acetone, a decomposition product of isopropanol, was confirmed.

Example 4 Confirmation of Growth Inhibition by Metabolite or Methanol

The cultivation was carried out by supplying only methanol and air at various concentrations (2 g / L, 3 g / L and 4 g / L) in order to check whether the methanol was used as a sole carbon source and inhibited the growth due to the metabolites or methanol . For comparison, experiments were carried out with methane and air at a volume ratio of 3: 7 and compared with these results when methanol was used as the sole carbon source.

As can be seen in FIG. 6, at low cell concentrations of initial OD 0.1 and final OD 1 and below, the total metabolite concentration was not high enough to appear to be unaffected by 4 g / L of methanol. However, -1 < / RTI > strain was inhibited. The effects of these two products were more apparent when the experiment was started at the initial OD 2.

The effect of initial cell concentration on the production of propanol was observed. Methanol was added at 2 g / L to the cells with initial OD of 10 and 4, and propanol, acetone and methanol concentrations were measured every 6 hours with 30% propane feed.

As can be seen from FIG. 7, when the initial OD was 10, no significant change was observed after the propanol concentration was recorded at 0.11 g / L at 6 hours of culture. OD decreased continuously from the beginning of culture and dropped to 6 at 24 hours.

As can be seen from Fig. 8, even when the initial OD was 4, propanol did not increase further after reaching the maximum value at 0.08 g / L at 6 hours of culture. From these results, it seems that the excess methanol inhibition effect has a greater effect than the initial cell concentration.

Since the excess methanol affects not only the cell activity but also the production of propanol, the initial methanol concentration was reduced to 1 g / L and the propanol production experiment was carried out.

As can be seen in FIG. 9, the concentration of propanol was 0.18 g / L, the highest concentration at 4 hours of culture, and was not further increased. The initial methanol concentration was increased about twice as much as that at 2 g / L, and it was once again confirmed that the excess methanol affects the cell activity. After 4 hours of incubation, methanol was depleted. Methanol was depleted and MDH enzyme was decomposed to produce propanol. No more propanol was produced and acetone was accumulated.

Example 5 Characterization of DH-1 Biocatalyst

Propanol and acetone were expressed as molar concentrations, and the characteristics of DH-1 biocatalysts related to methanol consumption and propane conversion were analyzed.

As can be seen from FIG. 10, the total amount of propanol and acetone after the depletion of methanol is partially increased but most of it is due to acetone. Even if methanol is depleted, reducing power can be provided by oxidation of formaldehyde or formic acid accumulated in the culture solution.

When methanol was depleted, additional feed was provided to increase the propanol continuously.

As can be seen in FIG. 11, at 4 hours after methanol was depleted, 1 g / L of methanol was further fed and propanol production was observed. As a result of addition of methanol after 4 hours of incubation, propanol decreased to 0.20 g / L, but the increase was not significant.

As can be seen in FIG. 12, the accumulation of acetone was rather more active, and the accumulation of acetone or the accumulation of formaldehyde, which is a byproduct of methanol, decreased the cell volume, indicating that the effect of addition of methanol was not significant.

Propanol production was observed by varying the concentration of the feed at 0.5 and 0.3 g / L, with the total methanol input being 1.5 g / L.

As can be seen from FIGS. 13 to 16, the influence of the methanol concentration was not significant, and in both cases, propanol was produced at about 0.17 g / L.

Example 6: Confirmation of inhibition of propane decomposition by simultaneous supply of methane and propane

Methane and propane were fed at the same time, so that the reducing power required for the production of propanol was obtained from methane and at the same time, propanol decomposition was prevented by the presence of methanol.

As can be seen from FIG. 17, it can be seen that propanol is produced when methane and propane are supplied at the same time. The final propanol concentration was 0.021 g / L and the yield was very low as compared with methanol when it was fed with reducing power. The acetone concentration was 0.08 g / L. Methane was not supplied with sufficient reducing power, and methanol concentration was low, indicating that the activity of MDH enzyme was difficult to prevent.

Example  7: Methylomicrobium alcaliphilum  Production of propanol using 20Z strain

The DH-1 strain has the advantage of high growth rate and methane consumption rate of 0.25 h ^-1 at non growth rate. However, as shown in the results of propane conversion, the consumption characteristic for methanol is not good and the specificity of MDH enzyme There was a problem of decomposing off propanol produced by acetone.

Methylomonas sp. The same experiment was carried out using Methylomicrobium alcaliphilum 20Z (DSM 19304), which consumes methanol well in the same type 1 strain, in order to examine the possibility of production of propanol from other strains other than DH-1.

First, to examine the effect of initial methanol concentration on the production of propanol, conversion of propane was carried out by varying the methanol concentration of 1 to 4 g / L as shown in Table 3 below.

The final OD was the highest at 3 g / L methanol, but the highest propanol concentration of 1.10 mM (65 mg / L) was recorded at 1 g / L in terms of propanol production and the initial methanol concentration was 1 g / L, Experiments were performed.

Methanol concentration 1 g / L 2 g / L 3 g / L 4 g / L Initial OD (g / L) 3.30 3.24 3.18 3.47 Final OD (g / L) 5.06 5.70 5.98 5.29 Initial propanol (mM) 0.13 0.18 0.15 0.16 The final propanol (mM) 1.10 0.55 0.42 0.38

When the initial methanol concentration was 1 g / L, the production of propanol by propane conversion was experimented.

As can be seen in Fig. 18, propanol continued to increase until the 16th hour of culture, and the final 0.15 g / L was recorded. The figure below shows that propanol continues to be produced after depletion of methanol. As mentioned above, it appears that the formate formed by the decomposition of methanol provided the reducing power required for propane oxidation.

Compared with DH-1, the remarkable point in the production of propanol using 20Z is the relatively low accumulation of acetone. It is suggested that the MDH specificity is different according to the methanogenic strain, and it is suggested that propanol productivity can be kept high if the methanol concentration is maintained optimally through proper methanol supply.

Claims (13)

Methylomonas, Methylomicrobium, Methylobacter, Methylococcus, Methylosphaera, Methylocaldum, Methylcyclohexyl, Methylcyclohexyl, Methylcyclohexyl, But are not limited to, Methyloglobus, Methylosarcina, Methyloprofundus, Methylothermus, Methylohalobius, Methylogelia, Methylogaea, Methylomarinum, Methylovulm, Methylomarinovum, Methylorubrum, Methyloparacoccus, Methylmercuric acid, Methylosinus, Methylocystis, Methylocella, Methylocapsa, Methylofurula, Methylacidiphilum, and Methylcyclohexylmethylsulfate are known in the art. Methylacidimicrobium Propanol for production of compositions comprising one or more cultures of the strain or strains are selected from the group consisting of. The propanol production composition according to claim 1, wherein the strain is methylolmonas DH-1 deposited with the deposit number KCTC 13004BP or Methylomicrobium alcaliphilum 20Z deposited with the deposit number DSM 19304 . The composition of claim 1, wherein the composition further comprises 0.5 to 4.0 g / L of methanol. The composition of claim 1, wherein the composition further comprises a gas comprising 5 to 30% (v / v) propane. 5. The composition for producing propanol according to claim 4, wherein the gas comprises 5 to 30% (v / v) methane. Methylomonas, Methylomicrobium, Methylobacter, Methylococcus, Methylosphaera, Methylocaldum, Methylcyclohexyl, Methylcyclohexyl, Methylcyclohexyl, But are not limited to, Methyloglobus, Methylosarcina, Methyloprofundus, Methylothermus, Methylohalobius, Methylogelia, Methylogaea, Methylomarinum, Methylovulm, Methylomarinovum, Methylorubrum, Methyloparacoccus, Methylmercuric acid, Methylosinus, Methylocystis, Methylocella, Methylocapsa, Methylofurula, Methylacidiphilum, and Methylcyclohexylmethylsulfate are known in the art. Methylacidimicrobium Propanol production method including a conversion step of inducing the conversion reaction from the culture medium of at least one strain or the strain is selected from the group consisting of. 7. The process according to claim 6, wherein the strain is Methylomonas genus DH-1 deposited with accession number KCTC 13004BP or Methylomicrobium alcaliphilum 20Z deposited with accession number DSM 19304. 7. The method for producing propanol according to claim 6, wherein the conversion step is carried out by adding 0.5 to 4.0 g / L of methanol to the culture liquid. The method for producing propanol according to claim 6, wherein the conversion step is carried out by adding 0.5 to 4.0 g / L of methanol to the culture broth when methanol in the culture liquid becomes depleted. 7. The method of claim 6, wherein the converting step is performed in the presence of a gas comprising 5 to 30% (v / v) propane. 11. The method of claim 10, wherein the gas comprises 5 to 30% (v / v) methane. 11. The method of claim 10, wherein the converting is performed by evacuating the gas for 10 to 900 minutes. 7. The process of claim 6, wherein said conversion is carried out at a temperature of 25-35 < 0 > C.

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