KR102201232B1 - Method for producing isovaleric acid and hexanoic acid simultaneously using Megasphaera hexanoica strain - Google Patents

Abstract

The present invention relates to a method for simultaneously producing isovaleric acid and hexanoic acid using a megasparera hexanoica strain. According to the present invention, isovaleric acid and hexanoic acid used as precursors of various bio-compounds by culturing the Megaspaera hexanoica strain under conditions in which yeast extract, fructose and leucine are added. Iksan can be produced with high yield.

Description

Method for producing isovaleric acid and hexanoic acid simultaneously using Megasphaera hexanoica strain}

The present invention relates to a method for simultaneously producing isovaleric acid and hexanoic acid using a megasparera hexanoica strain.

Syngas is generated when organic raw materials are partially oxidized in the presence of high-temperature steam. The main components are CO, H ₂ and CO ₂ , and raw materials include natural gas, coal, oil shale and bitumen sandstone. (tar sand) or the like may be used, and is mainly produced by reacting natural gas or coal with a metal catalyst.

In the technology of converting syngas to polycarbon compounds using a metal catalyst, the purity of the syngas (only CO and H ₂ is used) is very important. If impurities are included in the raw material, the reaction rate is greatly reduced, resulting in lower productivity. In particular, when sulfur compounds are included as impurities, the sulfur compounds irreversibly poison the metal catalyst, it is difficult to reactivate the catalyst, a separate manufacturing process is required to remove the sulfur compounds, and it is difficult to use CO or H2 concentrated. And not economical.

Recently, a technology for producing syngas by vaporizing biomass has been developed.The biomass utilization process can reduce the concentration of sulfur compounds contained in the synthesis gas or use it with less energy than when using coal, and at a lower temperature ( Coal: 1500-1800°C, biomass: 1100°C) is attracting attention because it can be synthesized in a relatively short reaction time.

As a microorganism that can use the biomass-derived synthesis gas, there is acetogen (Acethgens). The microorganisms, defined as Homoacetogen, are anaerobic microorganisms that grow CO or H ₂ as the only energy source through the Wood-Ljungdahl pathway.Acethyl-CoA synthease (or carbon monoxide dehydrogenase) on the metabolic pathway. , Hereinafter, CODH), which is involved in the oxidation of CO and reduction of CO ₂ . If the Wood-Yongdal route is used, synthesis gas can be converted to organic acids without a separate purification or concentration process, and it is performed at room temperature and pressure, and is not affected by sulfur impurities, so research using this is actively conducted worldwide. As bacteria known to ferment syngas to date, Clostridium ljungdahlii (Enzyme and Microbial Technology 14(8), 1992 602-608.; Applied Biochemistry and Biotechnology 45(6), 1994 145-157.; Biochemical Engineering Journal 27, 1992) 2005 110-119.), Clostridium autoethanogenum (Archives of Microbiology 161 (4), 1994345-351.), Clostridium carboxidivorans P7 (Biotechnology and Bioengineering 97 (5), 2007 1080-1086.; Biomass and Bioenergy 23(6), 2002 487-493.) and Clostridium ragsdalei P11 (US Patent No. 7,704,723).

On the other hand, isovaleric acid, which has 5 carbon atoms, is an organic acid used not only as a fragrance and pharmaceutical composition, but also for the purpose of various pathological studies.Valeriana, Origanum majorana, Hop, and animal lipids It can be obtained from, but there is a problem that the profitability is very low and it is difficult to obtain oil. Republic of Korea Patent Registration No. 0372218 relates to a method of culturing 55E of the genus Manheimia and an electrostrain under anaerobic or aerobic conditions to produce organic acids, and producing succinic acid, lactic acid, and formic acid under anaerobic conditions saturated with carbon dioxide. U.S. Patent Publication No. 2008/0311640 uses various microorganisms to produce hydrogen and organic acids, but conventional inventions as described above have 2 carbon atoms such as acetate, ethanol, butyrate, and butanol through the acethyl-CoA pathway. And only to produce four organic acids. Therefore, it is the time when a study on a strain in which the range of biological compound production has been expanded to produce organic acids having 2 to 5 carbon atoms, in particular, isovaleric acid, is required.

In addition, hexanoic acid is a linear fatty acid having 6 carbons and is used to make perfumes, medicines, lubricating greases, rubbers, and dyes, and can be converted to hexanol through a simple catalytic reaction. Since hexanol can be used as gasoline, diesel, or jet fuel through esterification, it is necessary to develop a technology to produce hexanoic acid as a precursor of biofuel. The production of hexanoic acid using microbial fermentation is one of the bacteria with the most research on the production of hexanoic acid using Clostridium kluyveri, an anaerobic bacterium published in 1942. The above strains can produce H2, butyric acid, and hexanoic acid using ethanol, acetate or succinate. As the acetic acid becomes excessive, hexanoic acid is mainly produced. In this relationship, it has been found that butyric acid is used as an intermediate material in the synthesis of hexanoic acid. Rodick et al. reported that in order to increase the production of hexanoic acid, the production yield of hexanoic acid was increased by culturing the fixed megaspera elsdenii in an incubator containing amberlite IRA-400, which is an ion exchange resin. It has been reported that an increase of 30% from L to 19 g/L has been reported, and in the conventional Korean Patent No. 10-1316732, a method of increasing the production of hexanoic acid through extraction and fermentation using Megaspera Elsdeni NCIMB 702410 was disclosed. However, due to insufficient production, it is insufficient to be applied to the production process of biofuels.

In addition, conventional Korean Patent No. 10-1745084 discloses a method of producing hexanoic acid using a megasparera hexanoica strain, but until now, isovaleric acid and hexanoic acid are simultaneously produced using the strain. There is no reported way to do this.

Under the above-described technical background, the present inventors are studying the production method of isovaleric acid, when using the leucine metabolic pathway of the megaspaera hexanoica strain known as a strain producing hexanoic acid, isovaleric acid and hexanoic acid are used. It was confirmed that it can be produced simultaneously in high yield, and the present invention was completed.

Roddick, F.A. and M.L. Britz, Journal of Chemical Technology and Biotechnology, 1997 69(3) 383-391.

It is an object of the present invention to provide a method for simultaneously producing hexanoic acid isovaleric acid using a megaspaera hexanoica strain and a culture solution for culturing a megasparera hexanoic acid strain.

In order to solve the above problems, the present invention is cultured by inoculating the Megasphaera hexanoica strain (KCCM11835P) in a culture solution to which yeast extract, fructose and leucine are added. It provides a simultaneous production method of isovaleric acid and hexanoic acid comprising a;

The present invention also provides a culture solution for cultivating a megasphaera hexanoica strain (KCCM11835P) containing yeast extract, fructose and leucine.

According to the present invention, the culture medium may further contain sodium acetate.

In this case, the concentration of the yeast extract may be 10 to 20 g/L.

In addition, the concentration of fructose may be 50 to 150 mM.

In addition, the concentration of the leucine may be 50 to 150 mM.

In addition, the concentration of sodium acetate may be 100 to 200 mM.

According to the present invention, the culture solution may be a culture solution containing K ₂ HPO ₄ , cysteine-HCl·H ₂ 0, a salt solution, or a mixture thereof.

According to the present invention, a mixed solvent of oleyl alcohol and alamin 336 may be added as an extraction solvent to the culture solution.

According to the present invention, isovaleric acid and hexanoic acid used as precursors of various bio-compounds by culturing the Megaspaera hexanoica strain under conditions in which yeast extract, fructose and leucine are added. Iksan can be produced with high yield.

1 shows the culture conditions of the Mega Spaera hexanoika strain according to Example 1 of the present invention.
Figure 2 shows the growth curve of the Mega Spaera hexanoica strain according to conditions according to Example 1 of the present invention.
3 shows the result of comparing the organic acid production amount by conditions according to Example 1 of the present invention.
Figure 4 shows the culture conditions of the Mega Spaera hexanoika strain according to Example 2 of the present invention.
Figure 5 shows the growth curve of the Mega Spaera hexanoica strain according to Example 2 of the present invention.
6 shows the result of comparing the organic acid production amount by conditions according to Example 2 of the present invention.
7 is a view showing the concentration of variables (yeast extract, leucine, sodium acetate) for applying the response surface analysis method according to Example 3-1 of the present invention and the production amount of isovaleric acid and hexanoic acid accordingly.
8 is a result showing the effect of each variable through a statistical method on the production concentrations of isovaleric acid and hexanoic acid obtained through the response surface analysis method according to Example 3-1 of the present invention.
FIG. 9 shows the results of digitizing the results according to FIG. 8.
10 is a view showing the concentration of variables (yeast extract, sodium acetate) for applying the response surface analysis method according to Example 3-2 of the present invention and the production amount of isovaleric acid and hexanoic acid accordingly.
11 is a result showing the effect of each variable through a statistical method on the production concentrations of isovaleric acid and hexanoic acid obtained through the response surface analysis method according to Example 3-2 of the present invention.
12 shows the growth curve of the strain when culturing the Mega Spaera hexanoica strain by applying the optimal conditions derived through the response surface analysis method.
13 shows the production amount of isovaleric acid and hexanoic acid according to the culture time when culturing the Megaspaera hexanoica strain by applying the optimum conditions derived through the response surface analysis method.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

In the present invention, it is intended to provide a method for simultaneously producing isovaleric acid and hexanoic acid in high yield by using the leucine metabolic pathway of the megaspaera hexanoic acid strain known as a strain producing hexanoic acid.

To this end, the present invention comprises the steps of inoculating and culturing the Megasphaera hexanoica strain (KCCM11835P) in a culture solution to which yeast extract, fructose and leucine are added; It provides a simultaneous production method of isovaleric acid and hexanoic acid containing.

In this case, the culture medium preferably contains K ₂ HPO ₄ , cysteine-HCl·H ₂ 0, a salt solution or a mixture thereof as a component of the basic medium.

In addition, in order to further improve the yield of isovaleric acid and hexanoic acid, the culture medium is sodium acetate in addition to the yeast extract, fructose and leucine added to the basic medium. It is preferable to further include ).

As can be seen from the results of the following examples, when yeast extract, fructose, and leucine are added to the above-described basic medium component, or additional sodium acetate is further added, the Megaspaera hexanoica strain according to the present invention is efficiently It has been shown that both isovaleric acid and hexanoic acid are produced best at the same time as they grow. Yeast extract, fructose, leucine, and sodium acetate are factors that have an important influence on the production of isovaleric acid and hexanoic acid. It was found that the production of isovaleric acid and hexanoic acid increased through the action.

In addition, the optimal concentration conditions of the factors for improving the production yield of isovaleric acid and hexanoic acid were derived through reaction surface analysis, and accordingly, the concentration of the yeast extract is preferably 10 to 20 g/L, and the The concentration of fructose is preferably 50 to 150 mM, the concentration of leucine is preferably 50 to 150 mM, and the concentration of sodium acetate is preferably 100 to 200 mM.

In addition, in order to maximize the production of isovaleric acid and hexanoic acid, it is most preferable that the concentration of the yeast extract is 18.83 g/L and the concentration of sodium acetate is 151.87 mM under the condition that the concentration of leucine is 150 mM.

In addition, in order to further improve the production amount of isovaleric acid and hexanoic acid by reducing the concentration of organic acids in the medium to reduce toxicity to microorganisms, oleyl alcohol and oleyl alcohol as an extraction solvent in the culture medium when culturing the Megaspaera hexanoic acid strain A mixed solvent of alamin 336 can be added.

In this case, the volume ratio of the oleyl alcohol and the alamin 336 is preferably 9:1 to 6:3. If the amount of the alarmin 336 is too large outside the above range, toxicity of microorganisms may be induced and growth may be inhibited. In addition, the volume ratio of the culture solution and the mixed solvent is preferably 1:0.5 to 1:2.

The present invention also provides a culture solution for cultivating a megasphaera hexanoica strain (KCCM11835P) containing yeast extract, fructose and leucine.

In relation to the culture medium for culturing the Megasphaera hexanoica strain (KCCM11835P), the Megasphaera hexanoica strain (KCCM11835P) was cultured to simultaneously produce isovaleric acid and hexanoic acid. The description of the method and the duplicated content is omitted in order to avoid excessive complexity of the present specification due to the duplicated description.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Mega Spaera Hexanoica Isolation of strains

In order to isolate the microorganisms that produce hexanoic acid, small intestines containing gastric juice were purchased at a cow market located in Majang-dong, Seoul, Korea and treated anaerobicly. The anaerobicly treated gastric juice was inoculated into Reinforced Clostridia medium (difco) containing 5 g/L hexanoic acid, and cultured for 3 days at 37°C. This enrichment culture solution was subcultured several times in the same medium. The enriched culture medium was plated on Reinforced Clostridia medium (difco) medium to form colonies.

Each of the selected colonies was inoculated into a Reinforced Clostridia medium (difco) liquid medium, incubated at 37° C. for 3 days, and then hexanoic acid production was measured using gas chromatography (GC). As a result, microorganisms that produce the largest amount of hexanoic acid were isolated. The separated microorganisms were deposited on April 28, 2016 at the Korea Microbial Conservation Center of the Republic of Korea, which is a microbial deposit organization under the Budapest Treaty, and was given the deposit number KCCM11835P.

Example 1. In a basic medium containing yeast extract Fructose , Leucine , Sodium acetate Depending on whether or not added Mega Spaera Hexanoic Growth and organic acid production trend analysis

Megaspaera In order to investigate the effect of the composition of the medium on the growth of the strain, the types and amounts of organic acids produced in the culture of hexanoica , fructose (F) and leucine (L) were added to the basic medium containing yeast extract. And the experiment was carried out by changing the presence or absence of sodium acetate (A). As shown in FIG. 1 below, a total of 8 types of media were prepared depending on whether or not pchuktose, leucine, and sodium acetate were added, and the amount of the media was all 20 mL, and 400 μl (2%) of Megaspaera hexanoica strain Was inoculated. Optical density (600 nm) was measured at intervals of 10 to 15 hours and sampling was performed until entering the extinction phase, and sampling was performed for a total of 72 hours, and the production and consumption of organic acids for each component were calculated in units of mmol/L (mM). .

Figure 2 shows the growth curve of the Mega Spaera hexanoica strain according to the above-described conditions, Figure 3 shows the result of comparing the organic acid production by conditions. 2 and 3, in the case of a basic medium containing only yeast extract and a medium containing only leucine (YL) or sodium acetate (YA) to the basic medium, megasparera hexanoica does not grow. Therefore, it was found that the target substances, isovaleric acid and hexanoic acid were not detected at all. In addition, when fructose and leucine are added to the basic medium containing yeast extract (YFL), or when fructose, leucine, and sodium acetate (YFLA) are added, megaspaera hexanoica is grown and isovaleric acid and hexano It was confirmed that Iksan was produced in high yield at the same time. In particular, in the case of YFLA, the concentrations of the produced isovaleric acid and hexanoic acid were 20.5 mM and 19.6 mM, respectively, showing the maximum production. As a result, it was confirmed that for the production of isovaleric acid, the addition of leucine must be accompanied under conditions in which megasparera hexanoica can grow well. When fructose and sodium acetate are added together with leucine, isovaleric acid It was confirmed that productivity was further improved and hexanoic acid could be simultaneously produced in high yield.

Example 2. Mega Spaera Hexanoica Identification of the components that have the greatest influence on the growth of strains and organic acid production

Based on the results derived in Example 1, using a medium (YFLA) containing all of fructose, leucine, and sodium acetate in a basic medium containing yeast extract, the growth of the strain and the production of organic acids including isovaleric acid An experiment was conducted to determine what the main ingredient has the greatest influence. Specifically, as shown in FIG. 3 below, a YFLA medium (control), a medium in which only the concentration of the yeast extract is added twice in the YFLA medium (A), a medium in which only the concentration of sodium acetate is added in the YFLA medium twice (B), a medium in which the concentrations of yeast extract, sodium acetate, fructose, and leucine in the YFLA medium were all added twice (C), a medium in which only the concentration of fructose in the YFLA medium was added twice (D), the In YFLA medium, a total of six types of medium such as medium (E) in which only the concentration of leucine was added twice was prepared, and the amount of the medium was all 20 mL, and 400 µl (2%) of the Megaspaera hexanoica strain was inoculated. Optical density (600 nm) was measured at intervals of 5 to 8 hours and sampling was performed until entering the extinction phase, and sampling was performed for a total of 72 hours, and the production and consumption of organic acids for each component were calculated in units of mmol/L (mM). .

Figure 5 shows the growth curve of the Mega Spaera hexanoica strain according to the above-described conditions, Figure 6 shows the result of comparing the organic acid production by conditions.

As shown in FIGS. 5 and 6, it was found that the megasparera hexanoica strain grows best in the medium (A, C) to which the yeast extract was further added in the YFLA medium. In addition, the production of isovaleric acid showed improved results in all other conditions compared to the YFLA medium, and in particular, a medium containing more leucine (E), leucine, yeast extract, fructose, and sodium acetate were all added further (C). It was confirmed that the production of isovaleric acid was significantly improved (45 mM for C, 34 mM for E), and the production amount of isovaleric acid in the medium (C) in which all of leucine, yeast extract, fructose, and sodium acetate were all further added. Confirmed that this is the highest. As a result, it was confirmed that yeast extract had the greatest effect on the growth of Megasparera hexanoica strain, and leucine had the greatest effect on the production of isovaleric acid. Yeast extract, fructose, and acetic acid along with leucine It was confirmed that the productivity of isovaleric acid further improved when sodium was added.

Example 3. yeast extract, Isovaleric acid And Sodium acetate Derived through response surface analysis as a variable Isovaleric acid Hexanoic acid Maximum production conditions

(One) Example 3-1

As a variable that affects the growth of the strain and the production of isovaleric acid and hexanoic acid when culturing the Megaspera hexanoica strain of the present invention, yeast extract, leucine, and acetate are selected as a response surface analysis method [Response Surface Methodology (RSM). , Software Design-Expert (Version 7.1.5, Sate-Ease Inc., Minneapolis, USA) was analyzed for optimal conditions. At this time, the concentration range of the yeast extract was set to 5-15 g/L, and the concentration range of leucine and sodium acetate was set to 50-150 mM.

FIG. 7 is a diagram showing the concentration of variables (yeast extract, leucine, sodium acetate) for applying the response surface analysis method according to Example 3-1 and the production amount of isovaleric acid and hexanoic acid accordingly, and FIG. 8 is the reaction It is a result showing the effect of each variable through a statistical method on the production concentration of isovaleric acid and hexanoic acid obtained through the surface analysis method, and FIG. 9 is a numerical result of the result according to FIG. 8.

As a result of the measurement, the production of isovaleric acid and hexanoic acid tended to increase as the concentrations of the above variables, namely yeast extract, leucine, and sodium acetate, increased, and when all of the above variables corresponded to the maximum concentration in the set range The production of isovaleric acid and hexanoic acid were found to be the highest. Through this, it was confirmed that the three variations, namely yeast extract, leucine, and sodium acetate, exert a synergistic effect with each other, and the productivity of isovaleric acid and hexanoic acid further improved as their concentrations increased.

(2) Example 3-2

Based on the observation that the organic acid production tends to increase as the concentration increases in the concentration range of the variables set in Example 3-1, in order to derive the optimum values of the variables, the concentration of leucine is 150 mM (for leucine The maximum dissolution concentration at 37° C., which is the hexanoica culture temperature), was fixed, and the concentration range of the yeast extract was set to 10-20 g/L, and the concentration range of sodium acetate was set to 100-200 mM. The reaction surface analysis experiment was conducted according to the same method as in 1.

10 is a view showing the concentration of variables (yeast extract, sodium acetate) for applying the response surface analysis method according to Example 3-2 and the production amount of isovaleric acid and hexanoic acid accordingly, and FIG. 11 is Example 3- This is a result showing the effect of each variable through a statistical method on the production concentrations of isovaleric acid and hexanoic acid obtained through the response surface analysis method according to 2.

As a result of the measurement, it was confirmed that the concentration of isovaleric acid produced under the conditions of 19.81 g/L of yeast extract, 151.87 mM of sodium acetate, and 150 mM of leucine was 63.24 mM, showing the maximum yield. However, as a result of considering the productivity of hexanoic acid together, the maximum production was shown as the concentration of hexanoic acid produced under the conditions of 18.83 g/L of yeast extract, 151.87 mM of sodium acetate, and 150 mM of leucine. It was confirmed that the concentration of isovaleric acid also showed a remarkably improved productivity to 62.9895 mM.

Example 4. Optimal condition verification experiment derived through response surface analysis method

Growth of the strain when culturing the Megaspaera hexanoica strain under the optimal conditions (yeast extract 18.83 g/L, sodium acetate concentration 151.87 mM, and leucine concentration 150 mM) derived according to Example 3-2. , An experiment was conducted to confirm whether the production of isovaleric acid and hexanoic acid actually improved.

12 shows the growth curve of the strain when culturing the Mega Spaera hexanoica strain by applying the optimum conditions derived through the response surface analysis method according to Example 3-2, and FIG. 13 is a diagram according to Example 3-2. It shows the production amount of isovaleric acid and hexanoic acid according to the cultivation time when culturing the Megasparera hexanoica strain by applying the optimal conditions derived through the response surface analysis method.

As a result of the measurement, the growth of the strain proceeds well when the Megasparera hexanoica strain is cultured under the optimal conditions derived through the response surface analysis method according to Example 3-2, and the production amount of isovaleric acid is 63.5 mM (6.5 g/L). ), it was confirmed that the result was almost consistent with the result according to the reaction surface analysis method, and the production amount of hexanoic acid was 50.9 mM (5.9 g/L), which was more improved than the result according to the reaction surface analysis method.

Korea Microorganism Conservation Center (overseas) KCCM11835P 20160428

Claims (15)

Isovaleric acid and hexanoica comprising: inoculating and culturing the Megasphaera hexanoica strain (KCCM11835P) in a culture solution to which yeast extract, fructose, and leucine are added. The simultaneous production method of Noiksan. The method of claim 1,
The culture medium is a method for simultaneously producing isovaleric acid and hexanoic acid further comprising sodium acetate. The method of claim 2,
The concentration of the yeast extract is 10 to 20 g / L of the simultaneous production method of isovaleric acid and hexanoic acid. The method of claim 2,
A method for simultaneously producing isovaleric acid and hexanoic acid in which the concentration of fructose is 50 to 150 mM. The method of claim 2,
The method for simultaneously producing isovaleric acid and hexanoic acid in which the concentration of leucine is 50 to 150 mM. The method of claim 2,
The method for simultaneously producing isovaleric acid and hexanoic acid in which the concentration of sodium acetate is 100 to 200 mM. The method of claim 1,
The culture solution is a method for simultaneous production of isovaleric acid and hexanoic acid comprising K ₂ HPO ₄ , cysteine-HCl·H ₂ 0, a salt solution or a mixture thereof. The method of claim 1,
Simultaneous production method of isovaleric acid and hexanoic acid, characterized in that a mixed solvent of oleyl alcohol and alamin 336 is added as an extraction solvent to the culture solution. Yeast extract (Yeast extract), fructose (fructose) and leucine (Leucine) containing megasphaera hexanoica (Megasphaera hexanoica) strain (KCCM11835P) culture medium for culture. The method of claim 9,
The culture medium is a medium for culturing the Megasphaera hexanoica strain (KCCM11835P), characterized in that it further contains sodium acetate. The method of claim 10,
The concentration of the yeast extract is a megasphaera hexanoica (Megasphaera hexanoica) strain (KCCM11835P) culture medium, characterized in that 10 to 20 g / L. The method of claim 10,
The concentration of fructose is 50 to 150 mM Megasphaera hexanoica (Megasphaera hexanoica) strain (KCCM11835P) culture medium for culturing. The method of claim 10,
The concentration of the leucine is 50 to 150 mM Megasphaera hexanoica (Megasphaera hexanoica) strain (KCCM11835P), characterized in that the culture medium for culture. The method of claim 10,
The concentration of the sodium acetate is 100 to 200 mM Megasphaera hexanoica (Megasphaera hexanoica) strain (KCCM11835P), characterized in that the culture medium for culture. The method of claim 9,
The culture medium is K ₂ HPO ₄ , cysteine-HCl·H ₂ 0, a salt solution or a mixture thereof, characterized in that it further comprises a megasphaera hexanoica strain (KCCM11835P) culture medium for culture.

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