JP6692232B2 - 3HB manufacturing method - Google Patents

Description

  The present invention relates to a method for producing 3-hydroxybutyric acid (3HB).

  3-Hydroxybutyric acid and its salts (hereinafter referred to as 3HB, and when referred to as 3HB, especially refer to its monomer) have a high biocompatibility because they are substances originally present in the human body, and sugar It is expected as an epoch-making energy source to replace quality. Further, it has been found that 3HB has not only a role as a simple energy source but also a function as a signal transduction substance that affects the expression of various genes and the activity of proteins. 3HB is known to improve cognitive function and long-term persistent memory by inhibiting histone deacetylase by, for example, a gene expression regulatory action, and its effectiveness in the prevention of Alzheimer has been confirmed. For example, 3HB produced by ingestion of medium-chain fatty acid contained in coconut oil and metabolism in the body has an effect of improving symptoms in patients with Alzheimer's disease and diabetes who cannot utilize sugar efficiently in the brain and body. It has been known. In addition, 3HB is converted into energy faster than carbohydrates in the body, and has the effect of suppressing the absorption of fats and sugars into cells. Can be expected.

  In addition to these uses, 3HB is known to be used as a raw material for biodegradable resins, and its utility value is increasing in industrial use.

  Such 3HB can be obtained by causing various microorganisms to produce poly-3-hydroxybutyric acid (hereinafter referred to as PHB) and then degrading the obtained PHB with an enzyme or the like (Patent Document 1). Further, it has been found that Halomonas bacterium as such a microorganism accumulates PHB under aerobic conditions and secretes and produces 3HB produced by decomposing PHB by shifting to anaerobic conditions in the culture medium. (Patent Document 2).

Japanese Patent Publication No. 2005-521718

Yutaka Tokiwa et al. J Biotechnol. (2007) 132, 264-72.

  It is difficult to recover 3HB or PHB derived from biological fermentation as a pure product from the culture solution, and in reality, it is recovered in a form containing various impurities. When the 3HB thus recovered is used for application, it is necessary to further purify it to increase its purity. However, in reality, 3HB having low purity (for example, 3HB derived from biological fermentation currently in circulation has a purity of about 70%) is used as it is, and 3HB having high purity is not yet stably supplied.

  In order to stably supply such highly pure 3HB, an efficient method for producing 3HB using a culture solution containing 3HB derived from biological fermentation is required.

  Therefore, in view of the above situation, it is an object of the present invention to provide a method for producing 3HB that can efficiently purify and recover highly pure 3HB from a culture solution containing biologically-derived 3HB.

The characteristic configuration of the method for producing 3HB of the present invention for achieving the above object is as follows.
A fermentation step of obtaining a fermented liquid in which 3HB is fermented and produced from a 3-hydroxybutyric acid (3HB) -producing bacterium;
From the fermentation liquor obtained in the fermentation step, a preliminary concentration step of distilling water off,
An extraction step of extracting a 3HB-containing solution containing 3HB from the fermentation liquid with an organic solvent,
A concentration step of distilling the organic solvent from the 3HB-containing solution extracted in the extraction step to obtain a 3HB concentrated solution;
A crystallization solvent containing at least one selected from ethyl acetate, diethyl ether, and t-butyl methyl ether as a main component was added to the 3HB concentrated liquid obtained in the concentration step, and 3HB crystals were obtained from the crystallization solvent in which 3HB was dissolved. A crystallization step for precipitation,
There is a point to do.

  It is considered that 3HB obtained by the fermentation process has an extremely high optical purity (close to 100%). By performing an extraction step of extracting this with an organic solvent, a 3HB-containing solution having high optical purity can be obtained from the fermentation broth.

  Here, in order to separate and recover 3HB from the fermentation broth, an extraction step of performing extraction with an organic solvent through a preconcentration step of distilling off water is performed, and 3HB is efficiently converted into a layer of the organic solvent with a small amount of the organic solvent. Can be transferred. By distilling off the organic solvent and concentrating in the concentration step, a 3HB concentrated solution containing 3HB at a high concentration is obtained. By performing a crystallization step using this 3HB concentrated solution, 3HB is crystallized very efficiently. It is possible to obtain highly pure 3HB.

By performing the crystallization step of precipitating 3HB in this way, 3HB can be recrystallized from the 3HB-containing solution just extracted from the fermentation broth to obtain 3HB with higher purity. Specifically, by performing a crystallization step after the concentration step, a recrystallization operation of 3HB can be performed to obtain highly pure 3HB.
Here, as a result of earnest research, the present inventors have found that when a crystallization solvent containing at least one selected from ethyl acetate, diethyl ether, and t-butyl methyl ether as a main component is used, the total amount of the fermentation liquid is increased. The inventors have found experimentally that 3HB can be crystallized very efficiently even if is used as a starting material as it is, and completed the present invention.

  That is, when any one of ethyl acetate, diethyl ether, and t-butyl methyl ether was used as the crystallization solvent, the gradient of the solubility curve of 3HB (temperature dependence of solubility) was large (difference in solubility depending on temperature). It is clear that 3HB can be easily crystallized by recrystallization and impurities can be removed. As a result, highly pure 3HB can be efficiently produced.

  Further, the 3HB-producing bacterium is preferably a 3HB-producing Halomonas bacterium, and in this case, the fermentation step comprises an aerobic fermentation step of aerobically fermenting the Halomonas bacterium and a polyhydroxybutyric acid by an aerobic fermentation step. An anaerobic fermentation step of anaerobically fermenting the Halomonas bacterium having accumulated (PHB) can be included.

  That is, when a 3HB-producing Halomonas bacterium is used as the 3HB-producing bacterium, 3HB can be produced with high productivity. In addition, the Halomonas bacterium performs aerobic fermentation step of aerobically fermenting and anaerobic fermentation step of anaerobic fermentation to remove polyhydroxybutyric acid (PHB) accumulated in the body by the aerobic fermentation step outside the body (in the fermented solution). Can be discharged. Therefore, by using Halomonas, 3HB can be relatively easily transferred to a form in which reaction is easy, and further, 3HB can be efficiently produced.

  Moreover, you may perform the said extraction process after performing the filtration process which filters the fermentation liquid obtained by the said fermentation process.

  The fermentation broth obtained in the fermentation step contains 3HB-producing bacterial cells in addition to 3HB. Since such cells do not migrate into the 3HB-containing solution when the extraction step is performed, it is considered that they exist as contaminants and cause a decrease in purity. Further, the extraction solvent is unnecessarily increased and used. Therefore, if ultrafiltration is performed in advance, the recrystallization can be performed in a state where the bacterial cells and the protein and other large molecular weight contaminants are not contained, so that the recrystallization purity can be further improved. Conceivable.

  Further, the extraction step may be performed after the pH adjustment step of adjusting the pH of the fermentation broth obtained in the fermentation step to an acidity of 1 to 6.

  Since 3HB contained in the fermentation liquor is acidic, a part of 3HB is dissolved as a salt in the fermentation liquor which is close to neutral. Such a form of 3HB is difficult to migrate into the 3HB-containing solution, and there is a concern that the extraction efficiency of 3HB may be reduced. Therefore, if a pH adjusting step of adjusting the pH of the fermentation liquor to pH 1 to 6 is performed, 3HB can be present in the fermentation liquor as a free acid, and 3HB easily migrates into the extracted 3HB-containing solution. It is considered that this contributes to the improvement of the recovery efficiency of 3HB.

  It is also possible to perform an activated carbon treatment step in which the 3HB-containing solution or the 3HB concentrated solution is brought into contact with activated carbon.

  By performing the above-mentioned filtration step, a contaminant component having a relatively large molecular weight can be removed, but a coloring component for 3HB having a relatively small molecular weight cannot be removed. Although such a coloring component has no great influence on the actual purity of 3HB, 3HB having a high degree of purification is a white crystal, while a brown to light brown colored crystal is obtained. It is inferior in appearance as a 3HB product. Therefore, if such a coloring component is removed by activated carbon, 3HB obtained in the crystallization step can be obtained as white crystals and can be provided as high quality 3HB.

  As the organic solvent, a solvent containing at least one selected from 1-butanol, 1-pentanol, ethyl acetate, isoamyl alcohol, and methyl ethyl ketone as a main component can be used.

  As the organic solvent, one having a high solubility in 3HB can be used regardless of the slope of the solubility curve. As such an organic solvent, 1-butanol, 1-pentanol, ethyl acetate, isoamyl alcohol, or methyl ethyl ketone can be used. Those having at least one selected from the following as a main component are preferable because of high solubility in 3HB.

  In the crystallization step, 3HB seed crystals may be added to a crystallization solvent in which 3HB is dissolved and cooled with stirring.

  Crystallization by cooling with stirring allows crystallization to start from a uniform solution without temperature unevenness, so crystals with an extremely narrow crystallization start temperature range can be taken out, which contributes to increasing the purity of 3HB. .. Further, when a seed crystal is used, crystallization can be started from a supercooled 3HB solution at an appropriate crystallization starting temperature, which contributes to providing 3HB with more stable quality.

  Therefore, it has become possible to efficiently purify and recover 3HB having a high purity from a culture solution containing 3HB derived from biological fermentation.

  Below, the manufacturing method of 3HB concerning embodiment of this invention is demonstrated. The preferred embodiments will be described below, but these embodiments are described for more specifically illustrating the present invention, and various modifications are possible without departing from the spirit of the present invention. However, the present invention is not limited to the following description.

The method for manufacturing 3HB according to the embodiment of the present invention is
A fermentation step of obtaining a fermented liquid in which 3HB is fermented and produced from a 3-hydroxybutyric acid (3HB) -producing bacterium;
From the fermentation liquor obtained in the fermentation step, a preliminary concentration step of distilling water off,
An extraction step of extracting a 3HB-containing solution containing 3HB from the fermentation liquid with an organic solvent,
A concentration step of distilling the organic solvent from the 3HB-containing solution extracted in the extraction step to obtain a 3HB concentrated solution;
A crystallization solvent containing at least one selected from ethyl acetate, diethyl ether, and t-butyl methyl ether as a main component was added to the 3HB concentrated liquid obtained in the concentration step, and 3HB crystals were obtained from the crystallization solvent in which 3HB was dissolved. The crystallization step of precipitating is performed.
Here, a 3HB-producing Halomonas bacterium is used as a 3HB-producing bacterium, and as a fermentation step, an aerobic fermentation step of aerobically fermenting the Halomonas bacterium, and polyhydroxybutyric acid (PHB) is accumulated by the aerobic fermentation step. And an anaerobic fermentation step of anaerobically fermenting Halomonas bacteria.
Further, prior to the extraction step, a filtration step of performing ultrafiltration on the fermentation solution obtained in the fermentation step is performed, and a pH adjustment step of adjusting the pH of the fermentation solution to an acidity of 1 to 6 is performed.
Further, an activated carbon treatment step of bringing the 3HB concentrated liquid into contact with activated carbon is performed.
As the organic solvent, a solvent containing at least one selected from 1-butanol, 1-pentanol, ethyl acetate, isoamyl alcohol, and methyl ethyl ketone as a main component is used. A seed crystal of 3HB can be added to the solvent and cooled with stirring. The solvent used in the crystallization step may be the same as the solvent used in the extraction step.

[Fermentation process]
The fermentation step is to obtain a fermented liquid in which 3HB is fermented and produced from 3HB-producing bacteria. Specifically, 3HB-producing Halomonas bacterium is added to a fermentation container containing a culture solution containing a sugar, and first, an aerobic fermentation step is performed while stirring and aerating. As a result, the carbohydrate can be assimilated by the Halomonas bacterium to produce PHB, and PHB is accumulated in the Halomonas bacterium. Next, when the sugars are almost completely consumed, aeration in the fermentation container is stopped and the anaerobic fermentation process is performed. As a result, the Halomonas bacterium decomposes and consumes PHB accumulated in the body and releases 3HB into the fermentation broth. As a result, a fermentation broth containing 3HB and bacterial cells can be obtained.

[Filtration process]
In the filtration step, bacterial components and contaminants such as high molecular weight proteins in the obtained fermentation broth are removed by filtration. Specifically, the obtained fermented liquor is filtered through an ultrafiltration membrane (UF membrane). Then, contaminants having a large molecular weight such as bacterial cells and proteins can be easily removed, and a fermentation broth containing mainly 3HB can be obtained. The fermented liquid obtained here was a fermented liquid containing 4.1% by mass of 3HB. In this embodiment, the concentration of 3HB is determined by a chromatographic method (the same applies hereinafter).

[PH adjusting step]
The pH adjustment step adjusts the pH of the fermentation liquor to pH 1 to 6 so that 3HB in the fermentation liquor can be easily transferred to the 3HB-containing solution in the form of free acid. Specifically, in the pH adjustment step, 1.4 kg of 96 mass% sulfuric acid was added to 64.3 kg of the fermentation solution obtained in the filtration step to adjust the pH to 3.0.
Here, various acids can be used in the pH adjusting step, but it is preferable to use an inorganic acid that is less likely to adversely affect the subsequent purification step. Moreover, the pH may be adjusted to an acidic region of 1 to 6, but it is preferable to adjust the pH by adding an acid until the pH becomes about 3 at which 3HB exists almost as a free acid. From the viewpoint of.

[Pre-concentration step]
The preconcentration step is a step of distilling off water as a solvent in the fermentation liquor to concentrate the fermentation liquor. Specifically, water was distilled off from the fermented liquid 65.7 kg after the pH adjustment by an evaporator for concentration. As a result, 12.4 kg of a 3HB-containing solution containing 20.8 mass% of 3-hydroxybutyric acid was obtained.

[Activated carbon treatment process]
In the activated carbon treatment step, powdery or granulated activated carbon is brought into contact with the 3HB-containing solution to remove impurities such as coloring substances. Here, the activated carbon treatment step is preferably performed for decolorizing the obtained 3HB to improve the quality, and can be omitted in the sense of avoiding a decrease in yield and an increase in man-hours.

[Extraction process]
Next, using 2 kg of the resulting 3HB-containing solution, the extraction step of extracting with the same weight of various organic solvents 1-butanol, 1-pentanol, ethyl acetate, isoamyl alcohol, or methyl ethyl ketone was repeated 3 times, and 3HB was contained. A solution was obtained. When the extraction process was repeated three times with each organic solvent, the yield of recovered 3HB was as shown in Table 1. As the organic solvent, other general-purpose solvents can be used, but 1-butanol, 1-pentanol, ethyl acetate, isoamyl alcohol, or methyl ethyl ketone is preferably used because of its high solubility in 3HB.

[Concentration process]
In the concentration step, the organic solvent is distilled off from the obtained 3HB-containing liquid by an evaporator. In this concentration step, a 3HB concentrated liquid containing 75% by mass of 3HB was obtained. The concentration step is preferably performed until the 3HB concentration of the 3HB concentrated liquid becomes about 50% or more and 90% or less. More preferably, the lower limit of concentration is 60% or more, or 70% or more, and the upper limit of concentration is 85% or less, and it is particularly preferable to concentrate to about 70% or more and 85% or less.

[Activated carbon treatment process]
Here, an activated carbon treatment step of removing impurities such as a coloring substance from a concentrated liquid using powdered or granulated activated carbon may be carried out. The activated carbon treatment step is preferably performed on either the 3HB-containing solution or the 3HB concentrated solution, but it can be omitted or performed on both.

[Crystallization process]
Subsequently, 60 g of ethyl acetate as a crystallization solvent was added to 200 g of a concentrated liquid containing 75% by mass of 3HB (extracted using 1-butanol as an organic solvent), and 3 HB was homogeneous at 40 ° C. A different solution was obtained. This 3HB solution was cooled to a saturated concentration of 3HB at 20 to 30 ° C. while being stirred and mixed in a crystallizer. As a seed crystal when the saturated concentration was reached, 3HB obtained as a crystal in advance by purification was added. Further, the mixture was cooled at a constant rate with stirring to deposit 3HB. The precipitated 3HB was suction filtered and dried under reduced pressure to isolate 85 g of white crystals. The yield for the crystallization step was 57%.

  Further, when ethyl acetate was changed to diethyl ether, t-butyl methyl ether, acetone and methyl ethyl ketone and 200 g of the concentrated liquid containing 75% by mass of 3HB was similarly subjected to the crystallization step, the results are shown in Table 2. It was

  As the crystallization solvent, an ester-based or ether-based substance having a large temperature-dependent solubility gradient can be used. The crystallization solvent selected from ethyl acetate, diethyl ether, and t-butyl methyl ether is methanol. It was found that it has a large solubility gradient and is extremely excellent as a crystallization solvent as compared with various well-known and easy-to-handle solvents such as ethanol, ethanol, propanol, butanol, acetone, and methyl ethyl ketone.

  According to the method for producing 3HB of the present invention, 3HB having high efficiency and high purity can be produced.

Claims (8)

A fermentation step of obtaining a fermented liquid in which 3HB is fermented and produced from a 3-hydroxybutyric acid (3HB) -producing bacterium;
From the fermentation liquor obtained in the fermentation step, a preliminary concentration step of distilling water off,
An extraction step of extracting a 3HB-containing solution containing 3HB from the fermentation liquid with an organic solvent,
A concentration step of distilling the organic solvent from the 3HB-containing solution extracted in the extraction step to obtain a 3HB concentrated solution;
A crystallization solvent containing at least one selected from ethyl acetate, diethyl ether, and t-butyl methyl ether as a main component was added to the 3HB concentrated liquid obtained in the concentration step, and 3HB crystals were obtained from the crystallization solvent in which 3HB was dissolved. A method for producing 3HB, which comprises a crystallization step of causing precipitation.   The method for producing 3HB according to claim 1, wherein the 3HB-producing bacterium is a 3HB-producing Halomonas bacterium.   The fermentation step comprises an aerobic fermentation step of aerobically fermenting the Halomonas bacterium, and an anaerobic fermentation step of anaerobically fermenting the Halomonas bacterium in which polyhydroxybutyric acid (PHB) is accumulated by the aerobic fermentation step. The method for producing 3HB as described above.   The method for producing 3HB according to any one of claims 1 to 3, wherein the extraction step is performed after performing a filtration step of filtering the fermentation liquid obtained in the fermentation step.   The method for producing 3HB according to any one of claims 1 to 4, wherein the extraction step is performed after the pH adjustment step of adjusting the pH of the fermentation liquid obtained in the fermentation step to an acidity of 1 to 6. ..   The method for producing 3HB according to any one of claims 1 to 5, wherein an activated carbon treatment step of contacting the 3HB-containing solution or the 3HB concentrated liquid with activated carbon is performed.   The production of 3HB according to any one of claims 1 to 6, wherein the organic solvent contains at least one selected from 1-butanol, 1-pentanol, ethyl acetate, isoamyl alcohol, and methyl ethyl ketone as a main component. Method.   The method for producing 3HB according to any one of claims 1 to 7, wherein in the crystallization step, a seed crystal of 3HB is added to a crystallization solvent in which 3HB is dissolved, and the mixture is cooled with stirring.