JP7797260B2 - Method for optical resolution of racemic 3-hydroxybutyric acid - Google Patents

Description

The present invention relates to a method for optically resolving racemic 3-hydroxybutyric acid.

3-Hydroxybutyric acid and its salts (hereinafter simply referred to as "3HB") are naturally occurring substances in the human body, making them highly biocompatible and promising as a revolutionary energy source to replace carbohydrates. Furthermore, 3HB has been shown to function not only as an energy source but also as a signaling substance that affects the expression of various genes and the activity of proteins. 3HB is known to improve cognitive function and long-term memory by inhibiting histone deacetylase through its gene expression regulation effect, and has been confirmed to be effective in preventing Alzheimer's disease. Furthermore, 3HB, which is produced through the ingestion and metabolism of medium-chain fatty acids, which are abundant in coconut oil, is known to improve the symptoms of patients with Alzheimer's disease and diabetes, who have difficulty effectively utilizing carbohydrates in the brain and body. Furthermore, because 3HB is converted into energy more quickly than carbohydrates in the body and has the effect of suppressing the absorption of fat and sugar into cells, it is actively used as an energy substance for athletes and in diet and health foods.

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

Not only for 3HB, but for many other compounds, optical activity is often advantageous when used for various purposes, and so there is a demand for technology that can optically resolve racemates to separate optically active compounds. For example, a method has been proposed for optically resolving racemic hydroxycarboxylic acid esters to produce optically active hydroxycarboxylic acid esters or optically active hydroxycarboxylic acids (Patent Document 1).

In the production method described in Patent Document 1, a racemic hydroxycarboxylic acid ester is subjected to the action of a microorganism belonging to the genus Enterobacter or Chitrobacter and having carboxylic acid ester decomposition activity, or bacterial cells obtained from the microbial culture medium. This stereoselectively hydrolyzes the carboxylic acid ester and optically resolves it, and one of the remaining optically active hydroxycarboxylic acid esters or the other optically active hydroxycarboxylic acid obtained by hydrolysis is collected.

Patent No. 4069742

However, the production method described in Patent Document 1 is a technology that is limited to racemic hydroxycarboxylic acid esters. Therefore, it is not possible to optically resolve racemic 3-hydroxybutyric acid or its salts to produce optically active 3-hydroxybutyric acid or its salts.

The present invention was developed in light of the above-mentioned circumstances, and its object is to provide a method for optically resolving racemic 3-hydroxybutyric acid or a salt thereof, thereby enabling the production of optically active 3-hydroxybutyric acid.

The method for optical resolution of racemic 3-hydroxybutyric acid according to the present invention for achieving the above object is characterized by the following features:
A method for optical resolution of racemic 3-hydroxybutyric acid or a salt thereof, comprising the steps of:
an aerobic culturing step of aerobically culturing a halophilic bacterium belonging to the genus Halomonas in a medium containing inorganic salts and racemic 3-hydroxybutyric acid or a salt thereof as an organic carbon source;
a separation step of separating bacterial cells from the culture solution after the aerobic culture step;
The bacterial cells separated in the separation step are microaerophilically cultured in a medium containing inorganic salts, and a microaerophilic culture step is carried out in this order to produce (R)-3-hydroxybutyric acid or a salt thereof in the culture solution.

As a result of extensive research, the present inventors discovered that when halophilic bacteria belonging to the genus Halomonas are aerobically cultured in a medium containing racemic 3-hydroxybutyric acid or a salt thereof as an organic carbon source, the halophilic bacteria preferentially take up (R)-3-hydroxybutyric acid and accumulate it as poly[(R)-3-hydroxybutyric acid] (hereinafter also referred to as "PHB"), thereby completing the present invention.

That is, according to the above-mentioned characteristic configuration, in the aerobic culture step, the halophilic bacterium preferentially takes up (R)-3-hydroxybutyric acid from racemic 3-hydroxybutyric acid, which accumulates as PHB within the bacterial cells, while most of the (S)-3-hydroxybutyric acid remains in the culture medium. Therefore, (S)-3-hydroxybutyric acid is present in the culture medium from which the bacterial cells were separated in the separation step. Then, by microaerophilically culturing the bacterial cells obtained in the separation step, (R)-3-hydroxybutyric acid is secreted and produced outside the bacterial cells (into the culture medium). Thus, according to the optical resolution method for racemic 3-hydroxybutyric acid having the above-mentioned characteristic configuration, racemic 3-hydroxybutyric acid can be separated into (R)-3-hydroxybutyric acid and (S)-3-hydroxybutyric acid.

A further characteristic feature of the method for optical resolution of racemic 3-hydroxybutyric acid according to the present invention is:
The present invention is characterized in that an R-isomer recovery step is carried out to recover (R)-3-hydroxybutyric acid or a salt thereof from the culture solution obtained in the microaerobic culture step.

As described above, the culture medium obtained in the microaerobic culture step contains (R)-3-hydroxybutyric acid and its salts secreted and produced by the bacterial cells. According to the above-described characteristic configuration, (R)-3-hydroxybutyric acid or its salts can be recovered from the culture medium by the R-isomer recovery step. Therefore, the optical resolution method having the above-described characteristic configuration makes it possible to produce (R)-3-hydroxybutyric acid as optically active 3-hydroxybutyric acid from racemic 3-hydroxybutyric acid.

A further characteristic feature of the method for optical resolution of racemic 3-hydroxybutyric acid according to the present invention is:
The difference is that an S-isomer recovery step is carried out to recover (S)-3-hydroxybutyric acid or a salt thereof from the culture medium from which the bacterial cells have been separated in the separation step.

As described above, the culture medium from which the bacterial cells were separated in the separation step contains (S)-3-hydroxybutyric acid, which is not easily taken up by halophilic bacteria. According to the above-described characteristic configuration, the S-isomer recovery step allows for recovery of (S)-3-hydroxybutyric acid or a salt thereof from the culture medium after bacterial cell separation. Therefore, the optical resolution method for racemic 3-hydroxybutyric acid having the above-described characteristic configuration makes it possible to produce (S)-3-hydroxybutyric acid as optically active 3-hydroxybutyric acid from racemic 3-hydroxybutyric acid.

A further characteristic feature of the method for optical resolution of racemic 3-hydroxybutyric acid according to the present invention is:
The halophilic bacterium is Halomonas sp. KM-1 strain.

The inventors of the present application have experimentally confirmed that when the halophilic bacterium is Halomonas sp. KM-1 strain, the Halomonas sp. KM-1 strain preferentially takes up (R)-3-hydroxybutyric acid from racemic 3-hydroxybutyric acid, accumulates PHB within the bacterial cell, and, upon transition to microaerobic culture, secretes and produces (R)-3-hydroxybutyric acid into the culture medium.

FIG. 1 is a diagram for explaining an outline of a method for optical resolution of racemic 3-hydroxybutyric acid according to an example. 1 is a graph showing analytical data during aerobic culture. 1 is a graph showing analytical data during microaerobic culture.

The method for optical resolution of racemic 3-hydroxybutyric acid according to the present invention is described below. While preferred examples are described below to more specifically illustrate the present invention, various modifications are possible without departing from the spirit of the present invention, and the present invention is not limited to the following descriptions.

[Outline of the method for optical resolution of racemic 3-hydroxybutyric acid]
The method for optical resolution of racemic 3-hydroxybutyric acid according to an embodiment of the present invention comprises the following steps.
(1) A step of aerobically culturing a halophilic bacterium belonging to the genus Halomonas in a medium containing inorganic salts and racemic 3-hydroxybutyric acid or a salt thereof as an organic carbon source.
(2) A separation step of separating the bacterial cells from the culture solution after the aerobic culture step.
(3) A microaerophilic culturing step in which the bacterial cells separated in the separation step are microaerophilically cultured in a medium containing inorganic salts to produce (R)-3-hydroxybutyric acid or a salt thereof in the culture solution.

Furthermore, in this embodiment, the following steps are further carried out.
(4) An R-isomer recovery step of recovering (R)-3-hydroxybutyric acid or a salt thereof from the culture medium obtained in the microaerobic culture step.
(5) An S-isomer recovery step of recovering (S)-3-hydroxybutyric acid or a salt thereof from the culture medium from which the bacterial cells have been separated in the separation step.

[Aerobic cultivation process]
The aerobic culturing step in the optical resolution method of the present invention is a step of aerobically culturing a halophilic bacterium belonging to the genus Halomonas in a medium.
<A: Halophilic bacteria belonging to the genus Halomonas>
The halophilic bacterium belonging to the genus Halomonas used in the present invention may be any bacterium that has the property of aerobically growing in a medium containing an inorganic salt and racemic 3-hydroxybutyrate (racemic 3HB), taking up (R)-3-hydroxybutyrate (R-3HB) into its cell body, accumulating it as poly[(R)-3-hydroxybutyrate] (PHB), and secreting and producing R-3HB or a salt thereof into the extracellular medium by changing the culture conditions.

The above-mentioned halophilic bacteria belonging to the genus Halomonas are bacteria that are halophilic and suitable for salt concentrations of 0.1 to 1.0 M, and can sometimes grow in salt-free media. Furthermore, the above-mentioned halophilic bacteria belonging to the genus Halomonas typically grow in media with a pH of approximately 5 to 12.

An example of the halophilic bacterium belonging to the genus Halomonas is the Halomonas sp. KM-1 strain. The Halomonas sp. KM-1 strain was deposited on July 10, 2007, with the International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (Chuo 6, 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture, 305-8566) under accession number FERM P-21316. This strain has also been transferred to an international depository with accession number FERM BP-10995. The 16S rRNA gene of the Halomonas sp. KM-1 strain has been registered with the DDBJ under accession number AB477015.

Furthermore, in consideration of the growth characteristics of the above-mentioned halophilic bacteria, the halophilic bacteria used in the aerobic culture step of the present invention are not limited to Halomonas sp. KM-1 strain, as long as they preferentially take up R-3HB into their cells, accumulate PHB, and then secrete R-3HB in the microaerobic culture step described below. Examples of such halophilic bacteria belonging to the genus Halomonas include Halomonas pantelleriensis (ATCC 700273) and Halomonas campisalis (ATCC 7000597).

Furthermore, analysis of 16S ribosomal RNA sequences has shown that in addition to the above-mentioned halophilic bacteria, Halomonas nitritophilus, Halomonas alimentaria, and the like may also be used as halophilic bacteria belonging to the genus Halomonas in the aerobic culture process.

A gene may be introduced into the halophilic bacterium belonging to the genus Halomonas. The gene to be introduced is not particularly limited, as long as it does not affect the properties of the bacterium, which preferentially take up R-3HB out of racemic 3HB, accumulate PHB, and then secrete R-3HB in the microaerobic culture step described below, in the optical resolution method of the present invention. Various common methods can be used to introduce recombinant DNA into the bacterium and to transform it using this method.

<B: Medium>
The medium used in the aerobic culture step contains inorganic salts and racemic 3HB or a salt thereof as an organic carbon source. The pH of the medium is not particularly limited as long as it satisfies the growth conditions of the halophilic bacteria, but specifically, it should be about pH 5 to 12, and more preferably pH 8.8 to 12. The use of an alkaline medium is preferable because it can effectively prevent contamination with other bacteria.

The culture medium may be a liquid medium or a solid medium.

The inorganic salts to be added to the medium used in the aerobic culture process are not particularly limited, and examples include phosphates, nitrates, carbonates, sulfates, and metal salts such as sodium, magnesium, potassium, manganese, iron, zinc, copper, and cobalt.

For example, when sodium is used as an inorganic salt, NaCl, NaNO ₃ , NaHCO ₃ , Na ₂ CO ₃ or the like may be used.

It is preferable to use these inorganic salts as compounds that serve as nitrogen and phosphorus sources for the halophilic bacteria.

The nitrogen source is not particularly limited and may be nitrate, nitrite, urea, ammonium salt, glutamate, or the like. For example, compounds such as NaNO ₃ , NaNO ₂ , and NH ₄ Cl may be used.

The amount of nitrogen source used can be set appropriately within a range that does not affect the growth of the bacterial cells and achieves the objective of optical resolution. Specifically, at the initial stage of cultivation, the amount should typically be at least about 500 mg of nitrate per 100 ml of medium, more preferably at least about 1000 mg, and even more preferably at least about 1250 mg.

The phosphorus source may be a phosphate, monohydrogen phosphate, dihydrogen phosphate, or the like, and is not particularly limited, but may be, for example, a compound such as K ₂ HPO ₄ or KH ₂ PO ₄ .

The amount of phosphorus source used can be determined appropriately from the same perspective as the amount of nitrogen source used above. Specifically, the amount of dihydrogen phosphate used is usually about 50 to 400 mg per 100 ml of medium, and more preferably about 100 to 200 mg.

These inorganic salts may be used alone or in combination of two or more.

The medium used in the aerobic cultivation process contains only racemic 3HB or a salt thereof as the organic carbon source. Racemic 3HB is a mixture of (R)-3-hydroxybutyric acid and (S)-3-hydroxybutyric acid, and is obtained, for example, by conventional organic synthesis, not asymmetric synthesis.

The concentration of racemic 3HB as an organic carbon source can be appropriately set within a range that allows for preferential uptake of R-3HB into the bacterial cell, promotes accumulation of PHB, and achieves the objective of optically resolving racemic 3HB.

In the aerobic culture step of the optical resolution method of the present invention, halophilic bacteria belonging to the genus Halomonas are cultured in a medium with a relatively high salt concentration, so there is almost no risk of contamination or proliferation of other bacterial cells. Therefore, the medium may or may not be sterilized, and culture can be carried out using simple equipment.

The halophilic bacteria in the aerobic culture step are cultured under aerobic conditions. The conditions for aerobic culture are not particularly limited, as long as they allow the bacteria to grow and take up R-3HB, which accumulates as PHB.

Specifically, the halophilic bacteria are inoculated into approximately 5 ml of medium and pre-cultured overnight with shaking at a specified stirring speed and temperature. The bacterial cells obtained from the pre-culture are then diluted approximately 100-fold into medium contained in an Erlenmeyer flask, fermenter, jar fermenter, etc., and subjected to main culture (corresponding to aerobic culture in this application).

The culture temperature for the main culture can usually be set within the range of about 20 to 45°C, but is preferably set within the range of about 30 to 37°C. Furthermore, when using an Erlenmeyer flask, the stirring speed can usually be set within the range of about 120 to 250 rpm, but is preferably set within the range of about 150 to 200 rpm. When using a fermenter or jar fermenter, it is preferable to supply oxygen at a rate comparable to the above.

In the aerobic culture step, halophilic bacteria belonging to the genus Halomonas are aerobically cultured under these culture conditions. Specifically, the dissolved oxygen concentration in the medium during aerobic culture is not particularly limited, but is usually 0 mg/L or higher, and preferably 7 mg/L or higher.

Cultivation methods in the aerobic culturing step include batch culture, semi-batch culture, continuous culture, and other culturing methods, and are not particularly limited. However, considering that the halophilic bacteria used in the optical resolution method of the present invention are highly unlikely to be contaminated with other bacteria, long-term continuous culture is also possible. The culture environment may be one in which the medium is exposed to air, and may be adjusted by actively blowing oxygen-containing gas onto the surface of the medium or by blowing such gas into the medium. The culture environment may be either a non-sterile environment or a sterile environment.

The aerobic culture time is the time required for R-3HB to disappear from the medium and for S-3HB to remain, and is, for example, 20 to 50 hours. It is preferable to continuously check for the presence of R-3HB in the medium using an analytical method such as HPLC, and, once R-3HB has disappeared, to carry out the separation process described below and stop the culture.

[Separation process]
The separation step in the optical resolution method of the present invention is a step of separating the bacterial cells from the culture medium after the aerobic culture step, and the aerobic culture step is terminated by separating the bacterial cells from the culture medium.

In the separation step, the bacterial cells are separated from the culture medium using a known method, and the aerobic culture step is stopped. For example, when a liquid medium is used for the aerobic culture step, the culture solution obtained in this step contains S-3HB or a salt thereof that has not been taken up by the halophilic bacteria. Therefore, by separating the halophilic bacteria from the culture solution using a separation means and stopping the aerobic culture step, a culture solution containing S-3HB or a salt thereof and containing no or almost no R-3HB can be obtained.

Specific separation techniques that can be used include known solid-liquid separation procedures such as centrifugation and filtration.

[Microaerobic culture process]
The microaerophilic culturing step in the optical resolution method of the present invention is a step of culturing the cells separated in the separation step in a medium microaerophilically to produce (R)-3-hydroxybutyric acid or a salt thereof in the culture medium.

The phrase "producing (R)-3-hydroxybutyric acid in the culture medium" means that the halophilic bacterium secretes 3HB into the culture medium. Furthermore, the phrase "producing a salt of (R)-3-hydroxybutyric acid in the culture medium" means not only that the halophilic bacterium secretes a salt of 3HB into the culture medium, but also that the 3HB secreted by the halophilic bacterium reacts with a cationic component present in the culture medium to form a salt of R-3HB. The cationic component that reacts with R-3HB to form a salt is not particularly limited as long as it is present in the culture medium, but examples include sodium ions, potassium ions, calcium ions, magnesium ions, cobalt ions, zinc ions, iron ions, copper ions, molybdenum ions, ammonium ions, manganese ions, etc.

Specifically, this process involves culturing halophilic bacteria by adjusting and/or maintaining the pH in the alkaline range of 8.0 or higher, and producing R-3HB or a salt thereof under conditions in which oxygen is not actively supplied to the halophilic bacteria in the culture medium. Continuing cultivation without actively supplying oxygen to the halophilic bacteria consumes oxygen within the system, resulting in a near-anoxic state. However, since an environment with an oxygen concentration of a few percent is maintained, which does not reach absolute anaerobic state, an environment suitable for microaerobic cultivation can be maintained.

The medium used in the microaerobic culture process contains inorganic salts but does not contain an organic carbon source. The same medium as that used in the aerobic culture process can be used, except that it does not contain an organic carbon source.

The conditions for microaerobic culture are not particularly limited, as long as they allow the PHB accumulated within the bacterial cells to be secreted into the medium as R-3HB.

When microaerobic cultivation is continued, the pH of the medium tends to decrease due to the production of organic acids. The pH of such a medium can be checked using a known pH measuring device or a jar fermenter equipped with such a device.

Note that "adjusting and/or maintaining" means maintaining an appropriate pH state by adding a pH adjuster while checking the pH as described above, or simply adding a pH adjuster to adjust the pH at the start of cultivation, without subsequently adjusting the pH.

The pH adjusted and maintained during the microaerobic culture process is preferably 8.0 or higher. This allows the productivity of R-3HB by halophilic bacteria to be maintained at a high level.

The halophilic bacteria used in the present invention can be cultured under moderately high salt concentrations and alkaline conditions, which reduces the risk of contamination with foreign bacteria. However, some lactic acid bacteria can grow in environments with moderately high salt concentrations and a pH of 8.4 or lower. If such bacteria contaminate the culture system of the present invention, they may consume the R-3HB or a salt thereof secreted by the halophilic bacteria as a substrate for lactic acid fermentation, further decreasing the pH of the medium.

For this reason, in the present invention, in order to culture halophilic bacteria in a medium without sterilizing it and/or in a non-sterile environment and secrete 3HB into the medium, it is preferable to adjust and maintain the pH of the medium at about 8.5 or higher during the microaerobic culture process.

pH adjusters are not particularly limited, but examples include hydroxides, carbonates, and bicarbonates. More specific examples include sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, sodium carbonate, potassium carbonate, calcium carbonate, magnesium carbonate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, and aqueous ammonia.

[R-isomer recovery step]
The R-isomer recovery step in the optical resolution method of the present invention is a step of recovering (R)-3-hydroxybutyric acid or a salt thereof from the culture medium obtained in the microaerobic culture step.

In the R-isomer recovery step, R-3HB or a salt thereof can be recovered using known techniques. For example, when R-3HB or a salt thereof is present in the culture medium, the culture in the microaerobic culture step can be stopped, the culture medium containing R-3HB or a salt thereof can be separated from the bacterial cells, and R-3HB or a salt thereof can be recovered from the culture medium. Furthermore, when the microaerobic culture step is performed using a liquid medium, the culture obtained in this step will contain secreted R-3HB or a salt thereof. Therefore, the culture in this step can be stopped, and if necessary, the culture medium can be separated from the halophilic bacteria using a separation means to obtain a culture medium from which R-3HB or a salt thereof can be recovered.

Specific separation techniques that can be used include known solid-liquid separation procedures such as centrifugation and filtration. Furthermore, there are no particular limitations on the method for terminating the culture. For example, after the microaerobic culture step, the culture solution containing the halophilic bacteria can be sterilized by heating, acid treatment, or other methods, or solid-liquid separation can be performed to separate the culture solution from the halophilic bacteria.

The method for confirming the presence of R-3HB or its salt in the culture medium may vary depending on the bacterial species, medium components, culture conditions, etc., and should be determined appropriately taking these factors into consideration. For example, the culture medium can be sampled periodically and subjected to analytical methods such as HPLC to determine the time to stop the culture.

In cases where R-3HB is present in the culture medium as an alkali metal salt formed by reacting with alkali metal or alkaline earth metal cations such as sodium or calcium based on inorganic salts contained in the culture medium, R-3HB can be obtained by subjecting the culture medium from which the bacterial cells have been separated to conventional methods such as crystallization.

The culture medium after bacterial cell separation may be purified by column chromatography using an appropriate column to recover R-3HB. Alternatively, the pH of the culture medium after bacterial cell separation may be appropriately adjusted to purify the desired R-3HB or a salt thereof. It is also possible to add lower alcohols to the culture medium after bacterial cell separation, subject the mixture to an esterification reaction, and then purify the resulting R-3HB ester by distillation or other methods.

[S-isomer recovery step]
The S-isomer recovery step in the optical resolution method of the present invention is a step of recovering (S)-3-hydroxybutyric acid or a salt thereof from the culture medium from which the bacterial cells have been separated in the separation step.

As described above, the culture medium from which the bacterial cells were separated in the separation step contains S-3HB or a salt thereof, but contains almost no R-3HB. In the S-isomer recovery step, (S)-3-hydroxybutyric acid (S-3HB) or a salt thereof can be recovered from the culture medium from which the bacterial cells were separated in this separation step using a known method.

When S-3HB exists in the culture medium as an alkali metal salt that has reacted with alkali metal or alkaline earth metal cations, such as sodium or calcium, based on inorganic salts contained in the culture medium, S-3HB can be obtained by subjecting the culture medium from which the bacterial cells have been separated to standard methods such as crystallization.

The culture medium after bacterial cell separation may be purified by column chromatography using an appropriate column to recover S-3HB. Alternatively, the pH of the culture medium after bacterial cell separation may be appropriately adjusted to purify the desired S-3HB or a salt thereof. Alternatively, lower alcohols may be added to the culture medium after bacterial cell separation, and the resulting mixture may be subjected to an esterification reaction, followed by purification as an S-3HB ester by distillation or other methods.

The present invention will be described in more detail below based on examples. Needless to say, the present invention is not limited to these examples.

As an example, racemic 3HB was optically resolved using the optical resolution method of the present invention using Halomonas sp. KM-1 strain as a halophilic bacterium. Figure 1 shows an outline of the optical resolution method used in this example.

Specifically, the KM-1 strain was added to a baffled flask culture vessel containing a medium of the composition shown in Table 1 below, containing SOT medium plus racemic 3-hydroxybutyrate (50% R-3HB, 50% S-3HB) as a carbon source. Aerobic cultivation was then performed while shaking at a speed of 200 rpm or higher. The concentrations of R-3HB and S-3HB in the culture medium and the amount of PHB accumulated in the bacterial cells were measured over time using known methods. When R-3HB disappeared from the culture medium (31 hours after the start of cultivation, as shown in Figure 2), the bacterial cells were separated from the culture medium and the aerobic cultivation was terminated.

Figure 2 is a graph summarizing analytical data from aerobic cultivation. As can be seen from the graph, the R-3HB concentration gradually decreased after the start of cultivation, reaching nearly zero after 31 hours, while no significant decrease was observed in the S-3HB concentration. Furthermore, the amount of PHB accumulated gradually increased in inverse proportion to the decrease in R-3HB concentration. This clearly demonstrates that during the aerobic cultivation process, R-3HB is preferentially taken up by the KM-1 strain, and is accumulated as PHB within the bacterial cells.

Next, the bacterial cells separated from the culture medium were subjected to a microaerobic culture step using SOT medium (a medium with the composition shown in Table 1 excluding (R)-3-hydroxybutyric acid and (S)-3-hydroxybutyric acid) under conditions where oxygen was not actively supplied. After confirming that the amount of PHB accumulated within the bacterial cells had become nearly constant, 39 hours after the start of culture, the bacterial cells were separated from the culture medium and the microaerobic culture was stopped.

Figure 3 is a graph summarizing analytical data from microaerobic cultivation. As can be seen from the graph, the concentration of R-3HB gradually increases for the first 15 hours after the start of cultivation and then remains nearly constant, while the concentration of S-3HB remains unchanged at nearly zero. Furthermore, the amount of PHB accumulated gradually decreases inversely proportional to the increase in R-3HB concentration and remains nearly constant after 15 hours from the start of cultivation. This clearly demonstrates that during the microaerobic cultivation process, PHB accumulated within the KM-1 strain cells is secreted and produced as R-3HB into the culture medium.

Furthermore, S-3HB with an optical purity of 95% or more could be recovered from the culture medium from which the bacterial cells were separated after the aerobic culture step, and R-3HB with an optical purity of 95% or more could be recovered from the culture medium from which the bacterial cells were separated after the microaerobic culture step. Therefore, it was confirmed that the optical resolution method of the present invention makes it possible to produce optically active 3-hydroxybutyric acid.

The present invention can be applied to a method for optically resolving racemic 3-hydroxybutyric acid.

Claims (4)

A method for optical resolution of racemic 3-hydroxybutyric acid or a salt thereof, comprising the steps of:
an aerobic culturing step of aerobically culturing a halophilic bacterium belonging to the genus Halomonas in a medium containing inorganic salts and racemic 3-hydroxybutyric acid or a salt thereof as an organic carbon source;
a separation step of separating bacterial cells from the culture solution after the aerobic culture step;
a microaerophilic culturing step in which the bacterial cells separated in the separation step are microaerophilically cultured in a medium containing inorganic salts to produce (R)-3-hydroxybutyric acid or a salt thereof in the culture solution; and The method for optical resolution of racemic 3-hydroxybutyric acid according to claim 1, further comprising an R-isomer recovery step of recovering (R)-3-hydroxybutyric acid or a salt thereof from the culture medium obtained in the microaerobic culture step. The method for optical resolution of racemic 3-hydroxybutyric acid according to claim 1 or 2, further comprising an S-isomer recovery step in which (S)-3-hydroxybutyric acid or a salt thereof is recovered from the culture medium from which the bacterial cells have been separated in the separation step. The method for optical resolution of racemic 3-hydroxybutyric acid according to any one of claims 1 to 3, wherein the halophilic bacterium is Halomonas sp. KM-1 strain.