JP6827242B2 - Method for producing oxaloacetate using Halomonas - Google Patents

Description

The present invention relates to a method for producing oxaloacetate using Halomonas.

In recent years, not only energy but also chemical refineries have become bio-based.

Oxaloacetic acid is a component of the citric acid cycle of aerobic respiration following glycolysis in vivo, and is produced by oxidizing malic acid by malic acid dehydrogenase, and oxaloacetate is acetylated by citrate synthase. It is an important compound that reacts with CoA to produce citric acid. Oxaloacetic acid is also used for gluconeogenesis via phosphoenolpyruvate.

Oxaloacetic acid is a compound that is rarely produced industrially, but is attracting attention for its future utility value because of its physiological effects as described above.

Regarding the production of substances other than oxaloacetate using halophilic bacteria belonging to the genus Halomonas, Patent Document 1 describes a method for producing 3-hydroxybutyrate (3-HB) using halophilic bacteria belonging to the genus Halomonas. Disclosed, and Patent Documents 2 and 3, a method for producing pyruvic acid using a halophilic bacterium belonging to the genus Halomonas is disclosed. Further, Patent Document 4 describes a method for producing oxaloacetate and an oxalo derivative using Gram-positive bacteria, but does not describe the amount of oxaloacetate produced.

International Patent Gazette 2013/051499 JP 2015-204769 JP 2016-202093 JP 2006-91

In general, when a substance is industrially produced using a microorganism, in many cases, the strain used is imparted with a trait by artificial mutation or the like, and thus such a trait is maintained. Or, there is a problem in that it takes a lot of labor to confirm it.

In addition, in order to use a microorganism for fermentative production, regardless of whether or not the microorganism has been genetically modified, it is generally contaminated with other bacteria such as adding an antibiotic to the medium and autoclaving the medium. It is necessary to take measures to prevent it, which is troublesome and costly.

As described above, Patent Document 1 discloses a simple and efficient 3-HB production method using wild-type Halomonas, and Patent Documents 2 and 3 each disclose Patent Document 1. A simple and efficient method for producing pyruvate using the same wild-type Halomonas as above is disclosed. However, the above-mentioned Patent Documents 1 to 3 do not mention anything about the production of oxaloacetate or a salt thereof, and there is no description or suggestion about the production method thereof.

Against this background, it is desired to develop a method for producing oxaloacetate or a salt thereof, which is simple and efficient, and can be applied to industrial mass production.

Therefore, an object of the present invention is to provide a method for easily and efficiently producing oxaloacetate or a salt thereof.

As a result of diligent studies to solve such a problem, the present inventors have cultivated halophilic bacteria belonging to the genus Halomonas in a medium containing a specific inorganic salt at a predetermined concentration. It has been found that oxaloacetate or a salt thereof can be produced with high efficiency.

The present invention has been completed based on these findings, and includes a wide range of inventions shown below.

[Item 1] A method for producing oxaloacetate or a salt thereof, which comprises the following steps 1, 2 and 3;
(1) A step of preparing a liquid medium containing (a) an organic carbon source and (b) an inorganic salt A composed of NaHCO ₃ , Na ₂ CO ₃ , and NaCl having a total concentration of 0.2 M to 2.5 M. 1
(2) Step 2 of culturing halophilic bacteria belonging to the genus Halomonas using the liquid medium prepared in step 1.
(3) A step 3 of recovering oxaloacetate or a salt thereof from the culture solution obtained in the above step 2.

[Item 2] The production method according to Item 1, wherein in the step 2, the inorganic salt B is added to the liquid medium when the growth state of the halophilic bacterium is in the induction phase or the logarithmic phase.

[Item 3] The production method according to Item 2, wherein the addition of the inorganic salt B is carried out when the OD600 value of the culture solution is 5 or more.

[Item 4] The item 2 or 3 above, wherein the inorganic salt B is at least one selected from the group consisting of NaHCO ₃ , Na ₂ CO ₃ , NaCl, KHCO ₃ , K ₂ CO ₃ , and KCl. Manufacturing method.

Item 5. The production method according to any one of Items 2 to 4, wherein the total concentration of the inorganic salt B and the inorganic salt A is in the range of 0.2M to 2.5M.

[Item 6] The production method according to any one of Items 1 to 5, wherein the culture temperature is 30 ° C to 40 ° C.

[Item 7] The production method according to any one of Items 1 to 6, wherein a nitrogen source is added to the culture solution in the step 2.

[Item 8] The halophilic bacterium is any one of Halomonas sp. KM-1 strain, Halomonas panterariensis, Halomonas campisaris, Halomonas nitritophilus, Halomonas alimentaria, or Halomonas meridiana. The production method according to any one of 7 to 7.

According to the present invention, a simple and efficient method for producing oxaloacetate or a salt thereof is provided.

The graph which shows the experimental result of Test Example 1. The vertical axis of the graph in the figure shows the concentration of oxaloacetic acid (g / L) in the culture solution, and the horizontal axis shows the time (h) from the start of the culture. The graph which shows the experimental result of Test Example 2. The vertical axis of the graph in the figure shows the concentration of oxaloacetic acid (g / L) in the culture solution, and the horizontal axis shows the time (h) from the start of the culture. The graph which shows the experimental result of Test Example 3. The vertical axis of the graph in the figure shows the concentration of oxaloacetic acid (g / L) in the culture solution, and the horizontal axis shows the time (h) from the start of the culture. The graph which shows the experimental result of Test Example 4. The vertical axis of the graph in the figure shows the concentration of oxaloacetic acid (g / L) in the culture solution, and the horizontal axis shows the time (h) from the start of the culture. The graph which shows the experimental result of Test Example 5. The vertical axis of the graph in the figure shows the absorbance in the culture solution (600 nm: OD ₆₀₀ value), and the horizontal axis shows the time (h) from the start of the culture. The graph which shows the experimental result of Test Example 5. The vertical axis of the graph in the figure shows the concentration of oxaloacetic acid (g / L) in the culture solution, and the horizontal axis shows the time (h) from the start of the culture. The graph which shows the experimental result of Test Example 6. The vertical axis of the graph in the figure shows the concentration of oxaloacetic acid (g / L) in the culture solution, and the horizontal axis shows the time (h) from the start of the culture.

The method for producing oxaloacetate or a salt thereof of the present invention includes the following steps 1, 2, and 3.
(1) A step of preparing a liquid medium containing (a) an organic carbon source and (b) an inorganic salt A composed of NaHCO ₃ , Na ₂ CO ₃ , and NaCl having a total concentration of 0.2 M to 2.5 M. 1
(2) Step 2 of culturing halophilic bacteria belonging to the genus Halomonas using the liquid medium.
(3) Step 3 of recovering oxaloacetate or a salt thereof from the culture solution obtained in step 2.

Process 1
Step 1 of the production method of the present invention contains (a) an organic carbon source and (b) an inorganic salt A consisting of NaHCO ₃ , Na ₂ CO ₃ and NaCl having a total concentration of 0.25 M to 2.0 M. This is a step of preparing a liquid medium to be prepared.

The liquid medium can be readily prepared by mixing commercially available substances in a conventional manner.

A commercially available liquid medium may be used to prepare the liquid medium.

The medium, the inorganic salt contained therein, and the organic carbon source will be described in the column of <B: Liquid medium> described later.

Process 2
Step 2 of the production method of the present invention is a step of culturing halophilic bacteria belonging to the genus Halomonas using the liquid medium.

<A: Halophile>
The halophilic bacterium used in step 2 is a halophilic bacterium belonging to the genus Halomonas. As the halophilic bacterium, the halophilic bacterium shown in either (i) or (ii) described later can be used.

(I) A halophilic bacterium that grows in a solid medium containing an inorganic salt and a single or a plurality of organic carbon sources to produce oxaloacetate or a salt thereof in a medium outside the cells.
(Ii) A halophilic bacterium characterized in that it grows in a liquid medium containing an inorganic salt and a single or a plurality of organic carbon sources, and then secretes and produces oxaloacetate or a salt thereof in a culture medium outside the cells.

The halophilic bacterium can use oxidative metabolism and anaerobic metabolism properly, can survive regardless of the presence or absence of free oxygen in the medium, and tends to grow more easily in the presence of free oxygen. It is a bacterial cell having the properties of a so-called facultative anaerobic bacterium.

The halophilic bacterium is a bacterium that has halophilicity in which a salt concentration in the range of about 0.2 M to 2.5 M is the optimum salt concentration for growth, and can sometimes grow even in a salt-free medium. The halophilic bacterium usually grows in a medium in the range of pH 5 to pH 12.

Examples of the halophilic bacterium include Halomonas sp. KM-1 strain. Halomonas SP KM-1 strain was entrusted to the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center (1-1-1, Higashi, Tsukuba City, Ibaraki Prefecture, 305-8566) on July 10, 2007. It has been deposited as number FERM P-21316. In addition, this strain has now been transferred to an international deposit, and its accession number is FERM BP-10995. The 16S rRNA gene of the Halomonas SP KM-1 strain is registered in DDBJ as Accession Number AB477015.

In view of growth characteristics and the like, as suitable halophilic bacteria used in step 2, in addition to Halomonas sp. KM-1 strain, Halomonas pantereriensis (ATCC 700273), Halomonas campisalis: ATCC 700597), Halomonas meridiana (NBRC15608) and the like can also be mentioned.

Furthermore, from the analysis by 16S ribosomal RNA sequence, not only the above-mentioned halophiles belonging to the genus Halomonas, but also halomonas nitritofilus, halomonas alimentaria, halomonas meridiana and the like are halophilics belonging to the genus Halomonas used in step 2. It may be adopted as a bacterium.

Among the halophilic bacteria, it is preferable to use Halomonas sp. KM-1 strain, Halomonas panterariensis, or Halomonas meridiana, and it is more preferable to use Halomonas sp. KM-1 strain or Halomonas panterariensis. ..

The halophilic bacterium belonging to the genus Halomonas is preferably a wild-type strain that has not been subjected to treatment such as gene transfer or mutation introduction, regardless of whether it is artificial or accidental, but these are appropriately introduced. May be. The gene and / or mutation to be introduced is not particularly limited as long as it expresses a function of improving the production efficiency of oxaloacetate or a salt thereof in the production method of the present invention. For example, a gene encoding an enzyme having catalytic activity that contributes to the production of oxaloacetate, a gene encoding a protein that exerts a function of increasing the secretion of oxaloacetate from the cells, and an increase in the expression of these genes. Examples include genes to be caused. As a method for introducing these genes into the cells, a general method can be adopted.

<B: Liquid medium>
The liquid medium used in the above step 2 contains (a) an organic carbon source and (b) an inorganic salt A composed of NaHCO ₃ , Na ₂ CO ₃ , and NaCl having a total concentration of 0.2 M to 2.5 M. To do.

The liquid medium used in step 2 contains the inorganic salt A and, if desired, other inorganic salts.

The inorganic salt contained in the liquid medium used in step 2 is not particularly limited as long as it contains at least the inorganic salt A.

Specific examples of the inorganic salt contained in the liquid medium used in step 2 include phosphate; nitrate; carbonate; sulfate; and sodium, magnesium, potassium, manganese, iron, zinc, copper, and the like. A metal salt such as cobalt can be used.

For example, when a sodium salt or a potassium salt is adopted as an inorganic salt, NaCl, KNO ₃ , KNO ₂ , NaNO ₃ , NaNO ₂ , NaHCO ₃ , Na ₂ CO ₃ , Na ₂ HPO ₄ , NaH ₂ PO ₄ , Compounds such as K ₂ HPO ₄ and KH ₂ PO ₄ can be used.

These inorganic salts are preferably those that can be used by the halophilic bacterium as a nitrogen source, a phosphorus source, or the like.

As the nitrogen source, nitrate, nitrite, ammonium salt, urea and the like may be used, and the nitrogen source is not particularly limited. For example, compounds such as NaNO ₃ , NaNO ₂ , NH ₄ Cl, and urea can be used. Preferred are nitrates, nitrites and the like.

The amount of the nitrogen source used can be appropriately set within a range in which the purpose of producing oxaloacetate or a salt thereof is achieved. Specifically, per 100 mL of the liquid medium at the initial stage of culturing (particularly at the start of culturing), usually, the amount of nitrate may be about 500 mg or more, more preferably about 1000 mg or more, still more preferably about 1250 mg or more.

As the phosphorus source, a phosphate, a monohydrogen phosphate, a dihydrogen phosphate or the like may be used, and the phosphorus source is not particularly limited. Specifically, for example, compounds such as Na ₃ PO ₄ , Na ₂ HPO ₄ , NaH ₂ PO ₄ , K ₃ PO ₄ , K ₂ HPO ₄ , and KH ₂ PO ₄ can be used.

The amount of the phosphorus source used can also be appropriately set from the same viewpoint as the amount of the nitrogen source used. Specifically, the dihydrogen phosphate may be in the range of about 50 mg to 400 mg per 100 mL of the liquid medium at the initial stage of culturing (particularly at the start of culturing), and more preferably about 100 mg to 200 mg.

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

Among the above-mentioned inorganic salts, the inorganic salt A (that is, NaHCO ₃ , Na ₂ CO ₃ , and NaCl) is contained in the medium used in step 2 of the present invention. The concentration of the inorganic salt A contained in the liquid medium prepared in step 1 (that is, the concentration of the total amount of NaHCO ₃ , Na ₂ CO ₃ , and NaCl) is 0.2 M to 2.5 M, preferably 0.5 M to 0.5 M. It is about 2.5M, more preferably about 0.8M to 1.5M. The other inorganic salts may be used at a concentration usually in the range of about 0.1 M to 2.5 M in total, preferably about 0.9 M to 2.0 M, and more preferably 0.9 M to 1.2 M. Degree. As is usually understood by those skilled in the art, the concentration of the inorganic salt A is not only the amounts of NaHCO ₃ , Na ₂ CO ₃ and NaCl used for preparing the liquid medium, but also other sodium salts, bicarbonates and carbonates. It is also determined in consideration of the amount of salt, chloro salt, etc.

These concentrations may change as step 2 progresses.

The organic carbon source prepared in step 1 and used in the liquid medium used in step 2 is not particularly limited. For example, trypton, yeast extract, soluble starch, ethanol, n-propanol, acetic acid, sodium acetate, propionic acid, waste glycerol, waste honey sugar, wood saccharified solution, psicose, fructose, sorbose, tagatose, allose, altrose, glucose, Six-carbon sugars such as mannose, growth, idose, galactose, and tarose; five-carbon sugars such as ribulose, xylulose, ribose, arabinose, xylose, lixose, and deoxyribose; disaccharides such as sucrose, lactose, maltose, trehalose, turanose, and cellobiose. ; Sugar alcohols such as erythritol, glycerin, mannitol, sorbitol, xylitol and the like can be mentioned.

The pH of the liquid medium used in the present invention is not particularly limited. For example, it is preferable that the pH satisfies the growth condition of the halophilic bacterium, and specifically, the pH can be in the range of about 5 to pH 12 at least at the time of introduction into the culture system. More preferably, the pH is about 8.8 to pH 12. By using an alkaline medium, it is possible to more effectively prevent contamination with bacteria other than halophilic bacteria belonging to the genus Halomonas. In addition, it is preferable to use an alkaline medium because it suppresses a significant decrease in pH caused by oxaloacetate or a salt thereof secreted from a halophilic bacterium belonging to the genus Halomonas.

In the production method of the present invention, halophilic bacteria belonging to the genus Halomonas can be suitably cultivated in a liquid medium under conditions having a relatively high salt concentration, so that contamination with other bacterial cells, risk of growth, and the like can be almost eliminated. Therefore, it is possible to culture the above-mentioned liquid medium in a simple facility without performing sterilization or the like.

In the present specification, the terms "culture medium" and the term "liquid medium" may be used interchangeably depending on the context.

Here, the term "liquid medium" basically means a substrate for culturing bacteria. In addition, the term "culture solution" is used with the intention of meaning a liquid containing a medium and a culture product, and is usually used with the intention of not containing cells, but depending on the context, It can be used with the intention of containing cells.

<C: Culture method>
The method for culturing the halophilic bacterium belonging to the genus Halomonas in the step 2 is a method for culturing the bacterium while secreting and producing oxaloacetate in the culture solution.

An example of a specific culture method is shown below. First, the halophilic bacterium is inoculated into an appropriate medium of about 5 mL, and pre-cultured at a stirring speed of about 120 rpm to 180 rpm at a predetermined temperature with shaking overnight. Subsequently, the cells obtained by pre-culture are diluted about 100-fold in the liquid medium contained in an Erlenmeyer flask, a fermenter, a jar fermenter, or the like, and the main culture (corresponding to the culture in the present specification). .).

The culture temperature of the main culture can be usually set in the range of about 20 ° C. to 45 ° C. In view of efficient secretory production of oxaloacetate, the culture temperature is usually preferably in the range of about 30 ° C. to 40 ° C. It is more preferably about 31 ° C. to 39 ° C., further preferably about 32 ° C. to 38 ° C., and most preferably about 33 ° C. to 37 ° C.

Examples of the culture method in step 2 include culture methods such as batch culture, semi-batch culture (fed-batch culture), and continuous culture, and are not particularly limited. Since the risk of contamination with other bacteria and proliferation is extremely low in the method of the present invention, long-term continuous culture can also be adopted. This method is preferable in that oxaloacetate or a salt thereof can be efficiently produced.

Step 2 includes a step of adding an inorganic salt to the culture broth before the growth state of the halophilic bacterium in the culture broth shifts to the stationary phase, that is, during the induction phase or the logarithmic phase. May be good.

The logarithmic period is a period in which the concentration of halophilic bacteria in the culture solution is observed to increase logarithmically when measured over time using the absorbance as an index. Such a logarithmic phase also includes a slow-growing phase. The induction period is a period from the start of culturing to the logarithmic period. Such an induction period also includes an acceleration period.

The time when the additional inorganic salt B is added to the culture solution is not particularly limited as long as the growth state of the halophilic bacterium is in the induction phase or the logarithmic phase as described above. For example, when the OD600 value in the culture solution reaches about 5 or more, it can be adopted as the timing for adding the inorganic salt B to the culture solution. The addition of the inorganic salt B into the culture solution is preferably, for example, preferably in the range of about 5 to 30, more preferably about 5 to 25 when the OD600 value in the culture solution is usually 5 or more. It can be carried out within the range, and more preferably within the range of about 5 to 20.

The addition can be continuous or discontinuous.

The number of such additions can be one or more.

The inorganic salt B to be added to the culture solution is not particularly limited, and can be appropriately selected from, for example, the inorganic salts described in detail in <B: Medium>. Among these inorganic salts, NaHCO ₃ , Na ₂ CO ₃ , NaCl, KHCO ₃ , K ₂ CO ₃ , and KCl can be mentioned. Of these, NaHCO ₃ , Na ₂ CO ₃ , and NaCl are preferred. The inorganic salts added to these culture broths may be used alone or in combination of two or more.

As for the inorganic salt B to be added to the culture solution, for example, the total amount of the inorganic salt A and the amount of the inorganic salt B used at the start of the culture is usually 0.2 M to 2.5 M in the solution concentration after the addition. Within the range of, preferably within the range of about 0.22M to 2.0M, more preferably within the range of about 0.23M to 1.8M, still more preferably within the range of about 0.24M to 1.5M, and Even more preferably, it can be added in an amount in the range of about 0.25M to 1.3M.

In the case of semi-batch culture and continuous culture, the concentration of halophilic bacteria present in the system decreases as a result of changing the culture medium solution, and the above-mentioned inorganic salt may be added at that time.

In the step 2, a nitrogen source can be added to the culture broth. As a specific nitrogen source, the one described in <B: Liquid Medium> described above can be appropriately adopted, and is not particularly limited, and for example, it is preferable to add nitrate to the culture solution.

The time when the nitrogen source is added to the culture broth is not particularly limited, and for example, the growth state of halophilic bacteria may be any state from the induction stage to the quiescent stage. The number of times the nitrogen source is added to the culture solution is not particularly limited, and can be usually about 1 to 10 times. When adding a nitrogen source to the culture broth multiple times, the addition interval may be determined appropriately. For example, it may be added according to the growth state of halophilic bacteria in the culture solution, or may be simply added at an appropriate time in time during the culture period.

The amount of the nitrogen source added to the culture solution is also not particularly limited. For example, the total amount of the nitrogen source to be added is usually in the range of about 0.1 M to 0.2 M after addition, more preferably. The range is about 0.12M to 0.18M, more preferably about 0.13M to 0.17M, and particularly preferably about 0.14M to 0.16M.

Step 2 can be preferably carried out without controlling the pH, but controlling the pH is not excluded. The pH can be controlled by adding an acidic substance (eg, hydrochloric acid) and / or an alkaline substance (eg, sodium hydroxide) to the culture system according to a conventional method.

The pH can be controlled, for example, so that the pH of the culture medium is the same as the pH described for the liquid medium.

Process 3
Step 3 of the production method of the present invention is a step of recovering oxaloacetate or a salt thereof from the culture solution obtained in the step 2.

For the recovery, a conventional method may be used. Specifically, in the case of batch culture or the like, when oxaloacetate or a salt thereof is present in the culture solution obtained in step 2, the culture in step 2 is stopped, and oxaloacetate or a salt thereof is added as needed. It can be recovered by providing the containing culture solution and the halophilic cell as a separation means and obtaining the culture solution.

It is also possible to recover biopolymer PHB, 3-hydroxybutyric acid, etc. from the isolated cells according to the method described in Patent Document 1 and the like.

The method for stopping the culture is not particularly limited, and examples thereof include a method for sterilizing halophilic bacteria belonging to the genus Halomonas obtained in the above step 2 by a method such as heating or acid treatment. Further, a method of separating the culture solution and the halophilic cells and stopping the culture may be adopted while appropriately combining known solid-liquid separation means such as centrifugation, filtration, and membrane separation.

The method for confirming the presence of oxaloacetate or a salt thereof in the culture solution can vary depending on the bacterial species, medium components, culture conditions, etc., and is appropriately determined in consideration of these factors. For example, the culture solution can be collected over time and used for analysis methods such as HPLC and an analysis kit to determine a preferable time for stopping the culture.

In addition, since oxaloacetate is a compound showing acidity, the presence of oxaloacetate can be confirmed by using the decrease in the pH of the medium during culture as an index while monitoring the pH of the culture solution over time. Good.

The recovered oxaloacetate salt should be recovered as a metal salt that has reacted with alkali metals such as sodium and calcium derived from inorganic salts contained in the culture solution, or cations such as alkaline earth metals. There is. Therefore, in order to produce oxaloacetate, the recovered culture solution may be subjected to a conventional method such as salting out.

In addition, the recovered culture solution may be subjected to a purification step by column chromatography using an appropriate column. As a method other than these, the pH of the recovered culture solution can be appropriately changed, and either the desired oxaloacetate or a salt thereof can be subjected to the purification step.

Since the production method of the present invention requires almost no consideration for contamination and consideration for keeping the culture environment in a sterilized state, oxaloacetate or a salt thereof can be easily obtained at low cost.

An experimental example for explaining the present invention in more detail is shown below. Needless to say, the present invention is not limited to the experimental examples shown later.

<Medium>
The SOT-modified 5 medium used in the test examples described below is that described in Patent Document 1. The specific medium composition is as shown in Table 1. The medium used in the following test examples was used without autoclaving.

<Measurement of oxaloacetate or its salt>
The measurement of pyruvic acid or a salt thereof produced in the culture solution in this experimental example was carried out in accordance with the analytical conditions described in the attachment to Aminex HPX-87H of BIO-RAD, and the detection was performed by Shimadzu RID- The method using a 10A differential refractometer detector was performed.

First, the culture solution obtained in the experiment described later was centrifuged. 50 μL of the obtained supernatant was collected, and 450 μL of water was added thereto to prepare a sample for measurement. This measurement sample was analyzed by the HPLC and compared with the oxaloacetate standard.

<Preculture of halophilic bacteria belonging to the genus Halomonas>
Halophiles belonging to the genus Halomonas were added from a plate culture to a 16.5 mm diameter test tube containing 5 mL of the SOT5 modified medium (in this case, one containing 1 w / v% glucose or the like as a carbon source). Then, the cells were cultured with shaking at 37 ° C. overnight.

<Culture of various halophilic bacteria belonging to the genus Halomonas, collection of various samples and their identification>
In the following, various experimental examples confirming the production of oxaloacetate using halophilic bacteria belonging to the genus Halomonas are shown.

Experimental Example 1
0.2 mL Halomonas sp. Belonging to the various Halomonas genus pre-cultured above. The KM-1 strain was mixed with 20 mL of the SOT-modified 5 medium in a 200 mL Erlenmeyer flask and inoculated to give silicosen. This was cultured with shaking at 33 ° C. under the condition that the stirring speed was 200 rpm, and 0.25 mL of the culture solution was collected every 6 hours or 12 hours from 12 hours after the start of the culture, and the culture supernatant was added. The amount of oxaloacetate or a salt thereof (hereinafter referred to as oxaloacetate) secreted was measured.

At the beginning of the culture, the cells were cultured under aerobic conditions with a stirring speed of 200 rpm, the culture solution was sampled, silicosen was added again, and shaking culture was continued at 33 ° C., and the culture was carried out in half batches.

At the start of culturing, glucose was added to the SOT-modified medium at a concentration of 20 wt% and used. As the nitrogen source, 12.5 g / L of sodium nitrate is used at the beginning of the culture as shown in the above-mentioned SOT modified 5 medium composition, but 18 hours, 24 hours, 36 hours, and 48 hours after the start of the culture, And after 60 hours, 2.5 g / L of sodium nitrate was added to the culture medium, respectively.

The SOT-modified 5 medium composition contains about 0.2 M of inorganic salts including NaHCO ₃ , Na ₂ CO ₃ , and NaCl. Here, those in which NaHCO ₃ was not added to the culture solution (Control), those in which 1.0 M NaHCO ₃ was added to the culture solution 18 hours after the start of the culture corresponding to the logarithmic phase, and 18 from the start of the culture. After 1 hour and 24 hours (24 hours after the start of culture also corresponds to the logarithmic phase), 1.0 M of NaHCO ₃ was added to the culture medium, respectively, but the amount of oxaloacetate secreted in a total of 3 culture patterns. Was compared. The result is shown in FIG.

As shown in FIG. 1, the secretion of oxaloacetate was confirmed 24 hours after the start of the culture. In particular, when NaHCO ₃ was added to the culture solution only 18 hours after the start of the culture, the amount of oxaloacetate secreted as much as 20 g / L was confirmed 42 hours and 48 hours after the start of the culture. Further, when 1.0 M NaHCO ₃ was added to the culture broth 18 hours and 24 hours after the start of the culture, the amount of oxaloacetic acid secreted was about 10 g / L.

From the above results, it was clarified that the amount of oxaloacetate produced in the culture medium was increased by adding NaHCO ₃ to the culture medium at an appropriate time during the logarithmic period during aerobic culture.

Experimental Example 2
Halomonas sp. Belonging to various pre-cultured Halomonas genus. 0.2 mL of the KM-1 strain cells was mixed with 20 mL of the SOT-modified 5 medium in a 200 mL Erlenmeyer flask and inoculated to obtain silicosen. This was cultured with shaking at 33 ° C. under the condition that the stirring speed was 200 rpm, and 0.25 mL of the culture solution was collected every 12 hours or 6 hours from 12 hours after the start of the culture, and the culture supernatant was added. The amount of oxaloacetate or a salt thereof (hereinafter referred to as oxaloacetate) secreted was measured.

At the beginning of the culture, the cells were cultured under aerobic conditions with a stirring speed of 200 rpm, the culture solution was sampled, silicosen was added again, and shaking culture was continued at 33 ° C., and the culture was carried out in half batches.

At the start of culturing, glucose was added to the SOT-modified medium at a concentration of 20 wt% and used. As the nitrogen source, 12.5 g / L of sodium nitrate is used at the beginning of the culture as shown in the above-mentioned SOT modified 5 medium composition, but 18 hours, 24 hours, 36 hours, and 48 hours after the start of the culture, And after 60 hours, 2.5 g / L of sodium nitrate was added to the culture medium, respectively.

The SOT-modified 5 medium composition contains approximately 0.2 M of salt, including NaHCO _3, Na ₂ CO ₃ , and NaCl. Here, those in which no additional inorganic salt was added (Control), those in which 0.8 M NaCl ₃ was added to the culture medium 18 hours after the start of the culture corresponding to the logarithmic phase, and 0 in 18 hours after the start of the culture. Although 0.8 M NaCl was added to the culture medium, the amount of oxaloacetate secreted in a total of three culture patterns was compared. The result is shown in FIG.

As shown in FIG. 2, a significant amount of oxaloacetate is secreted 24 hours after the start of the culture, and when NaHCO ₃ is added to the culture solution only 18 hours after the start of the culture, it is secreted 36 hours after the start of the culture. The maximum value of the amount reached 14 g / L. Further, when NaCl was added to the culture solution only 18 hours after the start of the culture, the maximum value of the secreted amount reached 20 g / L 48 hours after the start of the culture.

From the above results, at a given timing during log phase in aerobic culture, the addition of NaCl or NaHCO _3, and revealed that increased production amount in culture oxaloacetate.

Experimental Example 3
Halomonas sp. Belonging to various pre-cultured Halomonas genus. 0.2 mL of the KM-1 strain cells was mixed with 20 mL of the SOT-modified 5 medium in a 200 mL Erlenmeyer flask and inoculated to obtain silicosen. This was set to various temperatures of 30 ° C., 33 ° C., 37 ° C., and 40 ° C., and shake-cultured under the condition that the stirring speed was 200 rpm, and the culture was carried out approximately every 12 hours or 6 hours from 12 hours after the start of the culture. The liquid was collected by 0.25 mL each, and the amount of oxaloacetate or a salt thereof (hereinafter referred to as oxaloacetate) secreted in the culture supernatant was measured.

At the beginning of the culture, the cells were cultured under aerobic conditions with a stirring speed of 200 rpm, the culture solution was sampled, silicosen was added again, shaking culture was continued at the same temperature, and half-birth culture was performed.

At the start of culturing, glucose was added to the SOT-modified medium at a concentration of 20 wt% and used. As the nitrogen source, 12.5 g / L sodium nitrate is used at the beginning of the culture as shown in the above-mentioned SOT Kai 5 and medium composition, but 18 hours, 24 hours, 36 hours, and 48 hours after the start of the culture. After 60 hours, 2.5 g / L of sodium nitrate was added to the culture medium, respectively.

The SOT-modified 5 medium composition contains approximately 0.2 M of salt, including NaHCO _3, Na ₂ CO ₃ , and NaCl. Here, the amount of oxaloacetate secreted in the culture patterns under a total of eight temperature conditions was compared between those in which 0.8 M NaHCO ₃ was added to the culture solution 18 hours after the start of the culture and those in which this was not added. .. The result is shown in FIG.

As shown in FIG. 3, when NaHCO ₃ was added to the culture medium 18 hours after the start of the culture, secretion of oxaloacetate was observed 24 hours after the start of the culture in each case, especially at 37 ° C. and 40 ° C. In the case of culturing, the maximum value of secretion amount reached 25.7 g / L 42 hours after the start of culturing.

From the above results, by adding NaHCO ₃ at an appropriate time in the logarithmic period during aerobic culture, in the culture solution of oxaloacetate under any temperature condition of 30 ° C to 40 ° C. It was revealed that the production amount of was increased. In particular, it was clarified that the amount of oxaloacetate produced increased remarkably when cultured at 37 ° C. or 40 ° C.

Experimental Example 4
Halomonas sp. Belonging to various pre-cultured Halomonas genera. 0.2 mL each of the cells of KM-1 strain, Halomons panteleriensis: ATCC700273, and Halomons meridian: NBRC15608 was mixed with 20 mL of the SOT modified 5 medium in a 200 mL Erlenmeyer flask and inoculated with silicosen. .. This was cultured with shaking at 37 ° C. under the condition that the stirring speed was 200 rpm, and 0.25 mL of the culture solution was collected at intervals of about 12 hours or 6 hours 12 hours after the start of the culture, and the culture supernatant was added. The amount of oxaloacetate or a salt thereof, hereinafter referred to as oxaloacetate) was measured.

At the beginning of the culture, the cells were cultured under aerobic conditions with a stirring speed of 200 rpm, the culture solution was sampled, silicosen was added again, shaking culture was continued at the same temperature, and half-birth culture was performed.

At the start of culturing, glucose was added to the SOT-modified medium at a concentration of 20 wt% and used. As the nitrogen source, 12.5 g / L of sodium nitrate is used at the beginning of the culture as shown in the above-mentioned SOT modified 5 medium composition, but 18 hours, 24 hours, 36 hours, and 48 hours after the start of the culture, And after 60 hours, 2.5 g / L of sodium nitrate was added to the culture medium, respectively.

The SOT-modified 5 medium composition contains approximately 0.2 M of salt, including NaHCO _3, Na ₂ CO ₃ , and NaCl. Here, 18 hours after the start of culturing, 0.8 M of NaHCO ₃ was added to the culture broth, and although this was not added, the amount of oxaloacetate secreted in the culture patterns under a total of 6 cell conditions. Was compared. The result is shown in FIG.

As shown in FIG. 4, when the KM-1 strain and the BRC15608 strain were used, when 0.8 M of NaHCO ₃ was added to the culture medium, oxaloacetate secretion was observed 24 hours after the start of the culture, and the culture was started. After 48 hours, each showed a maximum value exceeding 23.5 g / L. On the other hand, when 0.8 M of NaHCO ₃ was not added, almost no oxaloacetate was secreted. Further, in the case of ATCC 700273, the amount of oxaloacetate secreted was as small as 9.1 g / L at the maximum, but the same tendency as that of the KM-1 strain and the BRC15608 strain was shown.

From the above results, it was suggested that the KM-1 strain or the BRC15608 strain is more suitable for the secretory production of oxaloacetate as a halophilic bacterium belonging to the genus Halomonas.

Experimental Example 5
Halomonas sp. Belonging to various pre-cultured Halomonas genus. 0.2 mL of the KM-1 strain cells was mixed with 20 mL of the SOT-modified 5 medium in a 200 mL Erlenmeyer flask and inoculated to obtain silicosen. This was cultured with shaking at 37 ° C. under the condition that the stirring speed was 200 rpm, and 0.25 mL of the culture solution was collected every 12 hours or 6 hours from 12 hours after the start of the culture, and the culture supernatant was added. The amount of oxaloacetate or a salt thereof (hereinafter referred to as oxaloacetate) secreted was measured.

At the beginning of the culture, the cells were cultured under aerobic conditions with a stirring speed of 200 rpm, the culture solution was sampled, silicosen was added again, shaking culture was continued at the same temperature, and half-birth culture was performed.

At the start of culturing, glucose was added to the SOT-modified medium at a concentration of 20 wt% and used. As the nitrogen source, 12.5 g / L of sodium nitrate is used at the beginning of the culture as shown in the above-mentioned SOT modified 5 medium composition, but 18 hours, 24 hours, 36 hours, and 48 hours after the start of the culture, And after 60 hours, 2.5 g / L of sodium nitrate was added to the culture medium, respectively.

The SOT-modified 5 medium composition contains approximately 0.2 M of salt, including NaHCO _3, Na ₂ CO ₃ , and NaCl. The amount of oxaloacetate secreted in a total of three culture patterns, one in which 0.8 M NaHCO ₃ was added to the culture solution at the start of the culture or 18 hours after the start of the culture, and the other in which this was not added (Control). Compared. The result is shown in FIG.

FIG. 5 shows Halomonas sp. It is an OD600 value (OD ₆₀₀ ) indicating the growth state of the KM-1 strain cells. 18 hours after the start of the culture, the control using only the SOT5 modified medium had an OD ₆₀₀ value of about 18, and 18 hours after the start of the culture, the OD value changed before and after that time, so that the halophilic bacteria in the medium were It can be seen from the induction period to the logarithmic period. In addition, when NaHCO ₃ was added to the SOT5 medium at the start of culturing, the growth was suppressed with an OD ₆₀₀ value of about 9, but halophilic bacteria were also in the induction phase or logarithmic phase as described above. It turns out that.

As shown in FIG. 6, oxaloacetate secretion was observed 24 hours after the start of the culture. When 0.8 M of NaHCO ₃ was added to the SOT5 modified medium at the start of the culture, the maximum value of oxaloacetate secretion of 14.8 g / L or more was shown 42 hours after the start of the culture. In addition, when 0.8 M of NaHCO ₃ was added to the culture solution 18 hours after the start of the culture, the maximum value of 13.4 g / L of oxaloacetate secretion was shown 18 hours after the start of the culture.

From the above results, it is clear that oxaloacetate can be efficiently secreted and produced by increasing the concentration of the inorganic salt used at the start of the culture without adding the inorganic salt to the culture solution during the logarithmic phase of Halomonas. It became.

Experimental Example 6
Halomonas sp. Belonging to various pre-cultured Halomonas genus. 0.2 mL of the KM-1 strain cells was mixed with 20 mL of the SOT-modified 5 medium in a 200 mL Erlenmeyer flask and inoculated to obtain silicosen. This was cultured with shaking at 37 ° C. under the condition that the stirring speed was 200 rpm, and 0.25 mL of the culture solution was collected every 12 hours or 6 hours from 12 hours after the start of the culture, and the culture supernatant was added. The amount of oxaloacetate or a salt thereof (hereinafter referred to as oxaloacetate) secreted was measured.

At the beginning of the culture, the cells were cultured under aerobic conditions with a stirring speed of 200 rpm, the culture solution was sampled, silicosen was added again, shaking culture was continued at the same temperature, and half-birth culture was performed.

At the start of culturing, glucose was added to the SOT-modified medium at a concentration of 20 wt% and used. As the nitrogen source, 12.5 g / L sodium nitrate is used at the beginning of the culture as shown in the above-mentioned SOT-modified 5 medium composition, but 18 hours, 24 hours, 36 hours, and 48 hours after the start of the culture, And 60 hours later, 2.5 g / L of sodium nitrate was added, respectively.

The SOT-modified 5 medium composition contains approximately 0.2 M of salt, including NaHCO _3, Na ₂ CO ₃ , and NaCl. Here, plus NaHCO ₃ in 0.3 M (final salt concentration 0.5M equivalent) supplemented with NaHCO ₃ in 0.8 M (corresponding final salt concentration 1.0 M), and NaHCO ₃ in 1.3M Was added (equivalent to a final salt concentration of 1.5 M), and the amount of oxaloacetate secreted in a total of three culture patterns was examined.

As shown in FIG. 7, except for the control, 0.3 M NaHCO ₃ was added, 0.8 M NaHCO ₃ was added, and 1.3 M NaHCO ₃ was added to the medium at the start of the culture. The maximum time of oxaloacetate secretion tended to shift to the late stage of culture in the order of the ones, and all of them secreted oxaloacetate of 8 g / L or more.

From the above results, it is clear that oxaloacetate can be efficiently secreted and produced by increasing the concentration of the inorganic salt used at the start of the culture without adding the inorganic salt to the culture solution during the logarithmic phase of Halomonas. It became.

Claims (8)

A method for producing oxaloacetate or a salt thereof, which comprises the following steps 1, 2, and 3;
(1) A step of preparing a liquid medium containing (a) an organic carbon source and (b) an inorganic salt A composed of NaHCO ₃ , Na ₂ CO ₃ , and NaCl having a total concentration of 0.2 M to 2.5 M. 1
(2) Step 2 of culturing halophilic bacteria belonging to the genus Halomonas using the liquid medium prepared in step 1.
(3) A step 3 of recovering oxaloacetate or a salt thereof from the culture solution obtained in the above step 2. The production method according to claim 1, wherein in the step 2, the inorganic salt B is added to the liquid medium when the growth state of the halophilic bacterium is in the induction phase or the logarithmic phase. The production method according to claim 2, wherein the addition of the inorganic salt B is carried out when the OD600 value of the culture solution is 5 or more. The production method according to claim 2 or 3, wherein the inorganic salt B is at least one selected from the group consisting of NaHCO ₃ , Na ₂ CO ₃ , NaCl, KHCO ₃ , K ₂ CO ₃ , and KCl. The production method according to any one of claims 2 to 4, wherein the total concentration of the inorganic salt B and the inorganic salt A is in the range of 0.2M to 2.5M. The production method according to any one of claims 1 to 5, wherein the culture temperature is 30 ° C to 40 ° C. The production method according to any one of claims 1 to 6, wherein a nitrogen source is added to the culture solution in the step 2. Claims 1 to 7, wherein the halophilic bacterium is either Halomonas sp. KM-1 strain, Halomonas panterariensis, Halomonas campisaris, Halomonas nitritofilus, Halomonas alimentaria, or Halomonas meridiana. The manufacturing method according to any one.