JP2015204769A - Production method of pyruvic acid using halomonas bacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fermentation production method of pyruvic acid, particularly to provide an inexpensive and simple fermentation production method of pyruvic acid.SOLUTION: A production method of pyruvic acid or a salt thereof comprises the following steps (1)-(3): (1) a step 1 of performing an aerobic culture of halobacteria belonging to Halomonas bacteria in a culture medium containing an organic carbon source and mineral salt, (2) a step 2 of adding at least one selected from the group consisting of a nitrogen source, metal salt, and borate into the culture medium, and (3) a step 3 of collecting pyruvic acid or a salt thereof from the culture medium obtained in the step 2.

Description

  The present invention relates to a method for producing pyruvic acid using Halomonas bacteria.

  In recent years, not only energy but also biorefinery of chemical refineries and conversion of industrial raw materials from petroleum to biomass have become issues.

  Pyruvate is an important compound in the metabolism of many organisms, and is a product of anaerobic metabolism of glucose known as a glycolytic system. During anaerobic metabolism, one glucose molecule is broken down into two pyruvate molecules, and in the subsequent aerobic metabolism process, pyruvate is a series of reactions known as the Krebs cycle (TCA cycle, citrate cycle). It is converted into acetyl coenzyme A, which is the main material. Pyruvate is also converted to oxaloacetate by a supplementary reaction. And oxaloacetate supplements the intermediate of the Krebs cycle and is also used for gluconeogenesis.

  In an environment where sufficient oxygen is not supplied, pyruvic acid is decomposed anaerobically to produce lactic acid, ethanol, and the like. Pyruvate produced from the glycolytic system is converted into acetaldehyde and further ethanol in lactate fermentation using lactate dehydrogenase and coenzyme NADH or in alcohol fermentation. In addition, pyruvic acid can be converted to fatty acid; energy; alanine; ethanol and the like via carbohydrate, acetyl-CoA and the like by gluconeogenesis. That is, pyruvic acid is a key compound in the metabolic pathway network.

  The use of pyruvic acid is highly reactive, so it is used as an important intermediate in fine chemicals such as synthetic substrates for pharmaceuticals and agricultural chemicals, and because it does not go through glycolysis when supplying energy to cells. Application to infusions and supplements is also expected.

  As a method for producing pyruvic acid, fermentative production of pyruvic acid by Candida sp. MC-P0101 strain (Candida sp. MC-P0101) FERM BP-7539 belonging to Candida is shown (Patent Document 6). .

  While examining the efficient culture method of the microalga Spirulina, the present inventors have recognized that, under certain conditions, halophilic bacteria belonging to the genus Halomonas grow as the only contaminating bacteria. The halophilic bacterium itself grows well in a medium containing a high concentration of sodium, usually under a pH of about 5 to 12, so that contamination with other bacteria is extremely difficult even under aerobic fermentation. It is known. Then, assimilation of various carbon sources of the halophilic bacteria was examined, it was clarified that a significant amount of PHB was accumulated in the cells of the halophilic bacteria (Patent Document 1). Furthermore, as the investigation proceeds, the Halomonas sp. Accumulates PHB in the cells under aerobic conditions and then shifts to microaerobic conditions, so that 3-hydroxybutyric acid, a monomer of PHB, is added to the culture solution. It has been found that it is secreted (Patent Documents 2 and 3). In addition, while proceeding separately, alpha ketoglutaric acid and glutamic acid are cultured in a culture solution by secreting lactic acid, acetic acid and the like into the medium in the early stage of growth (Patent Document 4) and using a medium containing biodiesel waste glycerol and the like. It was found that it is secreted and produced (Patent Document 5).

  In the course of diligent examination of fermentation techniques using genus Halomonas, we found a phenomenon in which the increase in the amount of PHB accumulated in the cells reached a peak, although glucose was consumed when the cell concentration increased.

International Publication No. 2009/041531 Pamphlet Japanese Unexamined Patent Publication No. 2013-081403 Japanese Patent Application No. 2014-030915 Japanese Patent No. 5229904 Japanese Patent Application No. 2012-170385 Japanese Patent Laid-Open No. 2003-024048

  A sterilizing environment is essential for culturing the Candida spp described in Patent Document 6 as described above, and it requires expensive medium components such as polypeptone and yeast extract. It is hard to say that the manufacturing method.

  An object of this invention is to provide the fermentative production method of pyruvic acid. In particular, an object is to provide an inexpensive and simple method for fermentative production of pyruvic acid.

  The inventors of the present invention have been studying the culture under a medium condition that accumulates a significant amount of PHB in the fermentation production process using a halophilic bacterium. In the presence of an excess carbon source, the cell concentration increased. It was confirmed that a decrease in the pH of the culture solution was observed. Thus, when this culture solution was analyzed, it was found that a significant amount of pyruvic acid and the like was accumulated in the culture solution.

  When polyhydroxyalkanoates (PHAs) are accumulated in the genus Halomonas by fermentation of halophilic bacteria, a part of the carbon source is secreted into the culture solution as lactic acid, acetic acid, etc. If it is taken up again by the cells and used as a nutrient source and the culture time is prolonged, lactic acid, acetic acid and the like are lost from the medium as a result.

  On the other hand, pyruvic acid continues to be secreted into the culture solution without progressing synthesis to PHB even if the cell concentration increases in the culture solution of Halomonas spp. It was confirmed that the pH of the solution was lowered.

  Furthermore, since the present inventors can produce pyruvic acid under high alkaline conditions without using expensive peptone or yeast extract, halophilic bacteria are less prone to culturing at low cost and contamination of other bacteria. It was found that pyruvic acid can be produced by culturing in an environment.

  The present invention has been completed by earnest studies based on these findings, and includes the inventions of the following broad aspects.

Item 1 A method for producing pyruvic acid or a salt thereof, comprising the following steps (1) to (3):
(1) Step 1 of aerobically cultivating a halophilic bacterium belonging to the genus Halomonas in a medium containing an organic carbon source and an inorganic salt,
(2) Step 2 of adding at least one selected from the group consisting of a nitrogen source, a metal salt, and a borate to the culture solution,
(3) Step 3 for recovering pyruvic acid or a salt thereof from the culture solution obtained in Step 2.

  Item 2 The method according to Item 1, wherein the nitrogen source in Step 2 is at least one selected from the group consisting of nitrate, nitrite, ammonium salt, and urea.

  Item 3. The method according to claim 1 or 2, wherein the metal salt in Step 2 is at least one metal selected from the group consisting of zinc salt, molybdenum salt, manganese salt, copper salt, and cobalt salt.

  Item 4 The above item 1 to item 3, wherein the time when at least one selected from the group consisting of a nitrogen source, a metal salt, and a borate in step 2 is added is a time when the OD600 of the culture solution is 30 or more. The manufacturing method of any one of these.

  Item 5 The method according to any one of Items 1 to 4, wherein the culture condition in Step 1 is 20 to 45 ° C.

  Item 6 The method according to any one of Items 1 to 5, wherein 10 g or more of pyruvic acid or a salt thereof is contained per liter of the culture solution obtained in Step 2.

  Item 7. The method according to any one of Items 1 to 6, wherein the halophilic bacterium is Halomonas sp. KM-1 strain (FERM BP-10995).

  The production method according to the present invention can produce pyruvic acid or a salt thereof in a culture solution.

  The production method according to the present invention includes a step of culturing a halophilic bacterium belonging to the genus Halomonas, and the culturing of the halophilic bacterium can be cultivated in an environment where contamination with other bacteria hardly occurs. In addition, culture in a non-sterile medium and / or a non-sterile environment is possible. Furthermore, it is an excellent manufacturing method because the air supply conditions can be easily changed.

  The halophilic bacteria used in the production method according to the present invention include, for example, waste glycerol by-produced in biodiesel production, wood saccharified liquid produced in the process of ethanol fermentation, etc .; An organic acid or the like obtained by fermentation can be used alone as an organic carbon source or in combination with other organic carbon sources. Furthermore, pentoses such as xylose and arabinose, which are obtained in ethanol fermentation using yeast cells and are difficult to use, can be effectively used as an organic carbon source.

  In the production method according to the present invention, pyruvic acid or a salt thereof can be produced in a culture solution. Since pyruvic acid is produced during the growth of a series of genus Halomonas, pyruvic acid can be produced in one stage of culture.

  In addition, it is possible to easily recover a fraction containing pyruvic acid or a salt thereof from a bacterial cell culture solution, and even if a purification step is performed, a simple purification method can be applied, and thus it is an excellent production method. .

  In this regard, in the production method according to the present invention, pyruvic acid or a salt thereof can be recovered from the culture solution under conditions that do not involve lysis of halophilic bacteria belonging to the genus Halomonas. When purifying the protein, there is an effect that the purification process for removing contaminant molecules such as nucleic acids, proteins, carbohydrates, lipids and the like accompanying lysis becomes very simple.

  In particular, according to the method of the present invention, the culture solution for recovering pyruvic acid is alkaline, and it is an environment in which pyruvic acid is difficult to form a salt, so that it is effective for recovering pyruvic acid.

  The pyruvic acid or salt thereof obtained by the production method according to the present invention is not only added to medical infusions, etc., but also used as health foods, etc., and is useful as a raw material for cosmetics, pharmaceuticals, etc. is there.

The graph which shows the dry cell weight CDM (vertical axis: g / L and culture | cultivation time (horizontal axis: h)) obtained by the method shown in the manufacture example 1 of an Example.

The black circles in the graph indicate the results of adding 2.5 g / L sodium nitrate at 18 hours, 24 hours, 36 hours, 48 hours, and 60 hours from the start of culture. The triangles (black triangles) in the graph indicate the results when sodium nitrate was not added after the start of culture.
The graph which shows the pyruvic acid density | concentration (vertical axis: pyruvic acid (g) / culture supernatant (L) and culture | cultivation time (horizontal axis: h)) obtained by the method shown in the manufacture example 1 of an Example.

The black circles in the graph indicate the results of adding 2.5 g / L sodium nitrate at 18 hours, 24 hours, 36 hours, 48 hours, and 60 hours from the start of culture. The triangles (black triangles) in the graph indicate the results when sodium nitrate was not added after the start of culture.
The graph which shows the dry cell weight CDM (vertical axis: g / L and culture | cultivation time (horizontal axis: h)) obtained by the method shown in the manufacture example 2 of an Example.

The black circles in the graph indicate the results of culturing at 33 ° C. from the start of culture. ▲ (black triangle) in the graph indicates the result of culturing at 37 ° C. from the start of the culture. The black squares in the graph indicate the results of culturing at 40 ° C. from the start of culture.
The graph which shows the pyruvic acid density | concentration (vertical axis: pyruvic acid (g) / culture supernatant (L) and culture | cultivation time (horizontal axis: h)) obtained by the method shown in the manufacture example 2 of an Example.

The black circles in the graph indicate the results of culturing at 33 ° C. from the start of culture. ▲ (black triangle) in the graph indicates the result of culturing at 37 ° C. from the start of the culture. The black squares in the graph indicate the results of culturing at 40 ° C. from the start of culture.
The graph which shows the dry cell weight CDM (vertical axis: g / L and culture | cultivation time (horizontal axis: h)) obtained by the method shown in the manufacture example 3 of an Example.

● (black circle) in the graph indicates Halomonos sp. The result of KM-1 is shown. The black triangles in the graph indicate the results of Halomons meridian: NBRC15608. The black squares in the graph indicate the results of Halomons pantererensis: ATCC 7000027. The diamonds (black diamonds) in the graph indicate the results of Halomonas campisalis: ATCC 700597.
The graph which shows the pyruvic acid density | concentration (vertical axis: pyruvic acid (g) / culture supernatant (L) and culture | cultivation time (horizontal axis: h)) obtained by the method shown in the manufacture example 3 of an Example.

  ● (black circle) in the graph indicates Halomonos sp. The result of KM-1 is shown. The black triangles in the graph indicate the results of Halomons meridian: NBRC15608. The black squares in the graph indicate the results of Halomons pantererensis: ATCC 7000027. The diamonds (black diamonds) in the graph indicate the results of Halomonas campisalis: ATCC 7000059.

  The manufacturing method of pyruvic acid or its salt which concerns on this invention is a manufacturing method including the following process (1)-(3).

(1) Step 1 of aerobically cultivating a halophilic bacterium belonging to the genus Halomonas in a medium containing an organic carbon source and an inorganic salt,
(2) Step 2 of adding at least one selected from the group consisting of a nitrogen source, a metal salt, and a borate to the culture solution,
(3) Step 3 for recovering pyruvic acid or a salt thereof from the culture solution obtained in Step 2.

  The salt of pyruvic acid produced by the production method according to the present invention is a cation derived from a component contained in a medium of a halobacterium belonging to the genus Halomonas used at the time of production and / or a cation present in the microbial cell. Although not particularly limited, for example, sodium salt, potassium salt, calcium salt, magnesium salt, cobalt salt, zinc salt, iron salt, copper salt, molybdenum salt, ammonium salt, lithium salt, silver Examples include salts, mercury salts, strontium salts, barium salts, cadmium salts, nickel salts, tin salts, lead salts, manganese salts, aluminum salts, and the like.

Step 1 Step 1 in the method for producing pyruvic acid or a salt thereof according to the present invention is a step of aerobic culture of halophilic bacteria belonging to the genus Halomonas in a medium containing an organic carbon source and an inorganic salt.

<A: halophilic bacteria>
The halophilic bacterium used in the production method step 1 of the present invention may be a halophilic bacterium belonging to the genus Halomonas represented by either (i) or (ii) below.
(I) A halophilic bacterium characterized by growing aerobically in a medium containing an inorganic salt and a single or a plurality of organic carbon sources to produce pyruvic acid or a salt thereof in a medium outside the cell.
(Ii) A halophilic salt characterized by secreting and producing pyruvic acid or a salt thereof in a culture solution outside the cell after aerobically growing in a medium containing an inorganic salt and a single or a plurality of organic carbon sources. Fungus.

  The “inorganic salt” and “organic carbon source” will be described later in the section <Medium>. Such halophilic bacteria belonging to the genus Halomonas can use both oxidative metabolism and anaerobic metabolism, can survive regardless of the presence of free oxygen, and grow in the presence of free oxygen. It is a microbial cell having the property of a so-called facultative anaerobic bacterium that tends to be easy.

  The above-mentioned halophilic bacteria belonging to the genus Halomonas are bacteria that have a halophilic property with a suitable salt concentration of 0.1 to 1.0 M and sometimes grow even in a medium containing no salt. And the halophilic bacteria which belong to the above-mentioned genus Halomonas usually grow on a medium of about pH 5-12.

  Examples of the halophilic bacteria belonging to the genus Halomonas include the Halomonas sp. KM-1 strain. Halomonas SP KM-1 was commissioned on July 10, 2007 to the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (Central 1-1, Higashi 1-1-1 Tsukuba City, 305-8586 Ibaraki Prefecture). Deposited under the number FERM P-21316. This strain has now been transferred to an international deposit and its deposit number is FERM BP-10995. The 16S rRNA gene of the Halomonas sp. KM-1 strain is registered as Accession Number AB477015 in DDBJ.

  Further, in view of the growth characteristics of halophilic bacteria belonging to the genus Halomonas as described above, as halophilic bacteria used in the production method according to the present invention, in addition to Halomonas sp. KM-1 strain, Halomonas pantheraliensis ( Halomonas pantelliensis (ATCC 700033), Halomonas campisalis (ATCC 7000059), Halomonas meridiana (NBRC15608), etc. can also be mentioned.

  Furthermore, from analysis by 16S ribosomal RNA sequence, not only the halophilic bacteria belonging to the genus Halomonas but also Halomonas nitritofilus, Halomonas amentitalia, Halomonas meridiana and the like are used in the production method according to the present invention. It may be used as a halophilic bacterium belonging to.

  A gene may be introduced into the halophilic bacteria belonging to the genus Halomonas described above. The gene to be introduced is not particularly limited as long as it can express the function of improving the production efficiency of pyruvic acid or a salt thereof in the production method according to the present invention. For example, a gene that increases the expression level of pyruvic acid, a gene that expresses the function of increasing the secretion of pyruvic acid outside the cells, a gene that increases the function of producing pyruvic acid or a salt thereof in a culture solution; Examples include genes that increase the amount of acid or its salt produced. Various general methods can be adopted as a method for introducing recombinant DNA into the cells and a transformation method using the method.

<B: Medium>
The medium used in step 1 contains an inorganic salt and an organic carbon source. The pH of the medium is not particularly limited, but is preferably a pH that satisfies the above-mentioned growth conditions for halophilic bacteria. Specifically, the pH may be about 5 to 12. A medium having a pH of 8.8 to 12 is more preferable. Use of an alkaline medium is preferable because contamination of other bacteria can be more effectively prevented, and a significant decrease in pH caused by secreted pyruvic acid is suppressed.

  The inorganic salt to be blended in the medium used in Step 1 is not particularly limited. For example, phosphate, nitrate, carbonate, sulfate, sodium, magnesium, potassium, manganese, iron, zinc, copper, cobalt And metal salts such as

For example, when sodium is used as the inorganic salt, NaCl, NaNO ₃ , NaHCO _3, Na ₂ CO ₃ or the like may be used.

  As these inorganic salts, it is preferable to use a compound that becomes a nitrogen source or a phosphorus source for the halophilic bacteria described above.

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

  What is necessary is just to set the usage-amount of a nitrogen source suitably in the range in which the production objective of the pyruvic acid of this invention or its salt is achieved, without affecting the growth of a microbial cell. Specifically, it is usually about 500 mg or more as nitrate, more preferably about 1000 mg or more, still more preferably about 1250 mg or more per 100 ml of culture medium at the initial stage of culture.

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

  The usage amount of the phosphorus source may be appropriately set from the same viewpoint as the usage amount of the nitrogen source, and specifically, it may be usually about 50 to 400 mg per 100 ml of the medium as dihydrogen phosphate, More preferably, it is about 100 to 200 mg.

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

  The inorganic salt including other compounds may be used at a concentration that is generally about 0.1 to 2.5 M, preferably about 0.2 to 1.0 M, more preferably 0.2 to 0. About 5M.

  The organic carbon source to be blended in the medium used in step 1 is not particularly limited. For example, tryptone, yeast extract, soluble starch, ethanol, n-propanol, acetic acid, sodium acetate, propionic acid, waste glycerol, and waste beet sugar. , Saccharified wood, psicose, fructose, sorbose, tagatose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, etc .; ribulose, xylulose, ribose, arabinose, xylose, lyxose, deoxyribose, etc. Pentose sugars; disaccharides such as sucrose, lactose, maltose, trehalose, tulanose, cellobiose; sugar alcohols such as erythritol, glycerin, mannitol, sorbitol, xylitol, and the like.

  In the production method according to the present invention, since halophilic bacteria belonging to the genus Halomonas are cultured in a medium having a relatively high salt concentration, there is almost no risk of contamination or growth of other bacterial cells. Therefore, the above-described medium may or may not be sterilized, and can be cultured with simple equipment.

<C: culture method>
The culture of halophilic bacteria belonging to the genus Halomonas in step 1 employs aerobic culture. The aerobic culture in step 1 is not particularly limited as long as the cells grow.

  Specifically, the halophilic bacteria are inoculated in a medium of about 5 ml, and preculture is usually performed at about 30 to 37 ° C. and a stirring speed of about 120 to 180 rpm while shaking overnight. Subsequently, the microbial cells obtained by pre-culture are diluted about 100 times in a medium in an Erlenmeyer flask, a fermenter, a jar fermenter, etc., and are subjected to main culture.

  The main culture is usually possible at about 20 to 45 ° C, but is preferably about 33 ° C or more, more preferably about 33 ° C to 40 ° C. The stirring speed at this time is usually about 150 to 250 rpm. The culture environment may be an environment in which the culture medium comes into contact with air, and a method of positively spraying a gas containing oxygen on the surface of the culture solution or a method of blowing the gas in the culture medium may be employed.

  In step 1, halophilic bacteria belonging to the genus Halomonas may be aerobically cultured under such culture conditions. Specifically, the dissolved oxygen concentration in the culture solution at the time of aerobic culture is not particularly limited, but is usually 0.5 mg / L or more, and preferably 2 mg / L or more.

  Examples of the culture method in Step 1 include culture methods such as batch culture, semi-batch culture, and continuous culture, and are not particularly limited, but in order to efficiently produce pyruvic acid or a salt thereof, the method according to the present invention is used. Considering that the possibility of halophilic bacteria to be mixed with other bacteria is extremely low, long-term continuous culture is also possible. The culture environment is not particularly limited, and may be a non-sterile environment or a sterilized environment.

Step 2 Step 2 in the method for producing pyruvic acid or a salt thereof according to the present invention is a step of adding at least one selected from the group consisting of a nitrogen source, a metal salt, and a borate to the culture solution. is there.

  In step 2, only the nitrogen source may be added, or a metal salt and / or borate may be added. The timing of addition of the nitrogen source and the metal salt and / or borate may be the same or different, and is not particularly limited, but if different, the metal salt and / or borate is added after the addition of the nitrogen source. Is preferably added.

  In addition, the addition time at the time of adding a metal salt and a borate may be the same, may differ, and is not specifically limited.

The nitrogen source may be as described in detail in Step 1 above. The metal salt is not particularly limited, and examples thereof include zinc salts, molybdenum salts, manganese salts, copper salts, and cobalt salts. These metal salts may be added in combination as appropriate, and are not particularly limited, but a combination containing at least a molybdenum salt is preferable.

  Specifically, metal salts including molybdenum salt, zinc salt, manganese salt, copper salt, and cobalt salt; metal salt including molybdenum salt and copper salt; metal salt including molybdenum salt and zinc salt; molybdenum salt and manganese salt Metal salt containing; Combinations, such as a metal salt containing a molybdenum salt and a cobalt salt, are mentioned.

The borate is not particularly limited. For example, boric acid (H ₃ BO ₃ ), metaboric acid (HBO ₂ ) _n , perboric acid (HBO ₃ ), hypoboric acid (H ₄ B ₂ O ₄ ), boronic acid (H ₃ BO ₂ ), borinic acid (H ₃ BO) and the like.

The timing of adding at least one selected from the group consisting of a nitrogen source, a metal salt, and a borate in the step 2 is not particularly limited. For example, when the OD600 value of the culture solution is 30 or more. do it. Preferably it is 50 or more.
The measuring method of OD600 is measured using a commercially available spectrophotometer.

  The amount of the nitrogen source added in step 1 is not particularly limited, but is usually about 0.1 to 0.5 parts by weight as sodium nitrate with respect to 100 parts by weight of the medium. The amount of the metal salt added in step 1 is not particularly limited, but is usually about 0.02 to 0.250 parts by weight with respect to 100 parts by weight of the medium. The amount of borate added in step 1 is not particularly limited, but is usually about 0.143 to 0.286 parts by weight with respect to 100 parts by weight of the medium. In addition, what is necessary is just to make the sum total of the addition amount of a metal salt and a borate normally about 0.02-0.286 weight part with respect to a 100 weight part culture medium.

  The number of times of adding at least one selected from the group consisting of a nitrogen source, a metal salt, and a borate is not particularly limited, but is usually about 1 to 5 times. In the case of adding a plurality of times, an interval of 3 to 24 hours may be left under the above-described culture conditions.

Step 3 Step 3 in the method for producing pyruvic acid or a salt thereof according to the present invention is a step of recovering pyruvic acid or a salt thereof from the culture solution obtained by the above steps 1 and 2. Here, the recovery refers to stopping the culture in the above step 2 when pyruvic acid or a salt thereof is present in the culture broth obtained in steps 1 and 2, and a culture solution containing pyruvic acid or a salt thereof, What is necessary is just to isolate a halophilic microbial cell.

  In addition, it is also possible to collect | recover biopolymer PHB, 3-hydroxybutyric acid, etc. from the isolate | separated microbial cell according to the method of patent documents 1-3.

  As a specific separation method, a known solid-liquid separation operation such as centrifugation or filtration may be employed. Further, the method for stopping the culture is not particularly limited. For example, a method for sterilizing halophilic bacteria belonging to the genus Halomonas obtained by the above steps 1 and 2 by a method such as heating and acid treatment, centrifugation, filtration, and the like are known. And a method for separating the culture solution from the halophilic cells using the solid-liquid separation means.

  Pyruvate secreted into the culture solution from the halobacteria belonging to the genus Halomonas, the more aerobic culture conditions are continued, while the culture solution contains pyruvic acid or its salt. Or the salt tends to be taken up again and used in the halophilic bacterium, and pyruvic acid or a salt thereof in the culture solution decreases and may eventually disappear from the culture solution. .

  Therefore, it is preferable to stop the culture when 10 g or more of pyruvic acid or a salt thereof is present in 1 L of the culture solution. That is, according to the method of the present invention, 10 g or more of pyruvic acid or a salt thereof can be produced per 1 L of the culture solution in the culture solution.

  The method for confirming the presence of pyruvic acid or its salt 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 subjected to an analysis method using HPLC, an analysis kit, or the like to determine the time for stopping the culture.

  In addition, since pyruvic acid is an acidic compound, the presence of pyruvic acid can be confirmed on the basis of a decrease in the pH of the medium during culture while monitoring the pH of the culture solution over time. Good.

  The recovered salt of pyruvic acid is recovered as an alkali metal salt reacted with an alkali metal such as sodium or calcium; an alkaline earth metal cation or the like based on an inorganic salt contained in the culture solution. Therefore, in order to produce pyruvic acid, the collected culture solution may be subjected to a conventional method such as salting out.

  Moreover, you may use the collect | recovered culture solution for a refinement | purification process by the column chromatography using a suitable column. As another method other than these, the pH of the collected culture solution may be changed as appropriate, and any desired pyruvic acid or a salt thereof may be subjected to the purification step.

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

  In this example, a method for producing pyruvic acid or a salt thereof using a halophilic bacterium belonging to the genus Halomonas will be specifically described in detail.

A medium based on SOT modified 5 (Spirulina platendium Medium modified 5) shown in Table 1 was used. This medium is Spirulina platendium Medium (HP of National Institute for Environmental Studies). The amount of NaHCO ₃ and Na ₂ CO ₃ is adjusted, the nitrogen source NaNO ₃ is increased 5 times, and the phosphorus source K ₂ HPO ₄ is 4 times. Adjusted to increase by a factor of two. The pH after adjusting the above medium was 9.4 ± 0.1, and it was used as it was without sterilization operation such as autoclave.

  During the culture, glucose was appropriately added to the above medium.

<Measurement of pyruvic acid or its salt>
The measurement of pyruvic acid or its salt produced in the culture solution in this example is based on the analysis conditions described in the attached to BIO-RAD Aminex HPX-87H, and Shimadzu RID-10A differential refraction is used for detection. This was performed by a method using a rate detector.

  Briefly, the culture broth was centrifuged to collect only the supernatant, 50 μL was collected, and 450 μL of water was added to dilute it 10 times. This was compared with the sample of pyruvic acid analyzed by the above HPLC. Moreover, since the measured value with the kit of "F-kit pyruvic acid" (JK International Co., Ltd.) and the measured value with a gas chromatograph apparatus corresponded, the analyzed peak may be pyruvic acid. confirmed.

<Pre-culture of halophilic bacteria belonging to the genus Halomonas>
From the plate culture, halophilic bacteria belonging to the genus Halomonas (Halomonas sp. KM-1 strain) were added to 5 ml of the above-mentioned SOT5 modified medium in a 16.5 mm diameter test tube as a carbon source instead of the above-mentioned glucose. Was added and cultured overnight at 37 ° C. with shaking.

<Cultivation of halophilic bacteria belonging to the genus Halomonas, sample recovery, etc.>
Production Example 1
The pre-cultured 0.2 ml of halophilic bacterium belonging to the genus Halomonas was mixed with 20 ml of the above-mentioned SOT modified 5 medium placed in a 100 ml Erlenmeyer flask and inoculated to produce silicosene. This was cultured with shaking at 33 ° C. under a stirring speed of 200 rpm. After 12 hours, 0.25 ml of the culture solution was collected approximately every 12 hours, and pyruvate or its salt in the supernatant was recovered. The amount was measured.

  At the beginning of the culture, the culture was performed under an aerobic condition with a stirring speed of 200 rpm, and the culture solution was subjected to silicocene again after sampling, followed by shaking culture at 37 ° C. and culturing in half batch.

  As the organic carbon source, 10% by weight of glucose was cultured at the beginning of the culture, and 5% by weight of glucose was added to each of the 18th, 24th and 36th hours.

  Furthermore, as a nitrogen source, 12.5 g / L sodium nitrate was added at the beginning of the culture, and 2.5 g / L was added at the 18th, 24th, 36th, 48th and 60th hours from the start of the culture. A comparison was made between the L sodium nitrate added and the one not added. The results are shown in FIGS.

  From the results of the cell weight shown in FIG. 1, there was no change in the cell weight whether or not sodium nitrate was added.

  On the other hand, from the result of the amount of pyruvate secreted in FIG. 2, when sodium nitrate was not added, the maximum amount of secretion was 18 g / L at 36 hours, whereas sodium nitrate was added sequentially. The maximum value of secretion amount exceeded 40 g / L in 60 hours.

  From the above results, it was revealed that the amount of pyruvic acid produced in the culture solution can be increased by adding an appropriate nitrogen source such as sodium nitrate at an appropriate time during the aerobic culture.

Production Example 2
At the beginning of the culture, the culture was performed under an aerobic condition with a stirring speed of 200 rpm, and the culture solution was sampled and then silicosene again, and the shaking culture was continued, followed by half-batch culture.

  As an organic carbon source, 15% by weight of glucose was cultured at the beginning of the culture, and 5% by weight of glucose was added at 24, 36, and 48 hours, respectively.

As a nitrogen source, 12.5 g / L sodium nitrate was added at the beginning of the culture, and 2.5 g / L was added at 24 hours, 36 hours, 48 hours, 60 hours and 72 hours from the start of the culture. Sodium nitrate was added.
Furthermore, the culture temperatures were 33 ° C., 37 ° C., and 40 ° C., respectively. The results are shown in FIGS.

  From the results of the cell weight shown in FIG. 3, after 36 hours, the dry cell weight is in the order of 33 ° C., 37 ° C., and 40 ° C. Especially, those cultured at 40 ° C. have almost no increase in cell weight. I couldn't see it.

  On the other hand, from the result of the amount of pyruvate secreted in FIG. 4, the secretion of pyruvate was observed in the order of 40 ° C., 37 ° C., and 33 ° C. after the 36th hour.

  From the above results, it was confirmed that the amount of pyruvic acid secreted was changed by the culture temperature, and especially at 40 ° C., growth was suppressed while pyruvic acid was secreted extremely rapidly. It became clear that there was an influence on the amount of secretion.

Production Example 3 At the beginning of comparative culture with Halomonas strains, culture was performed under aerobic conditions with a stirring speed of 200 rpm and a culture temperature of 37 ° C. Cultured. The strain used was Halomonos sp. KM-1, Halomons meridian: NBRC15608, Halomons pantererensis: ATCC7000027, Halomonas campisalis: ATCC7000059.

  The organic carbon source is a medium containing 15% by weight of glucose at the beginning of culturing. KM-1, Halomons meridian: NBRC15608 added 5% by weight glucose at 24 and 36 hours respectively, Halomons panthelerensis: ATCC70000273, Halomonas campalisis: ATCC7000059 at 5% by weight of glucose at 24 hours. Added.

  As a nitrogen source, 12.5 g / L sodium nitrate was added at the beginning of the culture, and 2.5 g / L sodium nitrate was added and compared at 24 hours, 36 hours, and 48 hours from the start of the culture. The results are shown in FIGS.

  From the results of the cell weight shown in FIG. KM-1, Halomons meridian: NBRC15608 exceeded 30 g / L at 24 hours. Halomons pantereriosis: ATCC 700033 and Halomonas campisalis: ATCC 7000059 did not exceed 20 g / L even at 84 hours. From the results of pyruvic acid secretion shown in FIG. It was found that not only KM-1 but also Halomons meridian: NBRC15608 secreted 30 g / L or more of pyruvic acid. Moreover, Halomons panteleriensis: ATCC70000273 and Halomonas campisalis: ATCC70000597 secreted a trace amount of pyruvic acid at 72 hours and 84 hours.

  From the above results, it was clarified that when culturing at 37 ° C. in a nitrogen-added and carbon-excess condition, a plurality of bacteria of the genus Halomonas secrete pyruvic acid.

Claims (7)

The manufacturing method of pyruvic acid or its salt including the following process (1)-(3):
(1) Step 1 of aerobically cultivating a halophilic bacterium belonging to the genus Halomonas in a medium containing an organic carbon source and an inorganic salt,
(2) Step 2 of adding at least one selected from the group consisting of a nitrogen source, a metal salt, and a borate to the culture solution,
(3) Step 3 for recovering pyruvic acid or a salt thereof from the culture solution obtained in Step 2.   The production method according to claim 1, wherein the nitrogen source in step 2 is at least one selected from the group consisting of nitrate, nitrite, ammonium salt, and urea.   The production method according to claim 1 or 2, wherein the metal salt in step 2 is at least one metal selected from the group consisting of a zinc salt, a molybdenum salt, a manganese salt, a copper salt, and a cobalt salt.   The time at which at least one selected from the group consisting of a nitrogen source, a metal salt, and a borate in Step 2 is added is a time at which the OD600 of the culture solution is 30 or more. 2. The production method according to item 1.   The manufacturing method of any one of Claims 1-4 whose culture conditions in the process 1 are 20-45 degreeC.   The method according to any one of claims 1 to 5, wherein 10 g or more of pyruvic acid or a salt thereof is contained per liter of the culture solution obtained in step 2. The method according to any one of claims 1 to 5, wherein the halophilic bacterium is Halomonas sp. KM-1 strain (FERM BP-10995).

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