WO2005045049A1 - 有機酸アンモニウム溶液の製造方法 - Google Patents
有機酸アンモニウム溶液の製造方法 Download PDFInfo
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- WO2005045049A1 WO2005045049A1 PCT/JP2004/016437 JP2004016437W WO2005045049A1 WO 2005045049 A1 WO2005045049 A1 WO 2005045049A1 JP 2004016437 W JP2004016437 W JP 2004016437W WO 2005045049 A1 WO2005045049 A1 WO 2005045049A1
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- magnesium
- organic acid
- ammonium
- ammonia
- carbonate
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/582—Recycling of unreacted starting or intermediate materials
Definitions
- the present invention relates to a method for producing an organic acid ammonium solution such as an ammonium succinate solution. More specifically, the present invention relates to a method for producing an organic acid ammonia solution, which is suitable for producing an organic acid by microbial conversion using biologically-derived glucose, glucose, cellulose, or the like as a raw material.
- Organic acids include fumaric acid, maleic acid, malic acid, succinic acid, etc.
- succinic acid or its derivatives are used as raw materials for polymers such as biodegradable polyesters and polyamides, or as foods and pharmaceuticals.
- Patent Document 1 As a method of separating and purifying an organic acid from an organic acid salt produced by fermentation, a method using electrodialysis (for example, see Patent Document 1), an ion exchange resin is used. (See, for example, Patent Document 2), a method of decomposing calcium succinate obtained by fermentation production while neutralizing with calcium hydroxide (see, for example, Patent Document 3) Salt using sulfuric acid (For example, refer to Patent Documents 4 and 5).
- An adaptive extraction method Patent Document 6 has been proposed!
- the neutralizing agent In the method using electrodialysis, the neutralizing agent must be a monovalent cation. Divalent cations precipitate as gypsum in electrodialysis membranes and significantly impair their membrane performance. Therefore, ammonia, sodium and potassium are preferred as neutralizing agents.
- the neutralizing agent In the method using the reaction extraction with amine, the neutralizing agent remains as a carbonate in the aqueous phase. If the solubility of the neutralizing agent in the water is too small, the neutralizing agent precipitates in situ, resulting in high pressure. The extraction tower cannot be operated. Therefore, ammonia, sodium and potassium are preferred as neutralizing agents.
- Patent Documents 4 and 5 disclose a method in which ammonium sulfate is thermally decomposed at a temperature of 300 ° C or more, reused as monoammonium sulfate, and ammonia is used as a neutralizing agent. Propose. To that end, we propose a method to convert sodium neutralization to ammonia neutralization.
- ammonia neutralization is a neutralizing agent that has the advantage that the most diverse purification methods can be applied and that the application of the method described in Patent Document 4 or 5 does not generate by-product salts. It can be said that.
- Sodium can be converted to ammonia by the method described in Patent Document 4 or 5, and various purifications can be used as they are depending on the environment.
- Calcium is a neutralizer with relatively good economic rationality as shown in Patent Document 3. Based on the above, magnesium, which is extremely limited and cannot be purified, is rarely considered to be used as a neutralizing agent, even though it is one of the metal ions required for cell reaction. Was.
- Patent Document 1 Japanese Patent Application Laid-Open No. 2-283289
- Patent Document 2 U.S. Patent No. 6,284,904
- Patent Document 3 Japanese Patent Application Laid-Open No. 3-030685
- Patent Document 4 JP 2001-514900 A
- Patent Document 5 U.S. Patent No. 5,958,744
- Patent Document 6 International Publication No.98Z01413 pamphlet
- An object of the present invention is to provide an efficient method for producing an organic acid ammonium solution.
- the present inventors have proposed the use of an ammonium salt of an organic acid in order to select the most suitable method for purifying an organic acid in various economic environments. We thought that it would be good if it could be converted to salt, and we studied diligently. As a result, they found that by adding a magnesium compound to a fermentation medium, culturing microorganisms, and replacing the generated magnesium salt of an organic acid with an ammonia compound, an organic acid ammonium solution can be efficiently produced. .
- magnesium carbonate containing almost no ammonia can be obtained by heating the by-produced magnesium carbonate 'ammonia-carbonate double salt.
- magnesium compounds can be recycled and ammonia salts of organic acids can be produced without producing waste.
- the present invention is as follows.
- a method for producing an organic acid ammonium solution A method for producing an organic acid ammonium solution.
- an ammonium carbonate produced by supplying carbon dioxide and ammonia to the fermentation liquor is used as an ammonia compound.
- a method for producing a solution of an organic acid ammonium is used.
- the separated magnesium carbonate is thermally decomposed into carbon dioxide and magnesium oxide, and water is added to the magnesium oxide to generate magnesium hydroxide.
- the organic acid ammonium salt of (1), wherein the magnesium compound is magnesium hydroxide or a mixture of magnesium hydroxide and magnesium carbonate, and ammonia is used in the salt exchange step. Method for producing a solution.
- FIG. 1 shows a procedure for constructing plasmid pKMB1 and a restriction enzyme map.
- FIG. 2 is a diagram showing a procedure for constructing a plasmid ⁇ LDH.
- FIG. 3 is a view showing a procedure for constructing a plasmid pTZ4.
- FIG. 4 is a view showing a procedure for constructing a plasmid pMJPCl.
- FIG. 5 is a view showing a procedure for constructing a plasmid pFRPCl.
- the production method of the present invention comprises a fermentation step of obtaining a fermentation solution containing an organic acid magnesium using a microorganism capable of producing an organic acid in the presence of a magnesium compound, and converting the organic acid magnesium contained in the fermentation solution into an ammonia compound.
- the type of organic acid ammonium is not particularly limited as long as it is an ammonium salt of an organic acid fermented and produced by a microorganism, but is preferably an ammonium salt of a dicarboxylic acid or a tricarboxylic acid.
- dicarboxylic acids include succinic acid, fumaric acid, maleic acid, malic acid, tartaric acid, aspartic acid, daltaric acid, glutamic acid, adipic acid, suberic acid, itaconic acid, and terephthalic acid, and tricarboxylic acids such as citric acid. Examples can be given.
- the “organic acid ammonium” includes an organic acid monoammonium and an organic acid polyvalent ammonium.
- a microorganism having organic acid-producing ability is used in the presence of a magnesium compound to obtain a fermentation liquid containing the organic acid magnesium.
- the microorganism used is "a microorganism capable of producing an organic acid".
- the term "organism capable of producing an organic acid” refers to a microorganism capable of producing and accumulating an organic acid in a medium when the microorganism is cultured in a medium containing a carbon source as described below.
- Examples of such microorganisms include bacteria belonging to the genus Anaerobiospirillum (US Pat. No. 5,143,833), bacteria belonging to the genus Actinobacillus (US Pat. No. 5,504,004), and Escherichia. Facultative anaerobic bacteria such as bacteria belonging to the genus Genus (US Pat. No.
- Corynebacterium glutamicunU (Brebinocterium)
- Brevibacterium flavum Brevibacterium flavum
- Brevibacterium ammoniagenes Brevibacterium 'lactofermentum
- Brevibacterium lactofermentum Brevibacterium lactofermentum
- Examples of the coryneform bacterium capable of producing succinic acid include the following. Brevibataterium 'Flavam MJ233 strain with reduced ratate dehydrogenase activity (JP-A-11 206385), or Brevibatatellium flavum MJ233 with enhanced pyruvate carboxylase or phosphoenol pyruvate carboxylase activity / pPCPYC strain (WO 01/27258 pamphlet), Brevibacterium 'Flavum MJ-233 (FERM BP-1497), MJ-233 AB-41 (FERM BP-1 498), Brevibataterimium' ammonia Genes ATCC6872, Corynebataterum 'Glutamicum ATCC31831, and Brevibataterim' Rat
- Brevibataterum 'Flavum MJ-233 was established on April 28, 1975, by the Research Institute of Microorganisms and Industrial Technology of the Ministry of International Trade and Industry (now the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology) ( 305-8566 Deposited at 1-1-1 Tsukuba-Higashi, Ibaraki Pref., Japan No. 1 under the accession number FERM P-3068, transferred to an international deposit based on the Budapest Treaty on May 1, 1981, and transferred to FERM Deposited under accession number BP-1497.
- Brevibacterium 'Ammoniagenes ATCC6872' etc. can be obtained from the American 'Type' Cultureya 'Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America).
- the microorganism used in the fermentation step may be a microorganism that has been modified so as to enhance the organic acid-producing ability.
- Microorganisms modified to enhance succinic acid-producing ability include, for example, microorganisms with enhanced pyruvate carboxylase gene expression (JP-A-11-196888) and microorganisms with lactate dehydrogenase gene disrupted (see Kaihei 11-206385).
- a microorganism having an enhanced expression of a fumarate reductase gene as described in Reference Examples below can also be used.
- microorganisms having an organic acid-producing ability that can be used in the present invention are not limited to those described above, and other succinic acid-producing bacteria, malic acid-producing bacteria obtained by known methods, fumaric acid-producing bacteria, and quencher Acid producing bacteria, isocunic acid producing bacteria and the like can be used.
- microorganisms having an organic acid-producing ability that can be used in the present invention are used. It may have the above organic acid producing ability.
- the liquid medium used for culturing microorganisms contains a carbon source.
- the carbon source is not particularly limited as long as it is a carbon source that can be assimilated by microorganisms, but carbohydrates such as galactose, ratatose, gnoleose, manoletose, funolectose, glycerolone, sucrose, saccharose, starch, and cellulose; Examples thereof include fermentable sugars such as polyalcohols such as mannitol, xylitol, and ribitol. Of these, glucose, fructose, and glycerol can be preferably used, and glucose can be particularly preferably used.
- cellulose which is a main component of paper
- starch syrup, molasses and the like containing the above-mentioned fermentable saccharide are also used. These carbon sources may be used alone or in combination of two or more.
- the concentration of the carbon source used is not particularly limited !, but it is advantageous not to inhibit the production of organic acids! It is advantageous to use as high as possible within the range, usually 5 to 30% (WZV). , Preferably within the range of 10-20% (WZV). Further, in accordance with the decrease of the carbon source with the progress of the reaction, the addition of the carbon source may be performed.
- the liquid medium preferably contains a nitrogen source, an inorganic salt, and the like in addition to the carbon source.
- the nitrogen source is not particularly limited as long as the microorganism can assimilate and produce an organic acid, but specifically, ammonium salt, nitrate, urea, soybean hydrolyzate, casein hydrolyzate , Peptone, yeast extract, meat extract, corn steep liquor and the like.
- the inorganic salts various phosphates, sulfates, and metal salts such as magnesium, potassium, manganese, iron, and zinc are used.
- a microorganism cultivated on a slant in a solid medium such as an agar medium may be directly used, but the microorganism is preliminarily cultured (seed culture) in a liquid medium. It is preferable to use the cells obtained by the method. In this case, the cells used for the reaction Although the amount is not particularly limited, it is usually 11 to 700 gZL, preferably 10 to 500 gZL, more preferably 20 to 400 gZL.
- a neutralizing agent is added to the culture solution.
- a compound of an alkaline earth metal preferably a magnesium compound is used.
- Magnesium compounds are advantageous because they increase succinic acid production and have a small range of pH fluctuations.
- the magnesium compound those which show alkalinity when ionized in an aqueous solution are preferable.
- magnesium hydroxide (Mg (Mg) magnesium hydroxide
- magnesium hydroxide is particularly preferable.
- two or more magnesium compounds may be used.
- the method of adding the magnesium compound is not particularly limited as long as it can be controlled to an appropriate pH.
- these magnesium compounds can be added as a powder.
- the magnesium compound may be added to the medium at the start of the culture, or may be added during the culture. Further, it may be added to the medium at the start of the culture, and further added during the culture as needed.
- the pH value adjusted by these magnesium compounds is adjusted to a range in which the organic acid generating activity is most effectively exerted, depending on the type of microorganism used. Generally, the pH value is adjusted to pH 4-10, preferably pH 6-10. Adjusted to about 9.
- Culture conditions such as temperature and pressure in the fermentation step vary depending on the microorganism used, but suitable conditions for obtaining an organic acid may be selected according to each case.
- the temperature during the cultivation is usually 25 ° C to 40 ° C, preferably 30 ° C to 37 ° C.
- the reaction time is preferably 1 hour to 168 hours, more preferably 3 hours to 72 hours.
- the fermented broth after completion of the cultivation contains an organic acid produced by a microorganism and an organic acid magnesium formed by magnesium contained in a neutralizing agent (the organic acid may be ionized into an organic acid ion and magnesium ion). ) Is included.
- the fermented liquid is preferably used in a salt exchange step after removing microbial cells and the like by centrifugation or the like.
- the fermented liquid from which the cells have been separated may be used in a salt exchange step after further purification.
- it after performing the concentration operation, it may be used in the salt exchange step.
- a conventional method can be used, for example, a kettle reboiler or an evaporator can be used. Energy consumption is important for large-scale production Then, a multi-effect can may be used.
- the organic acid magnesium contained in the fermentation liquor is subjected to salt exchange using an ammonia compound to produce an organic acid ammonium salt.
- This is a step of forming and depositing a magnesium compound such as magnesium.
- the organic acid ammonium produced in this step may be ionized into an organic acid ion and an ammonium ion.
- ammonia compound examples include ammonia and ammonium carbonate. Both ammonia and ammonium carbonate may be added.
- a device used in the salt exchange step for example, a commonly used crystallization tank such as a stirring tank, a draft tube, a crystal oslo-type crystallization tank, and a double propeller can be used. However, as long as the device can obtain crystals by the solid-liquid equilibrium phenomenon, its shape, method, and number of crystallization stages are not limited.
- the salt exchange using ammonium carbonate may be carried out by adding ammonium carbonate! (The following reaction formula (I)), and adding ammonia and carbon dioxide. (Reaction formula (II) below).
- the amount of ammonium carbonate is 0.3 times and 10 times, preferably 0.5 times and 5 moles of magnesium in the fermentation liquor supplied to the reaction tank. It is desirable to add so that the amount becomes 1 times, more preferably 1 to 4 times.
- magnesium in the fermentation broth means the total of magnesium and magnesium ions contained in the magnesium succinate and the magnesium compound in the fermentation broth.
- the amount of carbon dioxide supplied to the reaction tank is 0.3 times as much as 10 mol times, preferably 0.3 mol, of magnesium in the fermentation liquor supplied to the reaction tank. It is preferable that the five forces are also 5 times, more preferably 1 to 4 times.
- Ammonia is in a pH range such that the carbon dioxide can maintain a predetermined solubility, that is, the pH is 7-12. It is preferable to reduce the amount to be alkaline. More preferably, the pH is adjusted to 7.5 to 11, particularly preferably to 8 to 10.
- the time of the salt exchange reaction varies depending on the amount of the magnesium succinate reaction solution and is not particularly limited, but is preferably 0.1 to 14 hours.
- the salt exchange is preferably performed at a pH of 7 to 12, more preferably at a pH of 7.5 to 11, and even more preferably at a pH of 8 to 10, and is preferably performed with stirring.
- ammonium succinate and magnesium hydroxide are formed by the following reaction formula (III).
- the amount of ammonia to be added is preferably 2 to 15 times the amount of magnesium in the fermentation broth.
- the salt exchange time varies depending on the amount of the fermentation broth containing magnesium succinate and is not particularly limited, but is preferably 0.1 to 14 hours.
- the salt exchange is preferably carried out at a pH of 7 to 12, more preferably at a pH of 7.5 to 11, and even more preferably at a pH of 8 to 10.
- magnesium hydroxide is produced. Therefore, it is preferable to use magnesium hydroxide or a mixture of magnesium hydroxide and magnesium carbonate as the magnesium compound that can be added to the fermentation liquid.
- the concentration of magnesium dissolved in the aqueous solution of ammonium succinate is about 0.5 wt%.
- an ammonium carbonate is further added to the filtrate after the salt exchange of the reaction formula (III), and the reaction solution of the reaction formula (I) or (II) is further added.
- the produced magnesium carbonate, a double salt of magnesium carbonate This is a step of separating a magnesium compound such as magnesium hydroxide and obtaining an organic acid ammonium solution. Since these magnesium compounds are hardly soluble in water, they can be removed by a conventional method such as filtration. For complete filtration, pressure filtration, reduced pressure filtration, centrifugal filtration and the like can be used. Alternatively, it may be settled and separated and pumped as a supernatant and a highly concentrated slurry. By removing the magnesium compound in this way, an aqueous solution of an organic acid ammonium can be obtained.
- the removed magnesium compound can be recycled to the fermenter as a neutralizing agent. That is, it is economically preferable that the magnesium compound obtained in the magnesium separation step be reused as a magnesium compound in the fermentation step.
- heat sterilization is generally performed to prevent contamination by various bacteria.However, especially when transferred as a high-concentration slurry, heating with a normal heat exchanger, for example, a multi-tube heat exchanger or a plate-type heat exchanger is used. Sterilization becomes possible. Filtration may be performed after heat sterilization, or it may be directly supplied to the fermenter. Even when the filtration is complete, the steam can be sterilized by direct steam.
- Magnesium carbonate can also be reused in the form of carbon dioxide and magnesium hydroxide. That is, first, magnesium carbonate is thermally decomposed into diacid carbon and oxidized magnesium. The obtained carbon dioxide can be reused for the salt exchange of the above reaction formula (II). On the other hand, magnesium oxide can be reacted with water to obtain magnesium hydroxide, and can be reused as a magnesium-based neutralizing agent in the first step.
- Magnesium carbonate is known to be a double salt under conditions of high carbon dioxide, as shown in Japanese Patent Publication No. 1-133919 or reveu de Chimie minera let: 22, 1985, p. 692-698. I have. Therefore, when ammonium carbonate is used as the ammonium compound, part of the magnesium compound produced in the salt exchange step exists as magnesium carbonate and a double salt of ammonium carbonate. In this step, it is preferable to recover ammonium succinate in which magnesium has been reduced as much as possible, but it is clear from the solubility product that the amount of dissolved magnesium increases when the amount of carbon dioxide is reduced. Therefore, generation of double salts is inevitable. On the other hand, it is difficult to recycle magnesium as double salts. Therefore, the slurry containing the double salt is separated and heated or dried. By doing so, it is preferable to remove the double salt ammonium carbonate to obtain magnesium carbonate, and to circulate the magnesium carbonate to the fermentation step.
- the magnesium compound containing a double salt is separated by an ordinary method. In the case of complete filtration, pressure filtration, reduced pressure filtration, centrifugal filtration and the like are used. Alternatively, it may be settled and separated and pumped as a supernatant and a high-concentration slurry. Generally, it is preferable that the thus obtained crystals and slurries are washed with ammonia or the like to remove organic substances, and then supplied to a heating device for the next heating operation.
- Caro heat temperature is preferably 108 ° C-210 ° C, more preferably 120 ° C-180 ° C.
- the heating time varies depending on the amount of the double salt and the heating device and is not particularly limited. However, a 15-minute force is preferably 2 hours, and more preferably 30 minutes to 1 hour.
- Magnesium carbonate obtained by heating the double salt may not have to have a purity of 100% and a trace amount of ammonium carbonate may remain. In this case, it is preferable that the ammonia in the double salt is 1Z10 mol or less of magnesium, and it is more preferable that the ammonia in the double salt is 1Z30 mol or less of magnesium.
- the heating device to be used may be of any type as long as it can heat the crystal to a temperature exceeding a predetermined temperature. Kilns, dryers, heaters and the like can be mentioned. If the crystals may be in the form of flakes, a heating machine or baking machine such as a hot plate or belt type may be used. Generally, when used as a neutralizing agent, powder is easier to handle because it has better dispersibility. In such a case, it is preferable to use a rotary kiln or a fluid dryer.
- the efficiency of organic acid fermentation production can be improved by recycling the magnesium carbonate recovered after heating the double salt to remove the ammonium carbonate. That is,
- the shim may be reused as a neutralizing agent in the fermentation process, or may be converted into hydroxide and magnesium nitrate as described above, and magnesium hydroxide may be converted into carbon dioxide in the fermentation process. May be reused in the salt exchange step.
- Moyua may be contained, in which case it is easily evaporated and vaporized by heating to separate it, utilizing the property that the solubility of carbon dioxide and ammonia has a strong temperature dependence. be able to.
- carbon dioxide and ammonia are vaporized at the same time, when cooled, they are precipitated as ammonium carbonate, and the reaction tank may be blocked. Therefore, the temperature of the vaporized gas is more desirably higher than the melting point of ammonium carbonate, 108 ° C. However, even if the temperature is lower than 108 ° C, for example, at 80 ° C or higher, preferably 90 ° C at normal pressure, the ammonium carbonate can be sufficiently removed.
- the water is absorbed as an ammonium carbonate using a sufficient amount of water to dissolve the whole amount, and is stored in a buffer tank.
- a method such as supplying to a reaction tank is safe.
- the water required to dissolve the ammonium carbonate is simultaneously supplied to the salt exchange reactor. Therefore, from the viewpoint of energy saving, it is preferable to supply and reuse the gas as it is in the salt exchange reaction tank.
- Carbonic acid and ammonia, separated from the aqueous solution of the organic acid ammonium at a temperature of 108 ° C or higher, are supplied to the salt exchange reactor as gases under slightly pressurized conditions.
- An organic acid can be obtained by using the organic acid ammonium obtained by the method of the present invention.
- the method for obtaining an organic acid is not particularly limited also for the organic acid ammonia mud, but for example, a method using electrodialysis (JP-A-2-283289), a method using an ion-exchange resin (US Pat. No. 6,284,904 or WO01Z66508), a method in which calcium organic acid obtained by fermentative production while neutralizing with calcium hydroxide is decomposed with sulfuric acid. (Kaihei 3-030685) A method of performing reaction crystallization by salt exchange reaction using sulfuric acid (see Japanese Patent Application Laid-Open No. 2001-514900 or US Pat. No. 5,958,744), a reaction extraction method (WO98Z01413), a method using acetic acid (WO03Z95409), and the like.
- Example 2 shows the steps after the salt exchange step.
- aqueous ammonia (Wako reagent) was mixed with an aqueous solution of magnesium succinate (30 kg) to carry out salt exchange.
- 15 kg of aqueous ammonia (25%) was added over about 1 hour, and after the addition was completed, stirring was further performed for 60 minutes to complete the salt exchange reaction.
- a slurry liquid (45 kg) on which magnesium hydroxide was precipitated was obtained.
- a small amount of this slurry was collected and filtered through a 0.2 m membrane filter (Millipore).
- the Mg concentration in the filtrate containing the obtained ammonium succinate was analyzed by ion chromatography (electrical conductivity detector). As a result, the Mg concentration was 0.39 wt%.
- the slurry liquid obtained by the above operation was subjected to solid-liquid separation.
- a screw decanter (Sharpless Super Decanter Model P-660) manufactured by Tomoe Kogyo was used.
- the inner and outer cylinders of the decanter were set to 3900 and 5100 rpm. Under these conditions, the slurry was circulated at a rate of 30 L / h to continuously perform solid-liquid separation.
- the liquid coming out of the liquid outlet showed some powder.
- a portion of this solution was sampled, and a small amount of tartaric acid was added to dissolve the powder, and the Mg concentration was analyzed. The result was 0.49 wt%.
- the solids discharged were analyzed for Mg concentration.
- the solid was diluted 50-fold with distilled water and further made into a homogeneous solution by adding tartaric acid until the solid was completely dissolved.
- the Mg concentration was 14.6 wt%.
- Analysis of the succinic acid concentration for the recovered liquid and the recovered solid showed 6.79% and 7.89%, respectively.
- the final liquid recovered by solid-liquid separation was 42 kg and the solid was 3 kg.
- ammonium succinate aqueous solution (10 kg) was added to ammonium carbonate (10 kg).
- (Wako reagent) was mixed to perform salt exchange.
- 2.1 kg of ammonium carbonate was charged over about 1 hour, and after the addition was completed, stirring was further performed for 60 minutes to complete the salt exchange reaction.
- a small amount of the slurry was collected and filtered through a 0.2 m membrane filter (Millipore).
- the Mg concentration in the obtained filtrate containing ammonium succinate was analyzed by ion chromatography (electric conductivity detector). As a result, the Mg concentration was 0.01 wt%.
- the slurry liquid obtained by the above operation was subjected to solid-liquid separation.
- a 10 L pressure filter manufactured by Advantech was used for the filter paper.
- Advantech high-purity filter paper No. 5C was used for the filter paper. Filtration was performed twice in succession. The filtration pressure was 4 kg gauge pressure. When the liquid stopped flowing, the pressure was returned to normal pressure, and the next slurry liquid was charged. Under these conditions, solid-liquid separation of one slurry was performed. Solids were not found in the recovered filtrate. Mg analysis of the filtrate showed that the Mg concentration was 0.01 wt%. Similarly, the solids recovered were analyzed for Mg concentration.
- the solid was diluted 50-fold with distilled water and made into a homogeneous solution by adding tartaric acid until the solid was completely dissolved.
- the Mg concentration was 7.48 wt%.
- the succinic acid concentration of the recovered liquid and the recovered solid was 10.1% and 4.4%, respectively.
- the final liquid recovered by filtration was 9. lkg and the solid was 2.8 kg.
- Example 3 shows a step of obtaining magnesium carbonate 'ammonium-carbonate double salt strength magnesium carbonate obtained in the fermentation step, the salt exchange step, the magnesium separation step, and the magnesium separation step.
- Example 4 also shows the reuse of magnesium carbonate obtained from the double salt in the fermentation process.
- Urea 4 g, ammonium sulfate: 14 g, 1 potassium phosphate: 0.5 g, 2 potassium phosphate. 5 g, magnesium sulfate ⁇ heptahydrate: 0.5 g, ferrous sulfate ⁇ heptahydrate: 20 mg, manganese sulfate ⁇ hydrate: 20 mg, D-piotin: 200 ⁇ g, thiamine hydrochloride: 200 ⁇ g , Yeast extract: lg, power zamino acid: lg, and distilled water: lOOOOmL of medium lOOmL was placed in a 500mL Erlenmeyer flask and sterilized by heating at 120 ° C for 20 minutes.
- the LDH strain was inoculated and seed-cultured at 30 ° C for 24 hours. This strain is a strain in which the expression of the fumarate reductase and pyruvate carboxylase genes has been enhanced, and the ratate dehydrogenase gene has been disrupted.
- Urea 12 g, ammonium sulfate: 42 g, potassium monophosphate: 1.5 g, potassium diphosphate 1.5 g, magnesium sulfate ⁇ heptahydrate: 1.5 g, ferrous sulfate ⁇ 7 Hydrate: 60mg, manganese sulfate ⁇ Hydrate: 60mg, D-Piotin: 600 ⁇ g, Thiamine hydrochloride: 600 ⁇ g, Yeast extract 3g, casamino acid 3g, defoamer (Adekinol LG294: Asahi Denka ): 1 mL and distilled water: 2500 mL of the medium was placed in a 5 L fermentor, and sterilized by heating at 120 ° C for 20 minutes.
- Potassium phosphate 0.36g
- Potassium phosphate 0.36g
- magnesium sulfate ⁇ heptahydrate 1.8g
- ferrous sulfate ⁇ heptahydrate 72mg
- manganese sulfate 'hydrate 72mg
- a medium of D-Piotin: 720 g, thiamine hydrochloride: 720 g and distilled water: 2600 mL was put in a 5 L JAR, and sterilized by heating at 120 ° C. for 20 minutes. After cooling to room temperature, the cells harvested by the above culture were added to the cells and resuspended so that the OD (660 nm) became 60.
- the double salt obtained above was fractionated and used, and washing and heating operations were performed as follows. The study was performed by changing the number of washings, the heating temperature, and the heating time. Table 1 shows the conditions such as temperature, time, and number of washings in each experimental example.
- the magnesium carbonate and ammonium carbonate double salt obtained by the above-mentioned Solvay method salt exchange are separated into beakers, and 25% of industrial ammonia water (manufactured by Mitsubishi Chemical Corporation) of the same weight as the double salt is added. I washed it. The obtained suspension is subjected to suction filtration with a notch to separate solid and liquid. And the solid was collected. This washing and filtration operation was performed once or twice.
- the solid obtained by the washing operation was put into a 500 mL eggplant-shaped flask, and heated at a predetermined temperature and time using a rotary evaporator.
- the content of Mg, ammonia and succinic acid in the obtained heated solid was analyzed.
- Table 1 shows the analysis results of the obtained heated solid. From Experiment No.l-6, when the double salt was heated in the range of 120-180 ° C, the proportion of ammonia in the double salt decreased significantly and the proportion of magnesium increased. From this, it was evident that the double salt ammonium carbonate was efficiently removed and high purity magnesium carbonate was obtained. On the other hand, when heated at 90 ° C (Experiment No. 7), a considerable amount of ammonia remained in the double salt and could not be sufficiently removed. Also, succinic acid attached to the double salt could be removed by washing with 25% aqueous ammonia, but it is clear that succinic acid can be more removed by performing the washing twice in this example. Helped.
- Example 4 To fermentation process of magnesium carbonate obtained by double salt decomposition First, the cells to be used in the fermentation reaction were obtained by the method (1) described in Example 3. Next, a fermentation reaction was performed using magnesium carbonate obtained in the double salt decomposition step of Example 3 as a neutralizing agent. Potassium phosphate: 0.04 g, dipotassium phosphate.
- Reagents 1 and 2 show the experimental results when the above-mentioned succinic acid fermentation production was performed using commercially available magnesium carbonate. As can be seen from the column of reaction results, fermentative production of succinic acid using gnesium carbonate obtained by heating the double salt was similar to that using commercially available magnesium carbonate. Succinic acid was obtained. From this, it was proved that the magnesium carbonate obtained by heating the double salt can be efficiently reused.
- Bacillus subtilis ISW1214 was cultured in 10 mL of LB medium [composition: 10 g of tryptone, 5 g of yeast extratato, and 5 g of NaCl dissolved in 1 L of distilled water] until the late logarithmic growth phase, and the cells were collected. The obtained cells were suspended in 0.15 mL of 10 mM NaCl—20 mM Tris buffer (pH 8.0) —lmM EDTA ′ 2Na solution containing lysozyme at a concentration of 10 mgZmL. . Next, proteinase K was added to the above suspension so that the final concentration became 100 gZmL, and the mixture was kept at 37 ° C for 1 hour.
- sodium dodecyl sulfate was added to a final concentration of 0.5%, and the cells were lysed by keeping the temperature at 50 ° C for 6 hours.
- To this lysate add an equal volume of the phenol Z chloroform solution, gently shake at room temperature for 10 minutes, then centrifuge the whole (5,000 X g, 20 minutes, 10-12 ° C) and remove the supernatant. The fractions were collected, and sodium acetate was added thereto to a concentration of 0.3 M, and then twice the amount of ethanol was added to the mixture. The precipitate collected by centrifugation (15,000 ⁇ g, 2 minutes) was washed with 70% ethanol and air-dried.
- the Bacillus subtilis SacB gene was obtained by converting the DNA prepared in the above (A) into type II and designing a synthetic DNA (SEQ ID NO: 2) designed based on the previously reported nucleotide sequence of the gene (GenBank Database Accession No. X02730). This was performed by PCR using 1 and SEQ ID NO: 2). Reaction solution composition: Type 1 DNA 1 ⁇ L, Pfx DNA polymerase (Invitrogen) 0.2 ⁇ 1x concentration attached buffer, 0. Each primer, ImM MgSO, 0.25
- Reaction temperature conditions Using a DNA Thermal Cycler-PTC-200 (manufactured by MJ Research), a cycle consisting of 94 ° C for 20 seconds and 68 ° C for 2 minutes was repeated 35 times. However, the heat retention at 94 ° C in the first cycle was 1 minute and 20 seconds, and the heat retention at 68 ° C in the final cycle was 5 minutes.
- the amplification products were confirmed by separation by gel electrophoresis of 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts), and visualization by thidium bromide staining to detect a fragment of about 2 kb.
- the target DNA fragment was recovered from the gel using a QIAQuick Gel Extraction Kit (QIAGEN).
- the recovered DNA fragment was phosphorylated at the 5 'end with T4 polynucleotide kinase (T4 Polynucleotide Kinase: manufactured by Takara Shuzo), and then was ligated to an E. coli vector (pBluescriptll: Ligation Kit ver. 2 (Takara Shuzo)).
- Escherichia coli DH5a strain
- the recombinant Escherichia coli obtained in this manner was LB agar medium containing 50 ⁇ g ZmL ampicillin and 50 ⁇ g ZmL X-Gal [10 g tryptone, 5 g yeast extratato, 5 g NaCl and 15 g agar. Dissolved in 1 L of distilled water].
- the clone that formed a white colony on this medium was then transferred to an LB agar medium containing 50 ⁇ g ZmL ampicillin and 10% sucrose and cultured at 37 ° C. for 24 hours.
- those that could not grow on a medium containing sucrose were subjected to liquid culture by a conventional method, and then the plasmid DNA was purified.
- SacB gene in which the SacB gene is functionally expressed in E. coli should be unable to grow on sucrose-containing media.
- restriction enzymes Sail and Pstl an inserted fragment of about 2 kb was recognized, and the plasmid was named pBSZSacB.
- Escherichia coli plasmid vector PHSG396 (Takara Shuzo: chloramuecole resistance marker) 500 ng of the restriction enzyme PshBI 1 Ounits was reacted at 37 ° C for 1 hour, and then recovered by phenol Z chloroform extraction and ethanol precipitation. After blunting both ends with a tarenow fragment (K1 enow Fragment: manufactured by Takara Shuzo), Mlul linker (Takara Shuzo) was ligated and cyclized using a ligation kit ver. 2 (manufactured by Takara Shuzo), E. coli (DH5a strain) was transformed.
- K1 enow Fragment manufactured by Takara Shuzo
- Mlul linker (Takara Shuzo) was ligated and cyclized using a ligation kit ver. 2 (manufactured by Takara Shuzo), E. coli (DH5a strain) was transformed.
- the recombinant Escherichia coli thus obtained was spread on an LB agar medium containing 34 ⁇ g ZmL of chloramphene.
- Plasmid DNA was prepared from the obtained clones by a conventional method, and a clone having a restriction enzyme Mlul cleavage site was selected and named pHSG396 Mlu.
- the pBSZSacB constructed in the above was cut with restriction enzymes Sail and Pstl, and the ends were blunt-ended with a tarenow fragment. After ligation of the Mlul linker using Ligation Kit ver. 2 (Takara Shuzo), a DNA fragment of about 2.
- the colonies thus obtained were then transferred to a LB agar medium containing 34 / z gZmL Kualamu-Facol and 10% sucrose and cultured at 37 ° C for 24 hours.
- a medium containing sucrose were purified for plasmid DNA by a conventional method.
- the plasmid DNA thus obtained was digested with Mlul. As a result of analysis, it was confirmed that it had an insert fragment of about 2. Okb, which was named pCMBl.
- the kanamycin resistance gene was obtained by PCR using the DNA of Escherichia coli plasmid vector PHSG299 (Takara Shuzo: kanamycin resistance marker) as type III and the synthetic DNAs shown in SEQ ID NO: 3 and SEQ ID NO: 4 as primers.
- Reaction solution composition ⁇ type DNAlng, Pyrobest DNA polymerase (Takara Shuzo) 0.1 ⁇ l 1x concentration attached buffer, 0.5 ⁇ each primer and 0.25 ⁇ MdNTPs were mixed to make a total volume of 20 ⁇ L.
- Reaction temperature conditions Using a DNA Thermal Cycler PTC-200 (manufactured by MJ Research), a cycle consisting of 94 ° C for 20 seconds, 62 ° C for 15 seconds, and 72 ° C for 1 minute and 20 seconds was repeated 20 times. . However, the heat retention at 94 ° C in the first cycle was 1 minute and 20 seconds, and the heat retention at 72 ° C in the final cycle was 5 minutes.
- the amplified product was confirmed by separation by gel electrophoresis of 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts), followed by visualization by bromide staining, and a fragment of about 1. 1 kb was detected.
- the target DNA fragment was recovered from the gel using a QIAQuick Gel Extraction Kit (QIAGEN).
- QIAGEN QIAQuick Gel Extraction Kit
- the recovered DNA fragment was phosphorylated at its 5 ′ end with T4 Polynucleotide Kinase (Takara Shuzo).
- the approximately 3.5 kb DNA fragment obtained by cleaving the pCMBl constructed in the above (C) with restriction enzymes Van91I and Seal was separated and recovered by 0.75% agarose gel electrophoresis. This was mixed with the kanamycin resistance gene prepared in (D) above, ligated using Ligation Kit ver. 2 (Takara Shuzo), and Escherichia coli (DH5a strain) was transformed with the obtained plasmid DNA. .
- the recombinant E. coli thus obtained was spread on an LB agar medium containing 50 gZmL kanamycin. It was confirmed that the strain grown on the kanamycin-containing medium was unable to grow on the sucrose-containing medium.
- plasmid DNA prepared with the same strain was not mistaken for the structure shown in Fig. 1 because of the fact that fragments of 354, 473, 1807, and 1997 bp were generated by the IJ restriction enzyme Hindlll digestion!
- the plasmid was named pKMBl.
- Brevibataterum 'Flavum MJ-233 strain (FERM BP-1497) was obtained by a conventional method (Wolf H et al "J. Bacteriol. 1983, 156 (3) 1165-1170, Kurusu Y et al, Agric Biol Chem. 1990 The endogenous plasmid was removed (cured) according to the method described in J., 54 (2) 443-7), and the resulting plasmid-clearing strain Brevibataterium 'Flavam MJ233-ES strain was used for subsequent transformation.
- a medium (urea 2g, (NH) SO 7g, KH PO 0.5g, K HPO 0.5g, MgSO-7
- Genomic DNA was prepared from the cells by the method shown in (A) of Reference Example 1 above.
- the MJ233 strain ratatate dehydrogenase gene was obtained by using the DNA prepared in (A) above as a type III and synthesizing DNA (SEQ ID NO: 5 and SEQ ID NO: 5) designed based on the nucleotide sequence of the gene described in JP-A-11-206385. PCR was performed using No. 6). Reaction solution composition: Type I DNA1 / zL, Taq DNA polymerase (Takara Shuzo) 0.2 l 1x concentration attached buffer, 0.2 M each primer, 0.25 ⁇ M dNTPs were mixed to make a total volume of 20 ⁇ L.
- Reaction temperature conditions Using DNA Thermal Cycler PTC-200 (manufactured by MJ Research), a cycle of 94 ° C for 20 seconds, 55 ° C for 20 seconds, and 72 ° C for 1 minute was repeated 30 times. However, the heat retention at 94 ° C in the first cycle was 1 minute and 20 seconds, and the heat retention at 72 ° C in the final cycle was 5 minutes.
- the amplified product was confirmed by separation by gel electrophoresis of 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts), and visualization by titanium bromide staining to detect a fragment of about 0.95 kb.
- the target DNA fragment was recovered from the gel using a QIAQuick Gel Extraction Kit (QIAGEN).
- the recovered DNA fragment was mixed with the PCR product closing vector pGEM-TEasy (Promega), ligated using Ligation Kit ver. 2 (Takara Shuzo), and the resulting plasmid DNA was used for E. coli (DH5a strain). ) Was transformed.
- the recombinant Escherichia coli obtained in this way is weighed at 50 gZml. And LB agar medium containing 50 ⁇ g ZmLX-Gal. Clones that formed a white colony on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes Sacl and Sphl, an inserted fragment of about 1. Ok b was recognized and named pGEMTZCgLDH.
- the resulting plasmid DNA was cut with restriction enzymes Sacl and Sphl to select a clone in which an inserted fragment of about 0.75 kb was recognized, and this was named pGEMEM / ⁇ LDH.
- pGEMEM / ⁇ LDH a DNA fragment of about 0.75 kb generated by cutting the above pGEMTZ ⁇ LDH with restriction enzymes Sacl and Sphl was separated and recovered by 0.75% agarose gel electrophoresis, and the ratate dehydrogenase gene containing the defective region was recovered. Fragments were prepared. This DNA fragment was mixed with pKMB1 constructed in Reference Example 1 digested with restriction enzymes Sacl and Sphl, and ligated using Ligation Kit ver. 2 (Takara Shuzo).
- E. coli (DH5a strain) was transformed.
- the recombinant E. coli thus obtained was spread on an LB agar medium containing 50 ⁇ g ZmL kanamycin and 50 ⁇ g ZmL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified.
- the obtained plasmid DNA was digested with restriction enzymes Sacl and Sphl to select those having an inserted fragment of about 0.75 kb, which was named ⁇ 1 / ⁇ LDH (FIG. 2).
- Plasmid DNA used for the transformation of Brevibataterium 'Flavum MJ-233 strain was prepared by the calcium chloride method using pKMBlZ ALDH (Journal of Molecular Biology, 53, 159, 1970) was also prepared.
- LBG agar medium 10 g of tryptone, 5 g of yeast extratato, 5 g of NaCl, 20 g of glucose, and 15 g of agar dissolved in 1 L of distilled water] was smeared.
- the strain grown on this medium was a plasmid in which pKMBlZ ALDH was not replicable in the strain Brevibataterum 'Flavum MJ233-ES, and the ratate dehydrogenase gene and Brevibatatellium' flavum
- the kanamycin resistance gene and the SacB gene derived from the plasmid should have been inserted into the same genome.
- the homologous recombinant strain was liquid-cultured in an LBG medium containing 50 ⁇ g ZmL of kanamycin. An equivalent of about 100,000 bacterial cells in this culture was smeared on an LBG medium containing 10% sucrose. As a result, about 10 strains that were considered to be insensitive to sucrose due to the loss of the SacB gene by the second homologous recombination were obtained.
- the strains obtained in this manner include those that have been replaced with a mutant derived from the ratate dehydrogenase gene 3 ⁇ 410 ⁇ 1 ZALDH and those that have returned to the wild type.
- ratate dehydrogenase gene is a mutant or wild type
- directly detect the ratate dehydrogenase gene by subjecting the cells obtained by liquid culture in LBG medium to a PCR reaction. This can be easily confirmed.
- Analysis using the primers (SEQ ID NO: 7 and SEQ ID NO: 8) for PCR amplification of the ratate dehydrogenase gene should reveal a DNA fragment of 720 bp for the wild type and 47 lbp for the mutant having the deletion region. It is.
- a strain having only the mutant gene was selected, and the strain was named Brevibataterium 'Flavam MJ233Z ALDH.
- This promoter fragment was obtained using the Brevi battery technology prepared in (A) of Reference Example 2.
- Flavum MJ233 genomic DNA was type III, and PCR was performed using synthetic DNA (SEQ ID NO: 9 and SEQ ID NO: 10) designed based on the sequence described in SEQ ID NO: 4 of JP-A-7-95891.
- Reaction solution composition Type 1 DNA 1 ⁇ L, Pfx DNA polymerase (Invitrogen) 0.2 L, 1x concentration attached buffer, 0.3 M each primer, ImM MgSO
- Reaction temperature conditions Using DNA Thermal Cycler PTC-200 (manufactured by MJ Research), a cycle of 20 cereals at 94 ° C, 20 cereals at 60 ° C, and 30 seconds of power at 72 ° C was repeated 35 times. However, the heat retention at 94 ° C in the first cycle was 1 minute and 20 seconds, and the heat retention at 72 ° C in the final cycle was 2 minutes.
- the amplification products were confirmed by separation by gel electrophoresis on 2.0% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) and visualization by chidium bromide staining to detect a fragment of about 0.25 kb.
- the target DNA fragment was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).
- the recovered DNA fragment was phosphorylated at the 5 ′ end with T4 Polynucleotide Kinase (Takara Shuzo) and then ligated with E. coli vector PUC19 (Takara Shuzo) using Ligation Kit ver. 2 (Takara Shuzo).
- Escherichia coli (DH5 ⁇ strain) was transformed with the obtained plasmid DNA after binding to the Smal site.
- the recombinant Escherichia coli thus obtained was spread on an LB agar medium containing 50 ⁇ g ZmL ampicillin and 50 ⁇ g ZmL X-Gal.
- Six clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then the plasmid DNA was purified and the nucleotide sequence was determined.
- a clone in which the TZ4 promoter was inserted so as to have transcriptional activity in the reverse direction to the lac promoter of pUC19 was selected and named PUCZTZ4.
- a DNA fragment prepared by cleaving pUCZTZ4 with restriction enzymes BamHI and Pstl was added to a synthetic DNA (SEQ ID NO: 11 and SEQ ID NO: 12) whose 5 'end was phosphorylated.
- BamHI and a DNA linker having a sticky end to Pstl were mixed and ligated using a ligation kit ver. 2 (Takara Shuzo), and Escherichia coli (DH5 ⁇ strain) was transformed with the obtained plasmid DNA.
- This DNA linker contains a ribosome binding sequence (AGGAGG) and a clawing site located downstream thereof (Pad, NotI, Apal, in order from the upstream).
- pHSG298par-rep described in JP-A-12-93183 As a plasmid capable of autonomously replicating stably in coryneform bacteria, pHSG298par-rep described in JP-A-12-93183 is used.
- This plasmid contains the replication region and stabilizing function of the natural plasmid pBY503 carried by Brevibacterium 'statis IFO 12144 strain, the kanamycin resistance gene derived from the E. coli vector PHSG298 (Takara Shuzo) and the replication of E. coli. With an area.
- the amplification product was confirmed by separation by gel electrophoresis of 0.75% agarose (SeaKem GTG agarose: manufactured by FMCB Products) and visualization by chidium bromide staining to detect a fragment of about 3.7 kb.
- the target DNA fragment was recovered from the gel using a QIAQuick Gel Extraction Kit (manufactured by QIAGEN).
- the recovered DNA fragment was mixed with the PCR product closing vector pGEM-TEasy (Promega) and ligated using Ligation Kit ver. 2 (Takara Shuzo). Strain).
- the recombinant Escherichia coli thus obtained was spread on an LB agar medium containing 50 ⁇ g ZmL ampicillin and 50 ⁇ g ZmL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then plasmid DNA was purified. By cutting the obtained plasmid DNA with the restriction enzymes Pacl and Apal, an inserted fragment of about 3.7 kb was recognized and named pGEMZMJPC.
- the nucleotide sequence of the inserted fragment of pGEMZMJPC was determined using a nucleotide sequence decoding device (Model 377XL) manufactured by Applied Biosystems and a Big Dye Terminator One Cycle Sequence Kit ver3.
- the resulting DNA base sequence is set forth in SEQ ID NO: 15.
- the amino acid sequence predicted from this sequence shows extremely high homology (99.4%) with that derived from Corynebacterium 'daltamicum ATCC13032 strain, so that the inserted fragment of pGEMZMJPC contains the pill from Brevibataterimu' flavum MJ233 strain. It was determined to be a basic carboxylase gene.
- a pyruvate carboxylase gene fragment having a power of about 3.7 kb generated by cleaving pGEMZMJPC prepared in (A) above with restriction enzymes Pacl and Apal was separated and collected by 0.75% agarose gel electrophoresis.
- This DNA fragment was mixed with pTZ4 constructed in Reference Example 3 digested with restriction enzymes Pacl and Apal, and ligated using Ligation Kit ver.2 (Takara Shuzo). Was used to transform Escherichia coli (DH5 ⁇ strain). The recombinant Escherichia coli thus obtained was reconstituted with 50 gZmL kanamycin.
- MM A LB agar medium was smeared.
- the strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified.
- the plasmid DNA enzymes Pacl and Apal By cutting with restriction resulting plasmid DNA enzymes Pacl and Apal, and clone having an insert of about 3. 7 kb was observed, and was named P MJ PC I ( Figure 4).
- Plasmid DNA for transformation with pMJPCl capable of replication in Brevibatadium 'Flavum MJ233 strain was prepared from E. coli (DH5a strain) transformed in the above (B).
- Brevibacterium flavum strain MJ233Z ⁇ LDH was transformed by the electric pulse method (Res.Microbiol., Vol. 144, p. 181-185, 1993), and the resulting transformant was transformed into 50 ⁇ g ZmL Was spread on a LBG agar medium containing 10 g of tryptone, 5 g of yeast extratato, 5 g of NaCl, 20 g of glucose, and 15 g of agar in 1 L of distilled water.
- Escherichia coli JM109 strain was cultivated in 10 mL of LB medium until the late logarithmic growth phase, and genomic DNA was prepared from the obtained cells by the method shown in (A) of Reference Example 1 above.
- the Escherichia coli fumarate reductase gene was obtained using the DNA prepared in (A) above as type III and based on the sequence of the gene of Escherichia coli K12-MG1655 (GenBank Database Accession No. U00096), for which the entire genome sequence was reported. PCR was performed using the synthetic DNA (SEQ ID NO: 16 and SEQ ID NO: 17) designed in the above. Reaction solution composition: Type DN Al / zL, Pfx DNA polymerase (Invitrogen) 0.2 L, buffer attached at 1-fold concentration, 0.3 ⁇ Mix each primer, ImM MgSO, 0.25 ⁇ MdNTPs , Full amount
- the amplification products were confirmed by separating them by 0.75% agarose (SeaKem GTG agarose: manufactured by FMC BioProducts) gel electrophoresis and visualizing them by chidium bromide staining to detect a fragment of about 3.8 kb.
- the target DNA fragment was recovered from the gel using a QIAQuick Gel Extraction Kit (QIAGEN).
- QIAGEN QIAQuick Gel Extraction Kit
- the recovered DNA fragment was mixed with the PCR product closing vector pT7Blue T—Vector (Novagene) and ligated using Ligation Kit ver. 2 (Takara Shuzo). (DH5a strain).
- the recombinant Escherichia coli thus obtained was spread on an LB agar medium containing 50 ⁇ g ZmL ampicillin and 50 ⁇ g ZmL X-Gal. Clones that formed white colonies on this medium were subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. By cutting the obtained plasmid DNA with restriction enzymes Hindlll and Kpnl, an inserted fragment of about 3.9 kb was observed, which was named pFRD6.0.
- the nucleotide sequence of the inserted fragment of pFRD6.0 was determined using a nucleotide sequence decoding device (Model 377XL) manufactured by Applied Biosystems and a Big Dye Terminator One-Cycle Sequence Kit ver3.
- the resulting DNA base sequence is set forth in SEQ ID NO: 18.
- PMJPC1 constructed in Reference Example 3 was completely digested with the restriction enzyme Kpnl, and then reacted with alkaline phosphatase (Alkaline Phosphatase Calf intestine: Takara Shuzo). The fragments were mixed with a DNA linker that also had the ability to synthesize synthetic DNA (SEQ ID NO: 19 and SEQ ID NO: 20) with phosphorylated ends, and ligated using Ligation Kit ver. 2 (Takara Shuzo). Escherichia coli (DH5 ⁇ strain) was transformed with the DNA. The recombinant E. coli thus obtained was spread on an LB agar medium containing 50 ⁇ g ZmL kanamycin.
- the strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. From the obtained plasmid DNAs, those cleaved by the restriction enzyme Ndel were selected and named pMJPCl.1.
- the fragment containing the Escherichia coli fumarate reductase gene prepared in this manner was digested with the restriction enzyme Ndel of the pMJPCl.1 prepared in (A) above, the ends were blunted with a tarenow fragment, and then the restriction enzyme Kpnl
- the DNA was mixed with the DNA prepared by cleavage in step 1 and ligated using Ligation Kit ver. 2 (Takara Shuzo), and Escherichia coli (DH5a strain) was transformed with the obtained plasmid DNA.
- the recombinant E. coli thus obtained was spread on an LB agar medium containing 50 gZmL kanamycin.
- the strain grown on this medium was subjected to liquid culture by a conventional method, and then the plasmid DNA was purified. Based on the fact that fragments of 505, 2132, 2675, 3775, and 4193 bp were generated from the obtained plasmid DNA by digestion with the Hindlll restriction enzyme, it was judged that there was no mistake in the structure shown in FIG. 5, and the plasmid was converted into pFRPCl. Named 1.
- the present invention provides a method for producing a novel organic acid ammonia solution.
- a neutralizing agent and carbonic acid and ammonia used for salt exchange are reused.
- an organic acid ammonium solution such as an ammonium succinate solution can be efficiently produced.
- the succinic acid produced by the present invention which also provides an aqueous solution of ammonium succinate, is useful as a raw material for polymers such as biodegradable polyesters and polyamides, foods, pharmaceuticals, and cosmetics.
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BRPI0416274-9A BRPI0416274A (pt) | 2003-11-07 | 2004-11-05 | método para produzir solução de amÈnio de ácido orgánico |
EP04818202A EP1686183A1 (en) | 2003-11-07 | 2004-11-05 | Method for producing organic acid ammonium solution |
US11/429,049 US20070015264A1 (en) | 2003-11-07 | 2006-05-08 | Method for producing organic acid ammonium solution |
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JP5726745B2 (ja) * | 2008-12-02 | 2015-06-03 | ピュラック バイオケム ビー. ブイ. | 一価のコハク酸塩の製造方法 |
JPWO2011087062A1 (ja) | 2010-01-15 | 2013-05-20 | 三菱化学株式会社 | 含窒素組成物およびその製造方法 |
ES2432642T3 (es) * | 2010-02-12 | 2013-12-04 | Purac Biochem Bv | Proceso de producción de ácido succínico |
WO2012018699A2 (en) * | 2010-07-31 | 2012-02-09 | Myriant Corporation | Improved fermentation process for the production of organic acids |
US8829237B2 (en) | 2011-03-03 | 2014-09-09 | The Michigan Biotechnology Institute | Production of carboxylic acid and salt co-products |
US9315185B2 (en) * | 2011-03-31 | 2016-04-19 | Aisin Aw Co., Ltd. | Control device for transmission and braking torque generating determination method for transmission |
CN103797003B (zh) * | 2011-07-21 | 2016-10-26 | 阿彻丹尼尔斯米德兰德公司 | 通过发酵制备c4二酸的铵盐的方法及制备其c4衍生物的综合方法 |
US9550712B2 (en) | 2011-08-16 | 2017-01-24 | Purac Biochem B.V. | Acid/salt separation |
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US5168055A (en) * | 1986-06-11 | 1992-12-01 | Rathin Datta | Fermentation and purification process for succinic acid |
US5143834A (en) * | 1986-06-11 | 1992-09-01 | Glassner David A | Process for the production and purification of succinic acid |
US5766439A (en) * | 1996-10-10 | 1998-06-16 | A. E. Staley Manufacturing Co. | Production and recovery of organic acids |
US6667417B2 (en) * | 1997-02-21 | 2003-12-23 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Process for the recovery of lactic acid |
AU2976199A (en) * | 1998-03-02 | 1999-09-20 | Michigan Biotechnology Institute | Purification of organic acids using anion exchange chromatography |
MY137537A (en) * | 2002-05-10 | 2009-02-27 | Mitsubishi Chem Corp | Method for producing organic acid |
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- 2004-11-05 WO PCT/JP2004/016437 patent/WO2005045049A1/ja active Application Filing
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JPH07194387A (ja) * | 1993-12-08 | 1995-08-01 | Basf Ag | 乳酸塩の製法 |
WO1998033930A1 (en) * | 1997-01-31 | 1998-08-06 | Lockheed Martin Energy Research Corporation | A method for the production of dicarboxylic acids |
JP2001514900A (ja) * | 1997-08-18 | 2001-09-18 | アプライド カーボケミカルズ | コハク酸の製造方法および精製方法 |
JP2004196768A (ja) * | 2002-05-10 | 2004-07-15 | Mitsubishi Chemicals Corp | 有機酸の製造方法及びアンモニウム塩の分解方法、並びに有機酸及びポリマー |
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US20070015264A1 (en) | 2007-01-18 |
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