JP2014027887A - Producing method of organic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a producing method of an organic acid which requires no labor for neutralization in a fermentation process and being capable of efficient isolation of an objective organic acid.SOLUTION: The producing method of the organic acid includes: 1. a process of producing the organic acid by fermentation to obtain organic acid containing crude solution having pH 1-5; and 2. a process of isolating the organic acid from the organic acid containing crude solution obtained in process 1 with ion exchange chromatography method. In the ion exchange chromatography, an ion exchange resin is an amphoteric ion exchange resin and eluent is water.

Description

  The present invention relates to a method for producing an organic acid, and more particularly to a method for efficiently isolating an organic acid produced by fermentation from a fermentation broth.

  Organic acids such as lactic acid and succinic acid are used in various applications such as pharmaceuticals, agricultural chemicals and cosmetics. As an organic acid production method, a fermentation method has been used for a long time. In general, since organic acid-producing bacteria have fermentation inhibition by the organic acid produced, fermentation is often performed while adding alkali to the fermentation broth and adjusting the pH to near neutrality. However, such a neutralization operation is complicated, and a process for returning from an organic acid salt to an organic acid is also required, so that the production cost is high.

  Thus, it has been proposed to perform fermentation using acid-resistant bacteria that can be fermented under acidic conditions, for example, transformants using acid-resistant microorganisms as hosts (Patent Document 1). In this case, the organic acid produced is in the form of an acid rather than a neutralized salt.

  As a method for isolating an organic acid from a fermentation broth containing such an organic acid, a method using an amphoteric ion exchange resin is known (Patent Document 2). In this method, an ion exchange resin having a phthalamide group is converted to a sulfuric acid type and eluted with 0.2 W% sulfuric acid. According to this method, for example, citric acid can be separated cleanly from glucose in the fermentation broth.

International Publication No. 2011/021629 JP 2009-101353 A

  However, the amphoteric ion exchange resin is a special resin obtained by saponifying a crosslinked polymer bead after amide methylation with a phthalimide derivative, and is not suitable for industrial use. Moreover, the process of removing a sulfuric acid from an eluate is required. Furthermore, when elution is performed under such strong acidity, an organic acid may cause a reaction, and some may cause a polymerization reaction such as lactic acid.

  Therefore, the present invention provides a method for isolating an organic acid from a fermentation broth under conditions where a more general resin can be used and it is more chemically stable.

That is, the present invention is an organic acid production method described in the following [1] to [5]:
[1] A method for producing an organic acid,
1. Producing an organic acid by fermentation to obtain an organic acid-containing crude liquid having a pH of 1 to 5,
2. A step of isolating an organic acid from the organic acid-containing crude liquid obtained in step 1 by ion exchange chromatography,
And the ion exchange resin in the ion exchange chromatography method is an amphoteric ion exchange resin, and the eluent is water.

[2] The production method of the above-mentioned [1], wherein the organic acid is lactic acid, succinic acid, 3-hydroxypropionic acid, malic acid, or fumaric acid.

[3] The organic acid is lactic acid, and the microorganism that produces lactic acid has Schizosaccharomyces pombe as a host, a lactate dehydrogenase gene is integrated, and pyrubin of the Schizosaccharomyces pombe host The production method according to [1] or [2] above, which is a transformant in which a part of a gene group encoding acid decarboxylase is deleted or inactivated.

[4] The production method according to any one of [1] to [3], wherein the amphoteric ion exchange resin includes an ion exchange group represented by the following formula (1).
[Chemical 1]

_{^{- (CH 2) k-N}} + R 1 R 2 - (CH 2) m-COO - (1)

(Here, R ¹ and R ² are each independently an alkyl group having 1 to 3 carbon atoms, and k and m are each independently an integer of 1 to 4).
[5] The production method according to any one of the above [1] to [4], wherein the ion exchange chromatography is performed by a pseudo moving bed method.

  According to the production method of the present invention, it is not necessary to adjust pH in the fermentation process. Moreover, since it can carry out with general purpose resin, it is suitable for an industrial use. Further, since it can be eluted with water, no additional removal step is required and the organic acid is chemically stable.

FIG. 1 is an example of an elution curve of lactic acid, glucose, and ethanol by the method of the present invention. FIG. 2 is an example of elution curves of Na, K, Mg, and Ca ions in the same method as FIG.

  In the present invention, “fermentation” means that a target compound (organic acid) is produced by a microorganism.

  In the present invention, the organic acid may be an organic compound having a carboxyl group, such as lactic acid, 3-hydroxypropionic acid, pyruvic acid, malonic acid, succinic acid, malic acid, fumaric acid, maleic acid, glutaric acid, and Examples include adipic acid. Lactic acid, succinic acid, 3-hydroxypropionic acid, malic acid, fumaric acid, or maleic acid are preferred. Among them, lactic acid is preferred because of its wide use. These organic acids may be any of D-form, L-form and DL-form, and may form an oligomer, that is, a polymer having a polymerization degree of about 2 to 15.

  The fermentation in step 1 may be a homo-type fermentation in which only an organic acid is produced if an organic acid is produced by a microorganism, or a hetero-type in which ethanol or the like is produced in addition to the organic acid. .

  The microorganism may be either wild type or genetically modified. Examples of wild-type microorganisms include lactic acid-fermenting bacteria such as Streptococcus, Pediococcus, Leuconostoc, and Lactobacillus; and succinic acid-fermenting bacteria such as Anerobiospirilium and Corynebacterium It is done.

  Examples of the genetically modified microorganisms include genetically modified lactic acid-producing yeasts. For example, Schizosaccharomyces pombe is used as a host, a lactate dehydrogenase gene is integrated, and pyruvate decarboxylation of the host is incorporated. An acid-resistant microorganism such as a transformant (patent document 2) or a yeast of the genus Saccharomyces, in which a part of a gene group encoding the enzyme is deleted or inactivated, and lactate dehydrogenase is used as the host A transformant introduced with a gene encoding sucrose (Japanese Patent Laid-Open No. 2001-204464), and a gene encoding lactate dehydrogenase introduced, and the gene encoding pyruvate decarboxylase 1 being deleted or inactivated Saccharomyces cerevisiae (budding yeast) (Japanese Patent Laid-Open No. 2008-48726) and the likeAmong these, Lactobacillus pombe is used as a host, and a lactate dehydrogenase gene is integrated, and the host is capable of producing lactic acid with high productivity without the need to adjust the pH to around neutrality. A transformant in which a part of a gene group encoding the pyruvate decarboxylase is deleted or inactivated is preferable.

  The fermented liquor used for fermentation is not particularly limited, and may include basic inorganic salts such as Na and K suitable for the production of the target organic acid, and a carbon source. Moreover, components, such as a nitrogen source and an amino acid, may be included as needed. The fermentation broth may be any of natural, synthetic or semi-synthetic fermentation broth. Examples of the carbon source include sugars such as glucose, fructose, sucrose, and maltose. Examples of the nitrogen source include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, and ammonium acetate, peptone, casamino acid, and yeast extract. Examples of inorganic salts include magnesium phosphate, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate, and the like. Furthermore, fermentation promoting factors such as proteolipid can be blended.

  Preferably, glucose is used as the sugar. The glucose concentration in the fermentation liquid (100% by mass) in the early stage of fermentation is preferably 1% by mass or more, more preferably 1 to 25% by mass, and further preferably 2 to 16% by mass. Since the glucose concentration is reduced by fermentation, it is preferable to continue the fermentation by adding glucose as necessary. The glucose concentration at the end of fermentation may be 1% by mass or less. In addition, it is preferable to maintain the glucose concentration in the case where fermentation is continuously performed by isolating the organic acid while isolating the organic acid. By setting the glucose concentration to 2% by mass or more, the productivity of the organic acid is further improved. Moreover, the production efficiency of an organic acid improves more by making glucose in a fermentation liquid into 16 mass% or less.

  Moreover, in order to raise productivity, it is preferable to perform high-density fermentation. In high-density fermentation, the initial bacterial cell concentration of the transformant in the fermentation broth is preferably 0.1 to 50 g / L, expressed as a dry cell weight conversion value, and 0.2 to 40 g / L. Is more preferable. High productivity can be achieved in a short time by increasing the initial cell concentration. In addition, if the initial bacterial cell concentration is too high, there is a possibility that problems such as bacterial cell aggregation and reduction in purification efficiency may occur. In addition, the cell density | concentration shown by the below-mentioned Example etc. is the value converted from the light absorbency (OD660) of the light of wavelength 660nm measured by the JASCO Corporation visible ultraviolet spectrometer V550. OD = 1 at 660 nm corresponds to a yeast dry weight of 0.2 g / L and a wet weight of 0.8 g / L.

  For fermentation, a known yeast fermentation method can be used, and for example, it can be carried out by circulating fermentation, stirring fermentation or the like. It is preferable that fermentation temperature is 23-37 degreeC. Moreover, fermentation time can be determined suitably. The fermentation may be batch fermentation or continuous fermentation. For example, after performing fermentation by batch fermentation, the bacterial cells can be separated from the fermentation broth to obtain a fermentation broth containing an organic acid. In the continuous fermentation method, for example, a part of the fermented liquid is extracted from the fermenter during fermentation, and the organic acid is separated from the extracted fermented liquid, and glucose or new fermentation is separated into the remaining fermented liquid from which the organic acid has been separated. The method of adding a liquid and returning to a fermenter repeatedly and fermenting continuously is mentioned. By performing continuous fermentation, the productivity of the organic acid is further improved.

  The organic acid-containing crude liquid containing the produced organic acid has a pH of 1 to 5, preferably 1.5 to 4, particularly preferably 1.5 to 3.5. In the method for producing an organic acid of the present invention, the organic acid is produced without adjusting the pH even when the pH is lowered due to the accumulation of the organic acid in the fermentation broth. That is, the organic acid is produced by continuous fermentation that continues fermentation even after the pH of the fermentation broth is lowered. In order to increase the productivity of the organic acid, it is preferable to continue the fermentation even after the pH of the fermentation liquid becomes 3.5 or less. In particular, the above-mentioned transformant of Schizosaccharomyces pombe has excellent acid resistance, and thus can continue fermentation without adjusting the pH of the produced fermentation broth containing an organic acid.

  In step 2, the organic acid is isolated from the organic acid-containing crude liquid containing the organic acid obtained in step 1 by ion exchange chromatography. The organic acid-containing crude liquid is preferably subjected to a treatment such as centrifugation or filtration before being subjected to chromatography to separate solids such as bacterial cells. Moreover, you may concentrate by adding water, diluting, or removing water as needed.

  The ion exchange chromatography method uses an amphoteric ion exchange resin as a stationary phase and water as an eluent. The amphoteric ion exchange resin uses a cross-linked copolymer such as a styrene / divinylbenzene copolymer as a resin matrix, and has an anion exchange group and a cation exchange group bonded to the resin matrix. The resin matrix may be a gel type or a macroporous type. Examples of the anion exchange group include a trimethylammonium group, a dimethylhydroxyethylammonium group, and a dimethylamino group. Examples of the cation exchange group include a carboxyl group that is weaker than an organic acid ion and a group that forms a betaine structure in combination with the above-described anion exchange group. Further, the amphoteric ion exchange resin may be a resin called a snake cage type in which acrylic acid is impregnated into a strongly basic ion exchanger and polymerized.

The particle size of the ion exchange resin is an average particle size (D ₅₀ ), preferably ₅₀ μm to 2 mm, and more preferably 0.2 to 1.3 mm. The particle size distribution is preferably narrow in view of easy uniform ion exchange, and an ion exchange resin having a uniform particle size by sieving in advance may be used. Moreover, 0.1-10 meq / g resin is preferable and the exchange capacity of this ion exchange resin has more preferable 0.5-5 meq / g resin.
The amphoteric ion exchange resin is preferably an amphoteric ion exchange resin in which the following ion exchange group is bonded to a resin matrix of an acrylic or styrene cross-linked copolymer.
[Chemical formula 2]

^{_{- (CH 2) k-N}} + R 1 R 2 - (CH 2) m-COO - (1)

In the above formula (1), R ¹ and R ² are each independently an alkyl group having 1 to 3 carbon atoms, and k and m are each independently an integer of 1 to 4. The ion exchange group is bonded to the resin matrix through the leftmost alkylene group. More preferably, R ¹ and R ² are methyl groups, and k and m are 1. An example of such an amphoteric ion exchange resin is Diaion AMP03 (manufactured by Mitsubishi Chemical Corporation).

  The water used as the eluent may be deionized water, distilled water, or pure water. The specific resistance of water is preferably 1 to 10 MΩ · cm. Further, the elution temperature is preferably 5 to 80 ° C, more preferably 15 to 35 ° C.

  Chromatography may be any of a batch fixed bed method, a moving bed method, and a simulated moving bed method. In the batch fixed bed method, an organic acid-containing crude liquid is supplied to a column packed with an ion exchange resin, and then an eluent is supplied at an appropriate rate to separate components. In the moving bed system, the ion exchange resin is moved in the direction opposite to the flow of the eluent at a speed intermediate between the moving speed of the organic acid and the impurity component in the organic acid-containing crude liquid. The simulated moving bed method uses a plurality of columns connected in an annular shape, and moves the organic acid crude liquid supply and organic acid take-out port by one column in the eluent flow direction at a predetermined cycle. Do. The pseudo moving bed method is preferable in that the isolation can be continuously performed without adding an eluent.

The superficial velocity (SV) of the eluent is preferably 0.1 to 10 hr ^{−1 in} the case of the batch fixed bed method. Further, the liquid feeding pressure is preferably 0.1 to 2 MPa.

The organic acid-containing crude liquid is loaded by passing an amount of the organic acid in an amount of 0.1 to 0.2 times the total capacity of the ion exchange resin in the SV in the above range.
After loading, when eluent water is passed through SV in the above range, for example, as shown in FIG.
Non-electrolyte components such as glucose and neutral salts in organic acid-containing crude liquid at a effluent volume (BV), i.e., through volume with respect to the volume of the ion exchange resin bed, of about 0.4 to 0.9. Sodium chloride elutes and then the organic acid begins to elute. Multivalent ions such as phosphate ions are adsorbed on the ion exchange resin and hardly eluted. The fraction of the eluate is collected, and the elution of the organic acid can be detected and collected while monitoring the elution component with an analytical means such as an enzyme sensor, a conductivity meter, or ICP.

  Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples. However, the present invention is not limited to these examples.

<Preparation of lactic acid-containing crude liquid>
A transformant (ASP2782) was obtained by the method described in Patent Document 2 (WO2011 / 021629). When the transformant was inoculated into D12 liquid medium (glucose 12%) and fermented for 20 hours under the conditions of a temperature of 32 ° C. and a shaking speed of 100 rpm, a fermentation solution (pH 2. 3) was obtained. The fermentation broth was subjected to centrifugation (12000 G, 5 minutes), and the supernatant was obtained as a lactic acid-containing crude liquid.

<Ion exchange chromatography>
Add pure water to a chromatographic column with a diameter of 10 mm, add 10 ml of amphoteric ion exchange resin (Dianion AMP03, manufactured by Mitsubishi Chemical Corp.) swollen by immersion in pure water in advance to make a resin bed, and the level of pure water Was aligned with the top edge of the resin bed. After adding 1 ml of a lactic acid-containing crude liquid to the upper part of the resin bed, the SV was adjusted to ¹ hr ⁻¹ and pure water was passed through, and 1 ml of the eluate was fractionated. Each fraction was appropriately diluted with pure water, and lactic acid, glucose, and ethanol were quantified with each enzyme sensor, Na, K, Mg, and Ca were ICP, and phosphate ions were ion exchange chromatography. The elution curves for lactic acid, glucose, and ethanol obtained by plotting the effluent volume (BV) on the horizontal axis and the concentration on the vertical axis are shown in FIG. 1, and the elution curves for Na, K, Mg, and Ca ions are shown. 2 shows each. However, since no phosphate ion was detected, the plot was omitted.

  Chromatography was carried out in the same manner as in Example 1 except that 2 ml of the eluate was collected. As a result, the same elution curve as in Example 1 could be obtained with good reproducibility.

  As can be seen from FIGS. 1 and 2, lactic acid in the fermentation broth could be clearly separated from the other components. Moreover, when the eluate was analyzed by 1H-NMR, oligomeric lactic acid was not detected in any of the examples.

  According to the method of the present invention, an organic acid can be isolated from a fermentation broth under a chemically stable condition using a general ion exchange resin.

Claims (5)

A method for producing an organic acid, comprising:
1. Producing an organic acid by fermentation to obtain an organic acid-containing crude liquid having a pH of 1 to 5,
2. A step of isolating an organic acid from the organic acid-containing crude liquid obtained in step 1 by ion exchange chromatography,
And the ion exchange resin in the ion exchange chromatography method is an amphoteric ion exchange resin, and the eluent is water.   The production method according to claim 1, wherein the organic acid is lactic acid, succinic acid, 3-hydroxypropionic acid, malic acid, or fumaric acid.   The organic acid is lactic acid, and a microorganism that produces lactic acid has Schizosaccharomyces pombe as a host, a lactate dehydrogenase gene is integrated therein, and pyruvate decarboxylation of the Schizosaccharomyces pombe host. The production method according to claim 1 or 2, which is a transformant in which a part of a gene group encoding the enzyme is deleted or inactivated. The manufacturing method of any one of Claims 1-3 with which this amphoteric ion exchange resin is equipped with the ion exchange group represented by following formula (1).
[Chemical 1]

_{^{- (CH 2) k-N}} + R 1 R 2 - (CH 2) m-COO - (1)

(Here, R ¹ and R ² are each independently an alkyl group having 1 to 3 carbon atoms, and k and m are each independently an integer of 1 to 4). The production method according to any one of claims 1 to 4, wherein the ion exchange chromatography is performed by a pseudo moving bed system.

Images