Method for producing aspartic acid

Abstract

To provide a method for producing aspartic acid that enables improvement of the yield of aspartic acid in microbial production of aspartic acid.SOLUTION: A method for producing aspartic acid includes a step of generating aspartic acid by reacting an aspartic acid raw material with an aspartic acid-producing microorganism in a reaction liquid housed in a reaction vessel which allows control of an aeration rate and a stirring speed. The aeration rate and the stirring speed during the reaction are controlled so as to satisfy the following condition (i) and at least one of the following conditions (ii) and (iii): (i) the oxygen transfer capacity coefficient (kLa) is in the range of 30 / h to 60 / h; (ii) the agitation power per unit volume without aeration (P / V) is less than 2 kg m2 s-3; (iii) the aeration rate per unit volume is 0.2 vvm or more.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing aspartic acid.

Background Art

[0002] Aspartic acid is being considered for use as a superabsorbent polymer, a thickener for cosmetics, etc. by polymerization. Industrially produced aspartic acid is synthesized by an enzymatic method using petroleum-derived fumaric acid as a raw material.

[0003] As a method for producing aspartic acid, a method using microorganisms has also been proposed. For example, Patent Document 1 describes a method for producing aspartic acid from glucose using genetically modified Corynebacterium.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] In the production of aspartic acid using microorganisms, the yield of aspartic acid is greatly affected by the oxygen utilization conditions of the microorganisms.

[0006] Therefore, an object of the present invention is to provide a method for producing aspartic acid that can improve the yield of aspartic acid in the production of aspartic acid using microorganisms.

Means for Solving the Problems

[0007] The present invention includes the following aspects. [1] A method for producing aspartic acid, which includes a step of reacting an aspartic acid raw material with an aspartic acid-producing microorganism in a reaction solution contained in a reaction vessel capable of controlling the aeration rate and the stirring speed to produce aspartic acid, wherein the aeration rate and the stirring speed during the reaction are controlled to satisfy the condition of (i) below and at least one of the conditions of (ii) and (iii) below: (i) The oxygen transfer volumetric coefficient (k L a) is in the range of 30 / h to 60 / h; (ii) The stirring power without aeration per unit volume (P / V) is less than 2×10 -3 W / m 3 ; (iii) The aeration rate per unit volume is 0.2 vvm or more. [2] The method for producing aspartic acid according to [1], wherein the total oxygen consumption per unit volume from the start point of the reaction to the depletion point of the aspartic acid raw material is 250 mmol / L or more. [3] The method for producing aspartic acid according to [1] or [2], wherein the ratio of the acetic acid molar concentration to the aspartic acid molar concentration (acetic acid molar concentration / aspartic acid molar concentration) in the reaction solution at the depletion point of the aspartic acid raw material is 1.5 or less. [4] The method for producing aspartic acid according to any one of [1] to [3], wherein the aspartic acid raw material is glucose. [5] The method for producing aspartic acid according to any one of [1] to [4], wherein aeration is performed with air. [6] The method for producing aspartic acid according to any one of [1] to [5], wherein the aspartic acid-producing microorganism is a coryneform bacterium. [7] The method for producing aspartic acid according to [6], wherein the coryneform bacterium is a genetically modified microorganism. [Advantages of the Invention]

[0008] According to the present invention, there is provided a method for producing aspartic acid, which can improve the yield of aspartic acid in the production of aspartic acid using microorganisms. [Brief Description of the Drawings]

[0009]

Figure 1

Figure 2

BEST MODE FOR CARRYING OUT THE INVENTION

[0010] In one embodiment, the present invention provides a method for producing aspartic acid. The method for producing aspartic acid includes a step of reacting an aspartic acid raw material and an aspartic acid-producing microorganism in a reaction solution contained in a reaction tank capable of controlling the aeration rate and the stirring rate to produce aspartic acid (hereinafter, also referred to as the "aspartic acid production step"). In one embodiment, the aeration rate and the stirring rate during the reaction are controlled so as to satisfy the condition of the following (i) and at least one of the conditions of the following (ii) and (iii). (i) The oxygen transfer capacity coefficient (k L a) is in the range of 30 / h to 60 / h. (ii) The stirring power without aeration per unit volume (P / V) is less than 2×10 -3 W / m 3 . (iii) The aeration rate per unit volume is 0.2 vvm or more.

[0011] [Aspartic acid production step] In the aspartic acid production step, an aspartic acid raw material and an aspartic acid-producing microorganism are reacted in a reaction solution contained in a reaction tank capable of controlling the aeration rate and the stirring rate to produce aspartic acid.

[0012] [Reaction tank] The reaction tank is not particularly limited as long as it can control the aeration rate and the stirring rate. As the reaction tank, a culture tank of a culture device equipped with an aeration device and a stirring device can be used. Examples of such a culture device include a jar fermenter and the like.

[0013] Fig. 1 shows an example of a reaction tank. The reaction apparatus 1 shown in Fig. 1 includes a reaction tank 10, a stirring device 20, and a gas supply pipe 30. The upper part of the reaction tank 10 is closed by a lid 11.

[0014] The stirring device 20 includes a stirring shaft 21 and stirring blades 22. When the stirring shaft is rotated by a motor or the like, the stirring blades are rotated, and the reaction liquid M contained in the reaction tank 10 is stirred. The stirring device 20 can control the stirring speed, for example, by controlling the output of the motor that rotates the stirring shaft.

[0015] The gas supply pipe 30 is connected to a ventilation device, and a desired gas is supplied to the reaction liquid M in the reaction tank 10 through the gas supply pipe 30. The gas supplied by the gas supply pipe 30 is, for example, an oxygen-containing gas. Examples of the oxygen concentration in the oxygen-containing gas include 10 to 50% by volume, and 15 to 30% by volume is preferred. Examples of the oxygen-containing gas include air. The ventilation device includes, for example, a gas supply pump, and the ventilation speed can be controlled by controlling the output of the gas supply pump.

[0016] The capacity of the reaction tank 10 is not particularly limited, and examples include 1 L or more and 200 kL or less. Examples of the capacity of the reaction tank 10 include 1 L or more and 100 kL or less, 1 L or more and 10 kL or less, 1 L or more and 5 kL or less, 1 L or more and 1 kL or less, 1 L or more and 500 L or less, 1 L or more and 100 L or less, 1 L or more and 50 L or less, or 1 L or more and 10 L or less, etc.

[0017] The amount of the reaction solution accommodated in the reaction tank 10 is not particularly limited. For example, it may be an amount of 10 to 90% by volume with respect to the capacity (100% by volume) of the reaction tank 10. The amount of the reaction solution may be an amount of 40 to 90% by volume, 45 to 85% by volume, or 50 to 80% by volume with respect to the capacity (100% by volume) of the reaction tank 10. Specific examples of the amount of the reaction solution include 0.5 L or more and 100 kL or less, 0.5 L or more and 50 kL or less, 0.5 L or more and 10 kL or less, 0.5 L or more and 5 kL or less, 0.5 L or more and 1 kL or less, 0.5 L or more and 500 L or less, 0.5 L or more and 100 L or less, 0.5 L or more and 50 L or less, 0.5 L or more and 10 L or less, or 0.5 L or more and 5 L or less, etc.

[0018] <Aspartic acid-producing microorganism>

[0019] An aspartic acid-producing microorganism is a microorganism having the ability to produce aspartic acid. The aspartic acid-producing microorganism may be unicellular or multicellular. The aspartic acid microorganism may be a prokaryote or a eukaryote. Examples of prokaryotes include bacteria, archaea, cyanobacteria, etc. Examples of eukaryotes include fungi, etc. As the aspartic acid-producing microorganism, fungi or bacteria are preferred, and bacteria are more preferred.

[0020] Examples of fungi include Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris), Kluyveromyces lactis, Hansenula polymorpha, Yarrowia (e.g., Yarrowia lipolytica), Cryptococcus (e.g., Cryptococcus sp. S-2), Aspergillus (e.g., Aspergillus oryzae), Pseudozyma (e.g., Pseudozyma antarctica), etc. Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, etc. can be conveniently used because genetic recombination techniques and heterologous protein expression systems have been established for them.

[0021] Examples of bacteria include, for example, Escherichia (e.g., Escherichia coli), Bacillus (e.g., Bacillus subtilis), Lactobacillus (e.g., Lactobacillus acidophilus), Clostridium (e.g., Clostridium thermocellum, Clostridium acetobutylicum), Rhodopseudomonas (e.g., Rhodopseudomonas palustris), Rhodobacter (Rhodobacter capsulatus), and coryneform bacteria described below. As bacteria, since genetic recombination techniques and protein expression systems have already been established, Escherichia bacteria or coryneform bacteria are preferred, Escherichia coli or coryneform bacteria are more preferred, and Corynebacterium is even more preferred.

[0022] The bacterium may be a Gram-positive bacterium (e.g., actinomycetes) or a Gram-negative bacterium. Examples of Gram-negative bacteria include bacteria belonging to the phylum Proteobacteria. Bacteria belonging to the phylum Proteobacteria include bacteria belonging to the classes Alpha-, Beta-, Gamma-, Delta-, Epsilon- or Zeta-Proteobacteria, and bacteria belonging to the class Oligoflexia. Examples of Gram-negative bacteria include bacteria belonging to the family Enterobacteriaceae, the family Vibrionaceae or the family Pseudomonadaceae.

[0023] The term "coryneform bacterium" refers to a group of bacteria defined in Bergey's Manual of Determinative Bacteriology, 8th Edition, p. 599 (1974). Examples of coryneform bacteria include bacteria belonging to the genus Corynebacterium, the genus Brevibacterium, the genus Arthrobacter, the genus Mycobacterium, the genus Micrococcus, the genus Microbacterium, etc.

[0024] Examples of bacteria belonging to the genus Corynebacterium include the following species and bacterial strains. Corynebacterium glutamicum (e.g., FERM P-18976 strain, ATCC13032 strain, ATCC31831 strain, ATCC13058 strain, ATCC13059 strain, ATCC13060 strain, ATCC13232 strain, ATCC13286 strain, ATCC13287 strain, ATCC13655 strain, ATCC13745 strain, ATCC13746 strain, ATCC13761 strain, ATCC14020 strain); Corynebacterium acetoglutamicum (e.g., ATCC15806 strain); Corynebacterium acetoacidophilum (e.g., strain ATCC13870); Corynebacterium melassecola (e.g., strain ATCC17965); Corynebacterium efficiens (e.g., strain YS - 314, YS - 314 T strain (NBRC100395 T strain)); Corynebacterium alkanolyticum (e.g., strain ATCC21511); Corynebacterium callunae (e.g., strain ATCC15991, strain NBRC15359, strain DSM20147); Corynebacterium lilium (e.g., strain ATCC15990); Corynebacterium thermoaminogenes (Corynebacterium efficiens) (e.g., strain AJ12340, strain FERM BP1539); Corynebacterium herculis (e.g., strain ATCC13868); Corynebacterium ammoniagenes (Brevibacterium ammoniagenes) (e.g., strain ATCC6871, strain ATCC6872, strain DSM20306, strain NBRC12071 T strain, strain NBRC12072, strain NBRC12612 T strain); Corynebacterium pollutisoli; Corynebacterium marinum (e.g., strain DSM44953); Corynebacterium humireducens (e.g., strain NBRC106098); Corynebacterium halotolerans (e.g., strain YIM70093); Corynebacterium deserti (e.g., strain GIMN1.010); Corynebacterium doosanense (e.g., strains CAU212, DSM45436); Corynebacterium maris (e.g., strain DSM45190).

[0025] Examples of Brevibacterium bacteria include the following species and strains. Brevibacterium divaricatum (e.g., strain ATCC14020); Brevibacterium flavum [e.g., strains MJ-233 (FERM BP-1497), MJ-233AB-41 (FERM BP-1498), ATCC13826, ATCC14067, ATCC13826]; Brevibacterium immariophilum (e.g., strain ATCC14068); Brevibacterium lactofermentum (Corynebacterium glutamicum) (e.g., strain ATCC13869); Brevibacterium roseum (e.g., strain ATCC13825); Brevibacterium saccharolyticum (e.g., strain ATCC14066); Brevibacterium thiogenitalis (e.g., strain ATCC19240); Brevibacterium album (e.g., strain ATCC15111); Brevibacterium cerinum (e.g., strain ATCC15112).

[0026] Examples of bacteria belonging to the genus Arthrobacter include the following species and strains. Arthrobacter globiformis (e.g., strains ATCC8010, ATCC4336, ATCC21056, ATCC31250, ATCC31738, ATCC35698, NBRC3062, NBRC12137T), etc.

[0027] Examples of bacteria belonging to the genus Micrococcus include Micrococcus freudenreichii [e.g., strain No. 239 (FERM P-13221)]; Micrococcus luteus [e.g., strains NCTC2665, No. 240 (FERM P-13222)]; Micrococcus ureae (e.g., strain IAM1010); Micrococcus roseus (e.g., strain IFO3764), etc. Examples of bacteria belonging to the genus Microbacterium include Microbacterium ammoniaphilum (e.g., strain ATCC15354), etc.

[0028] Corynebacterium strains, for example, in the case of ATCC strains, can be obtained by license from the American Type Culture Collection (P.O. Box 1549 Manassas, VA 20108 USA). For other strains, they can also be obtained by license from each microorganism preservation institution that provides them.

[0029] The aspartic acid-producing microorganism may be a genetically modified microorganism. A "genetically modified microorganism" is one that has been subjected to some genetic recombination operation on a microorganism. Examples of genetically modified microorganisms include microorganisms that have been subjected to genetic recombination operations so as to improve the ability to produce aspartic acid. Examples of such genetically modified microorganisms include the genetically modified microorganisms described in International Publication No. 2020 / 208842, International Publication No. 2023 / 095896, etc.

[0030] The aspartic acid-producing microorganism may be live cells or dead cells, but preferably live cells. The aspartic acid-producing microorganism may be immobilized on a carrier. Examples of the carrier include porous materials, hydrogels, etc.

[0031] The aspartic acid-producing microorganism can be used alone or in combination of two or more.

[0032] <Reaction solution> The reaction solution is not particularly limited. The reaction solution preferably has an osmotic pressure such that the cells of the aspartic acid-producing microorganism do not rupture. Examples of the reaction solution include physiological saline, buffer solutions (phosphate buffer, phosphate buffered saline), media for microorganism culture, etc.

[0033] When using a medium for microorganism culture, the medium can be appropriately selected according to the type of aspartic acid-producing microorganism. For example, when the aspartic acid-producing microorganism is a bacterium, a medium for bacterial culture can be used. As the medium, for example, a natural medium, a semi-synthetic medium, or a synthetic medium containing an organic carbon source, a nitrogen source, inorganic salts, and optionally vitamins and other nutrients can be used.

[0034] Examples of the organic carbon source include carbohydrates. Specific examples include monosaccharides such as glucose, fructose, mannose, xylose, arabinose, and galactose; disaccharides such as sucrose, maltose, lactose, cellobiose, xylobiose, and trehalose; polysaccharides such as cellulose, starch, glycogen, agarose, pectin, and alginic acid; molasses (molasses), etc.; non-edible agricultural wastes such as rice straw, forest residues, bagasse, and corn stover, and non-edible biomass (resources made from non-edible herbaceous plants and woody plants); saccharified solutions obtained by saccharifying energy crops such as switchgrass, napier grass, and miscanthus with saccharifying enzymes; sugar alcohols such as mannitol, sorbitol, xylitol, and glycerin; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, and gluconic acid; alcohols such as ethanol, propanol, and butanol; hydrocarbons such as normal paraffin. The organic carbon source can be used alone or in combination of two or more.

[0035] Examples of the nitrogen source include inorganic ammonium salts such as ammonium carbonate ((NH4)2CO3), ammonium bicarbonate, ammonium chloride, ammonium sulfate, and ammonium nitrate; organic ammonium salts such as ammonium acetate; urea; aqueous ammonia; inorganic nitrates or organic nitrates such as sodium nitrate and potassium nitrate; nitrogen-containing organic compounds such as corn steep liquor, meat extract, protein hydrolysates (such as casamino acids, tryptone, peptone, and NZ-amine), and amino acids. The nitrogen source can be used alone or in combination of two or more. The concentration of the nitrogen source in the medium may be appropriately adjusted according to the type of aspartic acid-producing microorganism, the type of nitrogen compound, etc. Examples of the concentration of the nitrogen source include 0.1 to 10% (w / v).

[0036] Examples of the inorganic salts include phosphorus sources (such as inorganic phosphates like monopotassium phosphate and dipotassium phosphate), magnesium sources (such as inorganic magnesium salts like magnesium sulfate), iron sources (such as inorganic iron salts like iron(II) sulfate heptahydrate and ferrous nitrate), manganese sources (such as inorganic manganese salts like manganese sulfate), copper sources (such as inorganic copper salts like copper sulfate), zinc sources (such as inorganic zinc salts like zinc sulfate), cobalt sources (such as inorganic cobalt salts like cobalt sulfate), sodium chloride, calcium carbonate, etc. The inorganic salts can be used alone or as a mixture of two or more. It is preferable that the inorganic salts contain phosphates, magnesium salts, iron salts, manganese salts, and copper salts. The concentration of the inorganic salts in the medium may be appropriately adjusted according to the type of aspartic acid-producing microorganism, the type of inorganic salts, etc. Examples of the concentration of the inorganic salts include about 0.01 to 3% (w / v).

[0037] Examples of the vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, etc. The concentration of the vitamins in the medium may be appropriately adjusted according to the type of aspartic acid-producing microorganism, the type of vitamins, etc. Examples of the concentration of the vitamins include about 0.00001 to 3% (w / v).

[0038] Examples of the other nutrients include meat extract, peptone, polypeptone, yeast extract, dried yeast, casamino acid, corn steep liquor, skim milk powder, hydrochloric acid hydrolyzate of defatted soybeans, extracts of animals, plants or microbial cells, and their decomposition products, etc. The concentration of the other nutrients in the medium may be appropriately adjusted according to the type of aspartic acid-producing microorganism, the type of nutrients, etc. Examples of the concentration of the other nutrients include about 0.1 to 10% (w / v).

[0039] The medium may contain an antifoaming agent. Examples of the antifoaming agent include silicone-based antifoaming agents and polyether-based antifoaming agents.

[0040] The pH of the reaction solution can be appropriately set according to the type of aspartic acid microorganism. Examples of the pH of the reaction solution include 6 to 8.

[0041] When the aspartic acid-producing microorganism is a coryneform bacterium, as the medium, A medium (Inui, M. et al., J. Mol. Microbiol. Biotechnol. 7:182-196 (2004)), BT medium (Omumasaba, C.A. et al., J. Mol. Microbiol. Biotechnol. 8:91-103 (2004)), NA medium, etc. can be preferably used.

[0042] <Aspartic acid raw material> The aspartic acid raw material is a compound that is converted into aspartic acid by the action of an aspartic acid-producing microorganism. The aspartic acid raw material is preferably a compound that is converted into aspartic acid via the TCA cycle (citric acid cycle) possessed by the aspartic acid-producing microorganism. Examples of the aspartic acid raw material include reactants and products in the glycolysis system, reactants and products in the TCA cycle, and precursors of the reactants.

[0043] Specific examples of the aspartic acid raw material include monosaccharides such as glucose; oligosaccharides and polysaccharides containing glucose as a monosaccharide unit (maltose, starch, etc.); organic acids such as pyruvic acid, citric acid, fumaric acid, α-ketoglutaric acid, L-malate, oxaloacetic acid, succinic acid, phosphoenolpyruvic acid, etc. Glucose is preferred as the aspartic acid raw material. When the medium is used as the reaction solution, the aspartic acid raw material is used as an organic carbon source.

[0044] The concentration of the aspartic acid raw material in the reaction solution is not particularly limited. Examples of the concentration of the aspartic acid raw material include 1 to 30% (w / v), 3 to 25% (w / v), or 5 to 20% (w / v). Examples of the molar concentration of the aspartic acid raw material in the reaction solution include 50 to 1500 mM, 100 to 1300 mM, 200 to 1100 mM, or 300 to 1000 mM.

[0045] <Reaction conditions> (Condition (i)) During the reaction between the aspartic acid raw material and the aspartic acid-producing microorganism, the aeration rate and stirring speed in the reaction tank are controlled to satisfy Condition (i). Condition (i): The oxygen transfer volumetric coefficient (k L a) is in the range of 30 / h to 60 / h.

[0046] The oxygen transfer volumetric coefficient (k L a) is a numerical value indicating the oxygen transfer ability from the gas phase to the liquid phase under aeration and stirring conditions, and serves as an index of the oxygen supply capacity in the reaction tank. The oxygen transfer rate (OTR) is represented by Equation (1).

[0047] OTR [mol / m 3 / s] = N·A / V = k L a(C * -C) (1)

[0048] N: Total oxygen transfer flux [mol / m 2 / s] A: Gas-liquid contact area [m 3 V: Volume of the reaction solution [m 3 C: Dissolved oxygen concentration [mol / m 3 C * : Saturated oxygen concentration [mol / m 3

[0049] k L a can be measured by the sodium sulfite method. The sodium sulfite method utilizes the oxidation reaction of sodium sulfite (Na2SO3) to measure k L ​​​​It is a method for measuring a. When sodium sulfite is added to a reaction solution containing cobalt sulfate as a catalyst, the dissolved oxygen (DO) in the reaction solution is consumed by the oxidation of sodium sulfite. When an excessive amount of sodium sulfite is present, DO is 0, but when the sodium sulfite in the reaction solution is consumed, DO increases. When the addition rate of sodium sulfite and the oxygen supply to the liquid phase by aeration and stirring reach an equilibrium state, DO becomes constant. Therefore, k L a can be calculated by the following formula (2).

[0050] k L a = QC BF / {2V L C Ai (1 - C AS / C Ai )} (2)

[0051] Q: Amount of sodium sulfite aqueous solution added [mL / min] C BF : Concentration of sodium sulfite aqueous solution [mol / m 3 V L : Volume of reaction solution [mL] C Ai : Equilibrium concentration of dissolved oxygen [mol / m 3 C AS : Concentration of dissolved oxygen [mol / m 3

[0052] The equilibrium concentration of dissolved oxygen (C Ai ) is the DO when the addition rate of sodium sulfite and the oxygen supply to the liquid phase by aeration and stirring reach an equilibrium state. In the sodium sulfite method, pure water can be used as a virtual reaction solution.

[0053] Also, the relationship between k L a, the aeration rate, and the stirring speed is expressed by the following formula (3) (Cooper et al., Ind. Eng. Chem. 36, 504 (1944)).

[0054] k L a = a × n b × AF c ​​​(3)

[0055] n: Stirring speed [rpm] AF: Aeration rate [mL / min] a, b, and c are constants.

[0056] In a specific reaction vessel, at three or more conditions where both the aeration rate and the stirring speed are different, k L a is measured and applied to Equation (3) by the least squares method to obtain the constants a, b, and c in the reaction vessel. When using the culture tank of a commercially available culture device as the reaction vessel, the constants a, b, and c in the culture device can be obtained in the same way. By obtaining the constants a, b, and c in advance for the reaction conditions such as the reaction vessel, temperature, and solution to be used, based on the above Equation (3), k L a can be predicted from the stirring speed (n) and the aeration rate (AF).

[0057] Cooper et al. (Ind. Eng. Chem. 36, 504 (1944)) collected data by the sodium sulfite oxidation method regarding the oxygen transfer of aerated stirred algae, and proposed the following empirical equation (4) for the oxygen absorption rate Kd [kmol / (h·m 3 ·atm)] (Biochemical Engineering, Fundamentals and Applications of Bioprocesses, 2nd Edition, Chapter 9).

[0058] Kd = 0.635(Pg / V) 0.95 Vs 0.67 (4)

[0059] Pg: Stirring power required in the aeration system [W] V: Volume of the reaction solution [m 3 Vs: Superficial velocity [m / h]

[0060] Kd has a relationship with k L a, and Kd = k L a / H (H is the Henry's law constant of oxygen). The above Equation (3) is a transformation of the above Equation (4) assuming all conditions other than the aeration rate and the stirring speed are the same.

[0061] ​In one embodiment, k in condition (I) L a is a predicted value calculated by the above formula (3). Based on the above formula (3), by controlling the stirring speed (n) and the aeration rate (AF) during the reaction, k during the reaction L a can be adjusted to be within the range of 30 / h to 60 / h.

[0062] k during the reaction L a is preferably in the range of 34 / h to 60 / h, more preferably in the range of 35 / h to 60 / h, still more preferably in the range of 40 / h to 60 / h, still more preferably in the range of 45 / h to 60 / h, and particularly preferably in the range of 45 / h to 55 / h.

[0063] (Condition (ii)) During the reaction between the aspartic acid raw material and the aspartic acid-producing microorganism, the aeration rate and the stirring speed in the reaction tank are controlled so as to satisfy the following condition (ii) and / or the condition (iii) described later. Condition (ii): The stirring power per unit volume without aeration (P / V) is 2×10 -3 W / m 3 less than.

[0064] The stirring power per unit volume without aeration (P / V) is the stirring power per unit volume of the reaction liquid when assuming no aeration. P / V can be calculated by dividing the stirring power without aeration (P) by the reaction liquid volume (V) [m 3 . The stirring power without aeration (P) [kg·m 2 ·s -3 can be calculated by the following formula (5).

[0065] P = Npρn 3 d 5 (5)

[0066] Np: Power number ρ: Liquid density [kg / m 3 n: Impeller rotation speed [1 / s] d: Impeller diameter [m]

[0067] The power number Np can be expressed by the following formula (6).

[0068] Np = K L / Re + K T (6)

[0069] K L : laminar flow constant K t : turbulent flow constant Re: stirring Reynolds number

[0070] K in the main stirring blade L and K T are as follows (W.L.McCabe, J.C. Smith, P. Harriott. Unit Operations of Chemical Engineering 7th Ed.McGraw-Hill(2004),Chapter 9 Agitation and Mixing of Liquids (Table 9.2)). Flat plate turbine blade: K L 65; K T 5.75 Flat plate paddle blade: K L 36.5; K T 1.70 Propeller blade: K L 41.0; K T 0.32 Anchor blade: K L 300; K T 50.35

[0071] The stirring Reynolds number Re can be expressed by the following formula (7).

[0072] Re = ρnd 2 / μ (7)

[0073] ρ: liquid density [kg / m 3 n: blade rotation speed [1 / s] d: blade diameter [m] μ: liquid viscosity [Pa·s]

[0074] ​In the above formula, the liquid density ρ is the density of the reaction liquid. The impeller rotation speed n is the rotation speed of the stirring impeller. The impeller diameter d is the impeller diameter of the stirring impeller. The liquid viscosity μ is the viscosity of the reaction liquid. When the stirring impeller is a multi-stage impeller, it is only necessary to multiply Np by the number of impeller stages.

[0075] Based on the above formula (5), by controlling the rotation speed of the stirring impeller (i.e., the stirring speed), the P / V during the reaction can be adjusted to be less than 2×10 -3 W / m 3 .

[0076] The P / V during the reaction is preferably less than 2×10 -3 W / m 3 , more preferably in the range of not less than 0.1×10 -3 W / m 3 and less than 2×10 -3 W / m 3 , still more preferably in the range of 0.3×10 -3 W / m 3 to 1.8×10 -3 W / m 3 , even more preferably in the range of 0.4×10 -3 W / m 3 to 1.6×10 -3 W / m 3 , and particularly preferably in the range of 0.4×10 -3 W / m 3 to 1.5×10 -3 W / m 3 .

[0077] During the reaction, aeration is being carried out, so it is not actually non-aerated. Condition (ii) means that, based on the stirring speed during the reaction, when calculating the stirring power per unit volume (P / V) under non-aerated conditions, the stirring speed is controlled so that it is less than 2×10 -3 W / m 3 . Also, the stirring power per unit volume (P / V) under non-aerated conditions in condition (ii) is the liquid density (994.03 kg·m -3 ) and the liquid viscosity (0.000719 kg·m -1 ·s -1 when the reaction liquid is assumed to be pure water at 35°C.(Pa·s)) is calculated. That is, in condition (ii), when pure water at 35°C is used as the reaction solution and it is assumed that there is no aeration, the calculated P / V is 2×10 -3 W / m 3 is less than, and the stirring speed is controlled accordingly.

[0078] (Condition (III)) During the reaction between the aspartic acid raw material and the aspartic acid-producing microorganism, the aeration rate and stirring speed in the reaction tank are controlled so as to satisfy the above condition (ii) and / or the following condition (iii). Condition (ii) The aeration rate per unit volume is 0.2 vvm or more.

[0079] The aeration rate per unit volume (AF / V) [vvm] can be calculated by dividing the aeration rate (AF) [m 3 / min] by the volume (V) [m 3 of the reaction solution.

[0080] Q g / V during the reaction is preferably 0.1 vvm or more, more preferably in the range of 0.1 vvm to 10 vvm, still more preferably in the range of 0.2 vvm to 10 vvm, and particularly preferably in the range of 0.2 vvm to 6 vvm.

[0081] When the volume of the reaction tank is large, instead of Q g / V, the superficial gas velocity [m·s -1 can also be used. The superficial gas velocity can be expressed by the following formula (7).

[0082] Superficial gas velocity = aeration rate [m 3 ·s -1 / cross-sectional area of the reaction tank (m 2 ) (7)

[0083] For example, when the volume of the reaction tank is 1 kL or less, or when the oxygen concentration in the exhaust gas (the gas exhausted from the reaction tank) is sufficiently high (for example, 10% by volume or more), Q gInstead of / V, the superficial gas velocity may be used. The superficial gas velocity is preferably 0.0003 m·s -1 or more, and preferably 0.0003 m·s -1 to 0.1 m·s -1 is more preferable, and 0.0004 m·s -1 to 0.07 m·s -1 is more preferable, and 0.0004 m·s -1 to 0.05 m·s -1 is even more preferable, and 0.0004 m·s -1 to 0.01 m·s -1 is particularly preferable.

[0084] During the reaction, at least one of conditions (II) and (III) may be satisfied, but it is preferable to satisfy both conditions (II) and (III).

[0085] (Other conditions) The stirring speed during the reaction may be set so as to satisfy the above condition (I) and at least one of the above conditions (II) and (III). Examples of the stirring speed include 200 rpm or more and less than 500 rpm, preferably 250 to 490 rpm, more preferably 250 to 450 rpm, and particularly preferably 290 to 450 rpm.

[0086] The temperature during the reaction is not particularly limited and can be appropriately set according to the type of aspartic acid-producing microorganism. Examples of the reaction temperature include 10 to 50°C, preferably 15 to 40°C, more preferably 20 to 45°C, and particularly preferably 25 to 40°C. The reaction temperature may be switched according to the progress of the reaction time. For example, the reaction temperature may be gradually increased stepwise as the reaction time elapses.

[0087] The pH during the reaction is not particularly limited and can be appropriately set according to the type of aspartic acid-producing microorganism. Examples of the reaction pH include pH 3.0 to 9.0, preferably pH 4.0 to 8.5, more preferably pH 5.0 to 8.5, and even more preferably pH 6.0 to 8.0.

[0088] When the pH fluctuates during the reaction, an acidic pH adjusting solution or a basic pH adjusting solution may be added to adjust the pH. Examples of the acidic pH adjusting solution include hydrochloric acid, sulfuric acid, etc. Examples of the basic pH adjusting solution include sodium hydroxide, aqueous ammonia, ammonium carbonate, a mixed solution of ammonium carbonate and aqueous ammonia, etc.

[0089] The reaction may be carried out batchwise or continuously. When the reaction is carried out continuously, the components consumed during the reaction may be added in a timely manner.

[0090] When the reaction is carried out batchwise, the ratio of the molar concentration of acetic acid to the molar concentration of aspartic acid (acetic acid molar concentration / aspartic acid molar concentration) in the reaction solution at the time of depletion of the aspartic acid raw material is preferably 1.5 or less. That [acetic acid molar concentration / aspartic acid molar concentration] is below the above upper limit indicates that the generation of impurities is suppressed and aspartic acid is efficiently generated. In the reaction solution at the time of depletion of the aspartic acid raw material, [acetic acid molar concentration / aspartic acid molar concentration] is preferably 1.2 or less, more preferably 1.0 or less, and even more preferably 0.9 or less.

[0091] The total oxygen consumption per unit volume from the start of the reaction to the time of depletion of the aspartic acid raw material is preferably 250 mmol / L or more. That the total oxygen consumption per unit volume is above the above lower limit indicates that the aspartic acid production reaction has been carried out efficiently. The total oxygen consumption per unit volume is preferably 250 to 500 mmol / L, more preferably 260 to 450 mmol / L, and even more preferably 260 to 400 mmol / L.

[0092] The yield of aspartic acid at the time of depletion of the aspartic acid raw material (molar concentration of aspartic acid (mM) in the reaction solution at the end of the reaction / molar concentration of aspartic acid raw material (mM) in the reaction solution at the start of the reaction × 100) is preferably 70% or more, more preferably 75% or more, and even more preferably 80% or more.

[0093] The concentration of the aspartic acid-producing microorganism at the start of the reaction is not particularly limited. Examples of the concentration of the aspartic acid-producing microorganism at the start of the reaction include, for example, a cell turbidity at OD 610 of about 10 to 300, may be about 20 to 200, or may be about 30 to 100. "OD 610 " means the optical density of the bacterial solution measured at a wavelength of 610 nm.

[0094] The reaction can be terminated, for example, when the aspartic acid raw material in the reaction solution is depleted.

[0095] The term "reaction" in this step is intended to include the case where the aspartic acid-producing microorganism is a dead cell. When the aspartic acid-producing microorganism is a live cell, "reaction" may be read as "culture". For example, "reaction", "reaction solution", "reaction tank", and "reaction apparatus" may be read as "culture", "culture solution", "culture tank", and "culture apparatus", respectively.

[0096] [Other steps] In addition to the above aspartic acid production step, the method of this embodiment may include other steps. Examples of other steps include, for example, a step of recovering the aspartic acid-producing microorganism (aspartic acid-producing microorganism recovery step), a step of growing the aspartic acid-producing microorganism (aspartic acid-producing microorganism growth step), and the like.

[0097] <Aspartic acid-producing microorganism recovery step> After the aspartic acid production step, the aspartic acid-producing microorganism may be recovered. The recovery of the aspartic acid-producing microorganism can be performed, for example, by centrifugation, filter filtration, or the like.

[0098] The recovered aspartic acid-producing microorganism may be disrupted by physical treatment, chemical treatment, enzymatic treatment, or the like. Aspartic acid may be separated and / or purified from the disrupted cells of the aspartic acid-producing microorganism using known separation and / or purification techniques. Examples of separation / purification techniques include various crystallization methods; various filtration techniques such as ultrafiltration; various chromatography techniques such as ion exchange chromatography, affinity chromatography, hydrophobic chromatography, and reverse phase chromatography; concentration methods; dialysis; and activated carbon adsorption methods. These can be appropriately combined to separate and / or purify aspartic acid.

[0099] <Aspartic acid-producing microorganism growth step> Before the aspartic acid production step, the aspartic acid-producing microorganism may be grown. The grown aspartic acid-producing microorganism may be used as live cells as they are in the aspartic acid production step, or may be immobilized on a carrier or the like and used as dead cells, with or without immobilization, in the aspartic acid production step.

[0100] The medium used in this step is not particularly limited and can be appropriately selected according to the type of aspartic acid-producing microorganism. Examples of the medium include the same ones as described above. However, the medium used in this step does not need to contain an aspartic acid raw material.

[0101] The amount of the medium is not particularly limited and can be appropriately set according to the amount of the reaction solution in the aspartic acid production step. The amount of the medium can be, for example, 1 / 50 to 1.5 times the volume of the reaction solution in the aspartic acid production step.

[0102] Examples of the culture temperature include 10 to 50°C, preferably 15 to 40°C, more preferably 20 to 45°C, and particularly preferably 25 to 40°C.

[0103] The culture pH may be, for example, pH 3.0 to 9.0, preferably pH 4.0 to 8.5, more preferably pH 5.0 to 8.5, and even more preferably pH 6.0 to 8.0. When the pH fluctuates during the reaction, an acidic pH adjuster or a basic pH adjuster may be added to adjust the pH. Examples of the pH adjuster include the same ones as described above.

[0104] The culturing method is not particularly limited, and any method may be used as long as it allows the growth of the aspartic acid-producing microorganism. The culturing method may be static culturing, shaking culturing, or aeration stirring culturing, but from the viewpoint of oxygen supply, shaking culturing or aeration stirring culturing is preferred. As the culturing, batch culturing or fed-batch culturing is preferred.

[0105] When performing aeration stirring culturing, the k L a may be controlled, for example, so as to be 180 to 300 / h by controlling the stirring speed and the aeration rate.

[0106] When performing aeration stirring culturing, the stirring speed may be, for example, 200 to 900 rpm, may also be 300 to 800 rpm, may also be 400 to 800 rpm, or may also be 500 to 800 rpm.

[0107] When performing aeration stirring culturing, the aeration rate per unit volume may be, for example, 0.1 to 2 vvm, may also be 0.2 to 1 vvm, may also be 0.3 to 0.8 vvm, or may also be 0.3 to 0.5 vvm.

[0108] The culturing can be terminated, for example, when the growth of the aspartic acid-producing microorganism reaches the stationary phase.

[0109] After the completion of this step, the aspartic acid production step can be initiated by adding part or all of the culture solution after cultivation to the reaction solution of the aspartic acid production step. Alternatively, the aspartic acid production step can be initiated by adding an aspartic acid raw material to the culture solution after the completion of this step. Alternatively, the aspartic acid-producing microorganism can be recovered from the culture solution after the completion of this step, subjected to treatment such as immobilization, and then added to the reaction solution of the aspartic acid production step to initiate the aspartic acid production step.

[0110] In the method for producing aspartic acid according to this embodiment, the aeration rate and stirring speed during the reaction are controlled so as to satisfy the above condition (i) and at least one of the above conditions (ii) and (iii). Thereby, the production efficiency of aspartic acid by the aspartic acid-producing microorganism is improved, and the yield of aspartic acid can be increased. It is considered that by adjusting the aeration rate and stirring speed as described above, the oxygen supply to the aspartic acid-producing microorganism and the physical effects on the aspartic acid-producing microorganism due to aeration and stirring become conditions suitable for aspartic acid production.

[0111] As described above, the specific embodiments of the present invention have been described in detail, but the present invention is not limited to the above-described embodiments. Various modifications, corrections, and combinations can be adopted for each configuration, element, and feature without departing from the gist of the present invention. The terms "comprising" and "having" do not exclude the existence of elements other than the elements referred to as the object thereof, respectively, unless otherwise specified, and these terms are used interchangeably. The content of each document referred to in this specification is hereby incorporated herein as part of this specification.

Examples

[0112] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

[0113] <Oxygen transfer volumetric coefficient (k L a) measurement> Using a 2 L culture apparatus (MICRO TWIN, Marubishi Bioengine Co., Ltd.) equipped with a ventilation device and a stirring blade, the correlation between the measured value of ka by the sodium sulfite method and the predicted value of ka calculated based on the stirring speed and the ventilation volume was examined. L The measured value of ka by the sodium sulfite method and the predicted value of ka calculated based on the stirring speed and the ventilation volume were examined. L The correlation between the measured value of ka by the sodium sulfite method and the predicted value of ka calculated based on the stirring speed and the ventilation volume was examined.

[0114] (Measurement conditions) The sodium sulfite method is a method that utilizes the rapid progress of the oxidation reaction of sodium sulfite in the presence of catalysts such as copper and cobalt. Based on the materials provided by Marubishi Bioengine Co., Ltd. prepared with reference to Y. Imai et al. (Biotechnol Bioeng., (1987) 19(8):982-93), the measurement of ka by the sodium sulfite method was performed. L The measurement of ka by the sodium sulfite method was performed.

[0115] Regarding the 2 L jar fermenter (MICRO TWIN manufactured by Marubishi Bioengine Co., Ltd.) used in the aspartic acid production test, ka was measured. 1 L of water was poured into the container, and the measurement was performed with the temperature set at 35°C. Multiple-point measurements were performed with a ventilation volume of 0.14 to 0.42 L / min and a stirring speed of 300 to 500 rpm. The concentration of sodium sulfite added was set based on the approximate required amount from the value of ka to be measured. Sodium sulfite was subjected to nitrogen aeration and used in an unoxidized state. A peristaltic pump (EYELA SMP-21) was used for the addition of sodium sulfite. The flow rate for each dial was measured in advance, and the addition rate was calculated. For the stable dissolved oxygen (DO) value, those in the range of about 40 to 60% of the saturation concentration were adopted. The measurement was performed within a maximum of 10 minutes from the start of sodium sulfite addition. L Regarding the 2 L jar fermenter (MICRO TWIN manufactured by Marubishi Bioengine Co., Ltd.) used in the aspartic acid production test, ka was measured. 1 L of water was poured into the container, and the measurement was performed with the temperature set at 35°C. Multiple-point measurements were performed with a ventilation volume of 0.14 to 0.42 L / min and a stirring speed of 300 to 500 rpm. The concentration of sodium sulfite added was set based on the approximate required amount from the value of ka to be measured. Sodium sulfite was subjected to nitrogen aeration and used in an unoxidized state. A peristaltic pump (EYELA SMP-21) was used for the addition of sodium sulfite. The flow rate for each dial was measured in advance, and the addition rate was calculated. For the stable dissolved oxygen (DO) value, those in the range of about 40 to 60% of the saturation concentration were adopted. The measurement was performed within a maximum of 10 minutes from the start of sodium sulfite addition. L Regarding the 2 L jar fermenter (MICRO TWIN manufactured by Marubishi Bioengine Co., Ltd.) used in the aspartic acid production test, ka was measured. 1 L of water was poured into the container, and the measurement was performed with the temperature set at 35°C. Multiple-point measurements were performed with a ventilation volume of 0.14 to 0.42 L / min and a stirring speed of 300 to 500 rpm. The concentration of sodium sulfite added was set based on the approximate required amount from the value of ka to be measured. Sodium sulfite was subjected to nitrogen aeration and used in an unoxidized state. A peristaltic pump (EYELA SMP-21) was used for the addition of sodium sulfite. The flow rate for each dial was measured in advance, and the addition rate was calculated. For the stable dissolved oxygen (DO) value, those in the range of about 40 to 60% of the saturation concentration were adopted. The measurement was performed within a maximum of 10 minutes from the start of sodium sulfite addition.

[0116] (Calculation of ka by the sodium sulfite method) L (Calculation of ka by the sodium sulfite method) In the sodium sulfite method, ka was calculated by the following formula (2). L ka was calculated by the following formula (2). ka L = QC BF / {2V L C Ai (1 - C AS / CAi )} (2)

[0117] Q: Sodium sulfite aqueous solution addition amount [mL / min] C BF : Sodium sulfite aqueous solution concentration [mol / m 3 V L : Reaction solution volume [mL] C Ai : Dissolved oxygen equilibrium concentration 0.238 [mol / m 3 C AS : Dissolved oxygen concentration [mol / m 3

[0118] (Calculation of k L a based on stirring speed and aeration rate) k L The relationship between a and the aeration rate and stirring speed has been reported as an empirical formula by Cooper et al. (1994) (Kobayashi et al., 2019, Biochemical Engineering, Fundamentals and Applications of Bioprocesses, 2nd Edition, p. 80). This formula (the above formula (4)) shows the relationship between the aeration rate and stirring speed, the superficial velocity (aeration linear velocity), the input power per unit volume, and k L a. Assuming the same apparatus and the same solution, the length of the stirring blade and the viscosity of the solution are constant and can be treated as constants. Therefore, the formula is transformed to explain k L a in terms of the aeration rate and stirring speed, and can be expressed as the following formula (3). For this formula, the constants (a, b, c) were obtained by the least squares method using curve_fit (https: / / docs.scipy.org / doc / scipy / reference / generated / scipy.optimize.curve_fit.html) included in the Scipy package of python. Since the number of measurements varied depending on the experimental conditions, the average value of the measured values was used for the conditions measured multiple times so that the weight would not be biased towards specific conditions. According to the following formula (3), k L a was calculated. k L a = a × n b × AF c (3) ​​​

[0119] n: Stirring speed [rpm] AF: Aeration rate [mL / min] a, b, and c are constants and have the same numerical values in the same culture apparatus. In this test, the following numerical values were used. a = 2.50×10 -5 b = 1.78 c = 0.626

[0120] As shown in Figure 1, the measured value of k L a by the sodium sulfite method and the predicted value of k L a based on the stirring speed and aeration rate showed a high correlation. Therefore, in the following tests, k L a was calculated by the above formula (p).

[0121] [Experimental Example 1] <Aspartic acid-producing microorganism growth process: Process A> ≪Pre-culture≫ An aspartic acid-producing bacterium (Corynebacterium glutamicum) collected from a glycerol stock was inoculated onto a plate (1.5 wt% agar) of Medium A (Table 1) and cultured overnight in an incubator (IS-60, Yamato Scientific Co., Ltd.) at 33°C. The cells were scraped from the plate and subcultured into a test tube containing 16 mL of Medium A, and cultured at 33°C and 190 rpm for 12 hours using a shaking incubator (NR-300, Taitec Co., Ltd.). After culturing, the entire amount of the cells in the test tube was transferred to a 500 mL Erlenmeyer flask containing 100 mL of Medium A (containing 2 wt% glucose), and further cultured at 33°C and 190 rpm for 12 hours using a shaking incubator (NR-300, Taitec Co., Ltd.).

[0122] ≪Main culture≫ The main culture of the bacterial cells was carried out using a 2 L jar fermenter (MICRO TWIN, Marubishi Bio Engineering Co., Ltd.). 900 mL of the main culture medium (Table 2) was placed in a 2 L jar fermenter and autoclaved. To this, 100 mL of a 60 wt% glucose solution that had been autoclaved separately was added. To this, the bacterial cells cultured in 100 mL of Medium A were inoculated so that the initial OD 610 was 0.15, and the culture was started. The conditions for the jar culture were as follows. Air was used for aeration.

[0123] Temperature: 35 °C pH: 7.0 Agitation: 700 rpm Aeration: 430 mL / min pH adjustment solution: 16N NH3

[0124] The culture was carried out for about 18 hours and terminated at the stage when glucose was depleted.

[0125]

Table 1

[0126]

Table 2

[0127] In the preparation of the main culture medium, among the components shown in Table 2, components 1 to 12 were mixed before autoclaving. Component 13 was autoclaved separately from components 1 to 12. The mixed solution of components 1 to 12 that had been autoclaved and the autoclaved component 13 were mixed to prepare the main culture medium. In Table 2, "LG-126" represents Adekanol (registered trademark) LG-126 (ADEKA CORPORATION).

[0128] (Aspartic acid production step: Step B) To the culture broth of the aspartic acid-producing bacterium prepared as described above, 105 mL of a 60 wt% glucose solution, which serves as a substrate for aspartic acid production, was added, and the operating conditions of the jar fermenter were changed as follows to produce aspartic acid. Air was used for aeration.

[0129] Feed solution (also serving as a base for pH adjustment): A mixture of 2M NH4HCO3:15N NH3 water = 4:1 Temperature: 27 °C from the start of the reaction (0 hours) to 8 hours; 33 °C from 8 hours to 16 hours; 39 °C after 16 hours pH: pH 8 Stirring speed and aeration rate: Set as described in Table 4

[0130] The depletion of glucose added at the start of the reaction was confirmed using the semi-quantitative ion test paper QUANTOFIX (registered trademark) Glucose (MACHEREY-NAGEL). To analyze the solution composition at the end of the reaction, 1 mL of the culture broth from each test section was collected, centrifuged, and the supernatant was recovered. The recovered supernatant was used for measuring the glucose concentration, amino acid concentration, and organic acid concentration. The glucose concentration was measured using TM Glucose (FUJIFILM Wako Pure Chemical Industries, Ltd.). The amino acids were measured by the post-column OPA method using an amino acid analysis system Nexeta (Shimadzu). The organic acids were measured by UV detection using an HPLC system Nexera (Shimadzu) with a column of TSKgel OApak-A (TOSOH).

[0131] The growth of the aspartic acid-producing bacterium and each culture condition used for aspartic acid production are shown in Table 3. In Table 3, "Glc" represents glucose.

[0132]

Table 3

[0133] Table 4 shows the yield of aspartic acid and the culture conditions in the production of aspartic acid (Process B) for each example.

[0134] [Table 4]

[0135] In Table 4, the stirring power P / V without aeration per unit volume, the aspartic acid yield, and the gas superficial velocity were calculated as follows.

[0136] <Calculation of stirring power P / V without aeration per unit volume> The stirring power P / V without aeration per unit volume (kg·m 2 ·s -3 ) was calculated by dividing the stirring power P without aeration by the total volume V of the culture solution. The stirring power P without aeration was calculated by the following formula (5). P / V was calculated by dividing P by the volume V (1.0×10 -3 m 3 ) of the culture solution. Note that P / V was calculated assuming that the culture solution was pure water at 35°C. P = Npρn 3 d 5 (5)

[0137] Np: Power number ρ: Liquid density [kg / m 3 n: Impeller rotational speed [1 / s] d: Impeller diameter [m] In this test, the values shown in Table 5 were used.

[0138] [Table 5]

[0139] <Calculation of aspartic acid yield> The yield of aspartic acid was calculated by the following formula. Yield of aspartic acid (%) = Asp / Glc × 100 Asp: Molar concentration of aspartic acid in the culture broth at the end of Process B Glc: Molar concentration of glucose in the culture broth at the start of Process B

[0140] <Calculation of gas superficial velocity> The gas superficial velocity (m·s -1 ) was calculated by the following formula. The gas superficial velocity (m·s -1 ) = aeration rate [m 3 ·s -1 / cross-sectional area of the reaction tank (m 2 )

[0141] From the results shown in Table 4, it was confirmed that when k L a in the production of aspartic acid (Process B) is in the range of 30 - 60 / h, the yield of aspartic acid can be obtained at 80% or more.

[0142] [Experimental Example 2] The growth of aspartic acid-producing bacteria (Process A) and the production of aspartic acid (Process B) were carried out in the same manner as in Experimental Example 1, except that the culture conditions in Process B were changed as shown in Table 6 below.

[0143]

Table 6

[0144] In Table 6, the stirring power per unit volume without aeration P / V (kg·m 2 ·s -3 ) was calculated in the same manner as above using the numerical values shown in Table 7 below.

[0145]

Table 7

[0146] In Table 6, the total O2 consumption per unit volume (mmol / L) was calculated as follows.

[0147] <Calculation of total O2 consumption (mmol)> The total O₂ consumption was calculated by analyzing the oxygen concentration in the exhaust gas during aspartic acid production using an exhaust gas analyzer (DEX-1562A, ABLE Corporation). Assuming the oxygen concentration in the ventilated air was 20.7%, the integrated oxygen consumption from the start to the end of Process B was calculated as the difference between the oxygen concentration in the exhaust gas, which was considered to be consumed within the system. First, the oxygen uptake rate QO₂X (mmol / L / h) was calculated, and then the total O₂ consumption was calculated by multiplying the reaction liquid volume (1.1 L) and the time until all the substrate glucose was consumed.

[0148] From the results shown in Table 6, even when k in Process B L a was in the range of 30 - 60 / h, it was confirmed that the aspartic acid yield varied depending on the ventilation rate and stirring speed. The aspartic acid yield was good when the stirring power P / V without ventilation per unit area was less than 2, and the aspartic acid yield was good when the ventilation rate per unit volume was 0.2 vvm or more.

Industrial Applicability

[0149] According to the present invention, there is provided a method for producing aspartic acid that can improve the yield of aspartic acid in the production of aspartic acid using microorganisms.

Explanation of Symbols

[0150] 1... Reactor, 10... Reaction tank, 11... Lid, 20... Stirring device, 21... Stirring shaft, 22... Stirring blade, 30... Gas supply pipe, M... Reaction liquid, B... Bubble

Claims

1. including a step of reacting an aspartic acid raw material with an aspartic acid-producing microorganism in a reaction solution contained in a reaction tank capable of controlling the aeration rate and the stirring speed to produce aspartic acid, wherein the aeration rate and the stirring speed during the reaction are controlled to satisfy the condition of (i) below and at least one of the conditions of (ii) and (iii) below, A method for producing aspartic acid: (i) The oxygen transfer capacity coefficient (k Ｌ a) is in the range of 30 / h to 60 / h; (ii) The stirring power without aeration per unit volume (P / V) is less than 2 × 10 －３ W / m ３ ; (iii) The aeration rate per unit volume is 0.2 vvm or more.

2. The method for producing aspartic acid according to Claim 1, wherein the total oxygen consumption per unit volume from the start point of the reaction to the depletion point of the aspartic acid raw material is 250 mmol / L or more.

3. The method for producing aspartic acid according to Claim 1 or 2, wherein the ratio (acetic acid molar concentration / aspartic acid molar concentration) of the acetic acid molar concentration to the aspartic acid molar concentration in the reaction solution at the depletion point of the aspartic acid raw material is 1.5 or less.

4. The method for producing aspartic acid according to Claim 1 or 2, wherein the aspartic acid raw material is glucose.

5. The method for producing aspartic acid according to Claim 1 or 2, wherein aeration is performed with air.

6. The method for producing aspartic acid according to Claim 1 or 2, wherein the aspartic acid-producing microorganism is a coryneform bacterium.

7. The method for producing aspartic acid according to Claim 6, wherein the coryneform bacterium is a genetically recombinant microorganism.

Images