JPH0659228B2 - Amino acid continuous fermentation production method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a continuous fermentation production method of amino acids.

Amino acids have various uses such as additives to medicines, foods and feeds, and are used in a wide range of fields.

2. Description of the Related Art Conventionally, as a method for producing an amino acid using a microorganism, a production method by a batch method using various mutant strains and cell fusion strains is known.

Regarding the continuous culture method, for L-lysine, a method using Brevibacterium flavum [Folia Microbiol. 27 , 315-318 (198
2)], a method using Micrococcus glutamics (Proceedings of the 48th Annual Meeting of the Japanese Society of Fermentation Engineering p.170) is known. Regarding L-arginine, a method of using pure oxygen by immobilized cells of Serratia genus [Appl. Microbiol. Biotechnol. 19 , 79-84, ( 1984)] is known. Regarding L-phenylalanine, a method using Escherichia coli [Biotechnology Letters Vol. 4, 223-228 (198
2)], for L-glutamic acid, a method using Corynebacterium glutamicum [Biotechnology
And bioengineering (Biotechnology and B
ioengineering) Vol. 24, 2167-2174 (1982)], and L-tryptophan, a method using Escherichia coli [Biotechnology and Bioengineering Vol. 24, 1465-14].
68 (1982), ibid Vol.25, 1013-1025 (1983)] and the like are known.

Problems to be Solved by the Invention With the recent increase in demand for amino acids such as pharmaceuticals, foods, feeds, etc., development of industrially more advantageous amino acid production methods has been desired.

Means for Solving Problems In the fermentative production of amino acids, the main indicators of productivity are improvement of yield to carbon source and improvement of production rate. As a result of investigating the improvement of the production rate for the amino acid production method by the highly productive continuous fermentation method, as a result, a continuous feed solution with a carbon source substrate concentration of 10% or more was used to increase the oxygen supply rate to the culture solution. By culturing, it was found that the production rate can be improved as compared with the conventional batch culture method.

The present invention will be described in detail below.

INDUSTRIAL APPLICABILITY The present invention provides a method for continuous fermentation production of amino acids by a microorganism having amino acid producing ability, wherein the carbon source substrate concentration is 10
% Or more of the continuous feed solution, and the continuous culture is performed so that the redox potential of the culture medium during the continuous culture becomes -200 mV (saturated calomel electrode) or more, to produce and accumulate amino acids in the culture, A method for continuous fermentation production of amino acids, which comprises collecting amino acids from the culture.

Amino acids that can be produced by the method of the present invention include L-lysine, L-arginine, L-glutamic acid, L-glutamine, L-proline, L-histidine, L-ornithine,
L-threonine, L-isoleucine, L-leucine, L
-Valine, L-phenylalanine, L-tryptophan.

As the microorganism used in the present invention, any strain such as a wild strain and a mutant strain can be used as long as it has an amino acid-producing ability.
However, strains that can withstand continuous culture, that is, at least average residence time [fermentation liquid volume () / medium supply rate (/
A stable bacterial strain that does not change in titer and growth during 2 times or more of [h)] is preferable. Suitable examples include the following strains.

Corynebacterium glutamicum H-3055 (FERM BP-147) (JP 58-86094) Corynebacterium glutamicum H-3057 (FERM BP-148) (JP 58-86094) Corynebacterium glutamicum H -3119 (FERM BP-149) (JP-A-58-86094) Corynebacterium glutamicum H-3122 (FERM BP-150) (JP-A-58-86094) Corynebacterium glutamicum H-3290 (FERM BP- 156) (JP 59-88094) Corynebacterium glutamicum H-3149 (FERM BP-158) (JP 59-88095) Brevibacterium lactofermentum H-3291 (FERM BP-155) (special (Kaisho 59-88094) Corynebacterium glutamicum KY10671 (FERM P-3616) (Japanese Patent Publication No. 57-50479) Corynebacterium acetoside film H-4314 (FERM BP-1018) Corynebacterium glutamicum NRRL B-15531 (JP-A-60-66990) Corynebacterium glutamicum ATCC14752 Brevibacterium・ Lactofermentum H-3357 (FERM P-6828) (JP 59-109188) Corynebacterium glutamicum Ha (FERM P-8182) Corynebacterium glutamicum H-3797 (FERM BP-658) Escherichia coli H-4258 (FERM BP-985) Corynebacterium glutamicum H-3501 (FERM BP-848) Corynebacterium glutamicum ATCC21885 Corynebacterium glutamicum KY10628 (FERM P-2924) (JP-A-51-106788) Coryne C. glutamicum ATCC21674 Corynebacterium glutamicum H-3494 (FERM P-7427) (Japanese Patent Laid-Open No. 60-176593) As a continuous culture method, a one-tank method and a multi-tank method are known. In the method of the present invention, the one-tank method is sufficient, but when a large amount of the raw material remains in the extraction liquid, the multi-tank method can be used to improve the yield with respect to the carbon source. .

As the medium used in the present invention, either a synthetic medium or a natural medium can be used as long as it appropriately contains necessary nutrients such as a carbon source, a nitrogen source and an inorganic substance that can be utilized by the strain used.

Carbon sources include glucose, sucrose, molasses,
Carbohydrates such as fruit juice, decomposed products of starch and decomposed products of cellulose, organic acids such as acetic acid, alcohols such as ethanol can be used. As a nitrogen source, ammonia, ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium acetate, urea, amines, other nitrogen-containing compounds, and peptone, meat extract, yeast extract,
Corn, steep liquor, casein hydrolyzate, soybean meal hydrolyzate, various fermented bacterial cells and hydrolysates thereof can be used.

As the inorganic substance, potassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used. When the microorganism used in the present invention requires a specific nutrient for growth, the nutrient can be added as a preparation or a natural product containing the preparation. An antifoaming agent is also used if necessary.

In addition, various additives in the medium, such as aspartic acid,
In some cases, the production rate of amino acids can be improved by adding glutamic acid, leucine or a leucine fermentation solution, streptomycin, kanamycin, or the like.

The concentration of the carbon source substrate in the continuous feeding solution used for continuous culture is 10
% Or more is preferable. Even if the carbon source substrate concentration is 10% or less, the production rate is improved, but the amino acid accumulation concentration and the yield with respect to the carbon source are remarkably reduced, and there are many problems such as an increase in the amount of drainage, which makes it industrially practical. Few.

As the carbon source of the continuous feeding solution, molasses, glucose, sucrose, acetic acid or acetate, or a mixture thereof is used. As the nitrogen source, the inorganic substance, etc., any of those that can be used in the initial medium can be used as the feed solution component.

The culture is performed using a deep aeration stirring tank, a bubble column, a bubble column with a draft, or the like. The culture temperature is 20 to 40 ° C. It is desirable to maintain the pH of the medium in the range of 4 to 9, preferably around neutrality. The pH of the medium is adjusted with calcium carbonate, an acid or alkali solution, ammonia, or the like.

Conventional batch culture or semi-batch culture is performed prior to continuous culture, and the continuous culture is switched from the middle. Such a method is known per se, and known means can be applied to the method of the present invention.

That is, batch culture may be started, and the feed solution may be added at an appropriate time during the logarithmic growth phase, while the same amount of culture solution may be withdrawn. The feed rate of the fed-batch solution is determined by the amount of culture solution in the tank and the residence time.

Oxygen is supplied so that the redox potential becomes -200 mV or higher with the start of continuous culture. Redox potential is -200
In order to maintain mV, it is necessary to increase the supply rate of oxygen, and the culture is pressurized, for example, 1.5 to 3.0 kg / cm ² by supplying oxygen by adding oxygen to a gas having an oxygen concentration of 21% or more, for example, air.
The purpose can be achieved by applying means such as maintaining the temperature, increasing the stirring speed, and increasing the aeration rate, alone or in combination.

In the culturing process, it is necessary to pay more attention than usual batch culture or semi-batch culture to the equipment and operation so as to prevent contamination of bacteria in the fermenter. The longer the continuous culture period, the more labor-saving the process control, and the higher the productivity. Usually, 200-1000 to maintain sterility and prevent deterioration of production strains.
It will take about time, but if there is no problem, you can continue.

The production rate is calculated by the following formula.

From the above formula, the production rate can be improved by increasing the dilution rate [flow rate of fed-batch liquid (/ h) / operating liquid amount ()]. In other words, by feeding a larger amount of carbon source, substrate such as nitrogen source, and other nutrients per unit operating fluid volume, per unit time during continuous culture, amino acid-producing bacteria utilize them to produce amino acids, It only has to grow.

Precipitates such as bacterial cells are removed from the continuously extracted culture solution and the culture solution after completion of the culture, and known ion exchange treatment method, concentration method, adsorption method, salting out method, isoelectric point precipitation method, etc. By the method described above, the amino acid produced from the culture solution can be recovered.

Hereinafter, the present invention will be specifically described with reference to examples. Unless otherwise specified, the redox potential of the culture solution is -200 mV.
(Saturated calomel electrode) Culture is carried out as described above.

Example 1 Production of L-lysine by continuous fermentation method: (1) Corynebacterium glutamicum H-3149 (FERM BP-
158) was cultured for 30 hours at 30 ° C. using 250 ml of the following seed medium placed in a 2-volume Erlenmeyer flask. On the other hand, the following main fermentation medium 1.4 was placed in a 5-volume jar fermenter, and 100 ml of the above seed culture was inoculated into the jar fermenter. Culture temperature was adjusted to 34 ℃, pH was adjusted to 7.0, and aeration rate was 2.5 / mi.
The culture was carried out at n and a stirring speed of 600 rpm. After culturing for 15 hours,
The following feeding solution was fed, and when the addition of 1 was completed, the withdrawal of the culture solution was started, and the amount of the working solution was constantly controlled to 2.5. Molasses was used as the carbon source of the fed-batch liquid, and the concentration in terms of glucose shown in Table 1 was used. The flow acceleration is considered to have reached a steady state at that flow acceleration when a time at which the average residence time is three times or more is performed at a constant speed, and then the flow acceleration is gradually increased to increase the concentration of each carbon source. The results when the highest production rate of lysine was shown in Table 1. All lysines were represented by L-lysine hydrochloride.

[Seed medium]

0.5% glucose 4% yeast extract peptone 2% Urea _{0.3% MgSO 4 · 7H 2 O} 0.05% KH 2 PO 4 0.2% Biotin 100μg / pH7.0 (NaOH) sterilizing 120 ° C. 15 minutes [ Main fermentation medium] Initial culture medium Molasses 3.5% (glucose equivalent) Ammonium sulfate 2.5% KH ₂ PO ₄ 0.05% MgSO ₄ 0.05% Corn steep liquor 1% Defoamer 2 ml / pH 7.0 (NH ₄ OH) Sterilization 120 ° C. 15 minutes Fed-batch liquid Molasses 28-10% (glucose equivalent) Ammonium sulfate 4% KH ₂ PO ₄ 0.05% Corn steep liquor 0.5% Defoamer 1 ml / sterilization 120 ℃ 15 minutes As shown in Table 1, the lower the sugar concentration in the fed-batch solution, the higher the production rate, but the less the lysine accumulation amount, the industrially usable is that the sugar concentration of 10% or more is used. Addition.

(2) In the method shown in (1) of Example 1, H-31 was used as the microorganism.
49 and H-3057 (FERM BP-148), using a feeding solution with a carbon source concentration of 24%, and stirring speed in a 5-volume jar fermenter within the range shown in Table 2, as in (1). The maximum production rate of each L-lysine was determined by culturing in the following manner and shown in Table 2. When H-3057 strain was used, L-leucine 0.
5 g / was added to the medium.

(3) Using the carbon source concentration of 24% in the method shown in (1) of Example 1, continuous culture was carried out at a stirring rotation speed of 500 rpm at a dilution rate shown in Table 3. Table 3 shows the amount of L-lysine accumulated, the production rate, and the redox potential (saturated calomel electrode) at each dilution rate.

Further, similarly, continuous culture was performed using a fed-batch solution having a carbon source concentration of 24% at a dilution rate of 0.034 h ⁻¹ while changing the stirring rotation number as shown in Table 4. Table 4 shows the amount of L-lysine accumulated, the production rate, and the redox potential (saturated calomel electrode) at each stirring rotation speed.

(4) Of the methods described in (1) of Example 1, the fifth method is used as the microorganism.
Using the strains shown in the table, using a fed-batch solution with a carbon source concentration of 24%, a stirring rotation speed of 450 rpm, an aeration rate of 2.5 / min, and air containing 30% oxygen as aeration air. Continuous culture was performed, and the maximum production rate was compared with that when normal air (oxygen concentration 21%) was used. The carbon source in the fed-batch was molasses and glucose was used at a concentration of 19%. However, when using the H-3291 strain, L-leucine 0.6 was added to the medium.
g / added. Data showing the maximum production rate under each condition are shown in Table 5.

As shown in Table 5, when air containing 30% oxygen concentration is used, the amount of accumulated L-lysine is slightly reduced, but the production rate is significantly improved, and therefore industrial practicality is high.

(5) Among the methods shown in (1) of Example 1, H-3149 is used as the microorganism.
Using a fed-batch solution having a carbon source concentration of 24%, a stirring rotation speed of 450 rpm and a fermenter internal pressure of 1.5 and 1.9 kg / cm ²
The pressure was raised to a pressure and aeration was performed to determine the maximum production rate of L-lysine. In addition, the maximum production rate was also obtained when the aeration rate was increased while the tank pressure was normal pressure (1 kg / cm ² ). The maximum production rate under each condition is shown in Table 6.

(6) In the method shown in (1) of Example 1, the following was used as the main fermentation medium and the microorganism was continuously cultured using H-3055 (FERM BP-147).

[Main fermentation medium]

Initial medium Glucose 6% NH ₄ Cl 2.5% KH ₂ PO ₄ 0.2% MgSO ₄ 0.05% Urea 0.1% Corn steep liquor 2% Peptone 0.5% Biotin 300 μg / Thiamin hydrochloride 100 μg / FeSO ₄ · 7H ₂ O 10 mg / MnSO ₄ · 4H ₂ O 10 mg / CaCl ₂ 10 mg / CoCl ₂ · 6H ₂ O 1 mg / CuCl ₂ · 2H ₂ O 10 mg / NiCl ₂ · 6H ₂ O 1 mg / ZnCl ₂ 1 mg / molybdenum Ammonium acid 1 mg / [(NH ₄ ) ₆ Mo ₇ O ₂₄・ 4H ₂ O] Defoamer 2 ml / pH 7.0 (NH ₄ OH) Sterilization 120 ° C for 15 minutes Fed-batch Solution Initial medium except 20% glucose Similar composition to.

Maximum production speed when operated at a stirring speed of 450 rpm 2.
3.8 g / ・ h at 600 rpm for 3 g / ・ h
Improved.

(7) In the method shown in (1) of Example 1, the stirring rotation speed is 450.
As the rpm and the feeding solution, a mixed sugar solution having a carbon source concentration of 24% mixed at the mixing ratio shown in Table 7 was used. L- under each condition
The maximum production rate of lysine is shown in Table 7.

Example 2 Production of L-arginine by continuous fermentation method: (1) Corynebacterium acetoside film H-4314
(FERM BP-1018) was cultured for 48 hours at 30 ° C. using 250 ml of the following seed medium in a 2-volume flask. On the other hand, the following main fermentation medium 1.6 was put into a 5 volume jar fermenter and 250 ml of the above seed culture was inoculated into this. The culturing temperature was adjusted to 30 ° C. and pH 6.6, the aeration rate was 3 / min, and the culturing was performed at 600 rpm. Molasses was used as the carbon source of the medium. In the case of batch culture from the time when the molasses in the initial medium is consumed (after 18 hours), the following fed-batch medium (A) is used.
In the case of continuous culture, the fed-batch medium (B) was fed in 1.4 times each. At this point, in the latter case, continuous culture was started. The culture broth was withdrawn while adding the fed-batch medium (B) at various concentrations so that the volume of the culture broth during continuous culture was kept at 3. The flow acceleration was considered to have reached a steady state at that flow acceleration when it was fed at a constant speed for a time that was three times the average residence time or more, and the value at that time was used to calculate the production rate. Table 8 shows the L-arginine production rate and the sugar yield relative to the continuous culture, and the production rate and the sugar yield relative to the ordinary batch culture, which were obtained as described above.

Seed medium Glucose 50 g / Peptone 10 g / Yeast extract 10 g / NaCl 2.5 g / Biotin 50 μg / Urea 3 g / Corn steep liquor 5 g / pH 7.2 Main fermentation medium Waste molasses 70 g / KH ₂ PO ₄ 0.5 g / Ammonium sulfate _{38g / MgSO 4 · 7H 2 O} 0.5g / FeSO 4 · 7H 2 O 10mg / thiamine hydrochloride 100 [mu] g / pH 7.2 feed medium (A) molasses 400 g / ammonium sulfate 30 g / (B) molasses 80~250g / Ammonium sulphate 30g / Concentration of molasses is glucose equivalent concentration Sterilization was carried out at 120 ° C for 30 minutes.

As shown in Table 8, the lower the carbon source concentration of the fed-batch solution, the higher the production rate, but the accumulated amount of arginine and the yield with respect to sugar decrease, so that it can be industrially advantageously used. It is a fed-batch solution having a carbon source concentration of 10% or more.

(2) As a microorganism in the method shown in (1) of Example 2, H-4314
The culture was performed using a fed-batch solution with a carbon source concentration of 20% and changing the stirring number as shown in Table 9 in a 5-volume jar fermenter. The production rate of L-arginine and the yield of sugar, the redox potential (saturated calomel electrode) at that time
Shown in the table.

(3) As a microorganism, H-4314 in the method shown in (1) of Example 2
A stirring rate of 450 rp using a feed solution with a carbon source concentration of 20%
Continuous culturing was performed using normal air (21% oxygen concentration) and air containing 30% oxygen concentration as ventilation air at m, and the production rates were compared. The results are shown in Table 10.

(4) In the method shown in (1) of Example 2, H-43 was used as the microorganism.
14, using a fed-batch solution with a carbon source concentration of 20%, and stirring at 450 rpm, the internal pressure of the fermenter was 1.5 and 1.9 kg / cm ^2.
The pressure was raised to aeration and ventilation was performed to determine the maximum production rate of L-arginine. In addition, the maximum production rate was also obtained when the aeration rate was increased while the tank pressure was normal pressure (1 kg / cm ² ). The maximum production rate under each condition is shown in Table 11.

(5) In the method shown in (1) of Example 2, the stirring number 450 rp
m, continuous culturing was performed using a mixed sugar solution having a total carbon source concentration of 20% mixed at a mixing ratio shown in Table 12 as a feed solution.
The production rate of L-arginine at this time is shown in Table 12.

Effect of the Invention According to the present invention, an amino acid can be industrially advantageously obtained at a high production rate.

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Claims (2)

[Claims] 1. A continuous fermentation production method of L-lysine or L-arginine by a microorganism belonging to the genus Corynebacterium or Brevibacterium which has the ability to produce L-lysine or L-arginine.
% Or more of the continuous feed solution and continuously culturing at a redox potential of -200 mV (saturated calomel electrode) or higher during continuous culturing to produce L-lysine or L-arginine in the culture. L-lysine or L-arginine collected from the culture
-A method for continuous fermentation production of lysine or L-arginine. 2. The method according to claim 1, wherein molasses, glucose, sucrose, acetic acid or acetate, or a mixture thereof is used as the carbon source.