JP4032765B2 - Method for producing organic acid - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to the production of organic acids using bacteria.
[0002]
[Prior art]
When producing succinic acid and the like by fermentation, anaerobic bacteria such as the genus Anaerobiospirillum and the genus Actinobacillus are usually used and studied.
When anaerobic bacteria are used, the yield of the product is high, but on the other hand, in order to require a lot of nutrients to grow, it is necessary to add a large amount of organic nitrogen source such as CSL to the medium. . Addition of a large amount of these organic nitrogen sources not only leads to an increase in medium cost, but also increases the purification cost when taking out the product, which is not economical.
[0003]
There is also a method of culturing aerobic bacteria once under aerobic conditions, growing the cells, collecting and washing, and using this as stationary cells to produce organic acids without aeration of oxygen. Are known. In this case, the amount of organic nitrogen added may be small when growing the cells, and it is economical because it can be sufficiently grown on a simple medium, but the target organic acid production rate and production rate per cell are sufficient. Is still insufficient, and the establishment of a better method has been desired.
[0004]
[Problems to be Solved by the Invention]
The subject of this invention is providing the manufacturing method of organic acid with higher fermentation efficiency.
[0005]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventor has shown that the production rate of organic acid is increased by setting the reaction temperature after the growth of the bacterial cells to the stationary phase to a specific temperature. As a result, the present invention has been completed.
That is, the present invention is characterized in that, in producing an organic acid by a bacterial cell reaction, the reaction temperature after the growth of the bacterial cell reaches a stationary phase is set to a temperature higher than the optimum growth temperature of the bacteria. In producing an organic acid by a method for producing an organic acid or a bacterial cell reaction, after the treatment at the optimum growth temperature of the bacteria ± 2 ° C., the reaction is subsequently performed at a temperature higher than the temperature. The present invention resides in a method for producing an organic acid.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the invention will be described in detail.
Bacteria used in the present invention are not particularly limited as long as they have the ability to produce organic acids. Among these, the genus Bacillus, the genus Rizobium, the genus Corynebacterium, the genus Brevibacterium ( Aerobic bacteria such as microorganisms belonging to the genus Brevibacterium and Arthrobacter are preferred.
[0007]
Among the aerobic bacteria, a coryneform bacterium is preferable, and the coryneform bacterium is preferably a microorganism belonging to the genus Corynebacterium, a microorganism belonging to the genus Brevibacterium, or a microorganism belonging to the genus Arthrobacter. Among them, preferred are those belonging to the genus Corynebacterium or Brevibacterium, and more preferred are Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium ammoniagenes Brevibacterium. Or the microbe belonging to Brevibacterium lactofermentum (Brevibacterium lactofermentum) Examples include living organisms.
[0008]
Particularly preferred specific examples of the microorganism include Brevibacterium flavum MJ-233 (FERM BP-1497), MJ-233AB-41 (FERM BP-1498), Brevibacterium ammoniagenes. ATCC6872, Corynebacterium glutamicum ATCC31831, Brevibacterium lactofermentum ATCC 13869.
[0009]
The microorganism used in the production method of the present invention is not only a wild strain, but also a mutant obtained by normal mutation treatment such as UV irradiation or NTG treatment, or induced by genetic techniques such as cell fusion or gene recombination. Any strain such as a recombinant strain may be used. The host of the genetically modified strain may be the same genus species as the parent strain or may be different from the genus species as long as it is a transformable microorganism. Bacteria are preferred as hosts.
[0010]
Among these, it is more effective to use a mutant lacking lactate dehydrogenase in this reaction. As a specific method for producing a mutant strain lacking lactate dehydrogenase of a coryneform bacterium, the method described in JP-A-11-205385 can be mentioned, which can be easily prepared according to this method.
In using the bacterium in this reaction, a slant culture in a solid medium such as an agar medium may be directly used for the reaction, but it is preferable to use a bacterium previously cultured in a liquid medium (seed culture). .
[0011]
The main carbon source of the medium used for culturing these bacteria is not particularly limited as long as it is a carbon source that can be assimilated by the microorganism, but usually, galactose, lactose, glucose, fructose, glycerol, sucrose, Carbohydrates such as saccharose, starch and cellulose; fermentable carbohydrates such as polyalcohols such as glycerin, mannitol, xylitol and ribitol are used, among which glucose, fructose and glycerol are preferable, and glucose is particularly preferable.
[0012]
Moreover, the starch saccharified liquid, molasses, etc. containing the said fermentable saccharide | sugar are also used. These fermentable carbohydrates can be used alone or in combination.
The concentration of the carbon source used is not particularly limited, but it is advantageous to make it as high as possible within the range that does not inhibit the production of organic acid, and is usually 30% (W / V) or less, preferably 25% (W / V). V), more preferably 20% (W / V) or less.
[0013]
On the other hand, if the concentration of the raw sugar is too low, it is not preferable from the viewpoint of the pot efficiency during industrial production. Therefore, it is usually 5% (W / V) or more, preferably 10.5% or more, more preferably 11%. Reaction is performed at (W / V) or higher.
It is also preferable to add the carbon source as the carbon source decreases with the progress of the reaction in order to improve the amount of accumulated product.
[0014]
The nitrogen source is not particularly limited as long as it is a carbon source that can be assimilated by the microorganism, but specifically, ammonium salt, nitrate, urea, soybean hydrolysate, casein degradation product, peptone, yeast extract, meat extract, Examples include various organic and inorganic nitrogen compounds such as corn steep liquor.
As the inorganic salt, various phosphates, sulfates, magnesium, potassium, manganese, iron, zinc, and other metal salts are used.
[0015]
In addition, factors that promote growth such as vitamins such as biotin, pantothenic acid, inositol, and nicotinic acid, nucleotides, and amino acids are added as necessary.
In order to suppress foaming during the reaction, it is desirable to add an appropriate amount of a commercially available antifoaming agent to the culture solution.
The pH of the culture solution is usually adjusted to pH 5-9, preferably pH 6.5-8.5, and the pH of the culture solution is kept within the above range by alkaline substances, carbonates, urea, etc. as necessary during the reaction. Adjust.
[0016]
When this reaction is carried out using aerobic bacteria, oxygen is required for the growth of the cells. In this reaction, after bringing the culture medium into contact with the bacterial cells, the bacterial cells first grow logarithmically and then reach a stationary phase. Therefore, since the amount of oxygen required varies between the logarithmic growth phase and the stationary phase, it is necessary to adjust the aeration amount and the stirring amount in consideration of the difference in stirring efficiency due to the difference in reaction scale and feather shape. .
[0017]
In the method of the present invention, the reaction temperature after the growth of the bacterial cells reaches the stationary phase is set to a temperature higher than the optimum temperature for growth of the bacteria, preferably 2 ° C. or higher. Here, if the temperature is raised too much, there is a possibility that the enzyme activity may be lowered. Therefore, the temperature is usually appropriately selected within a range up to 10 ° C. above the temperature.
The optimum growth temperature refers to the temperature at which the growth rate is fastest under the conditions used for the production of organic acid. The optimum temperature for growth varies depending on various conditions during production of the organic acid, such as the type of medium, aeration rate, and stirring speed. Therefore, it is necessary to confirm the optimum growth temperature in advance according to each reaction condition. There is.
[0018]
Further, it is preferable that the temperature in the logarithmic growth phase is set to a temperature of optimal growth temperature ± 2 ° C. of the cells, and the reaction is carried out by raising the temperature from the temperature after reaching the stationary phase.
The optimum growth temperature of the microorganism used in this reaction is usually 25 ° C to 35 ° C.
This reaction is usually terminated when the main raw material such as glucose in the culture medium is consumed. At this time, organic acids such as malic acid, fumaric acid, and succinic acid are generated in the reaction solution. Of these, succinic acid has the highest accumulation and is preferred as a product.
[0019]
The organic acid thus accumulated in the culture solution is separated and purified from the culture solution according to a conventional method. Specifically, after removing solids such as bacterial cells by centrifugation, filtration, etc., desalting with an ion exchange resin or the like, and separating and purifying the organic acid from the solution by crystallization or column chromatography it can.
[0020]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
Example 1
A strain lacking lactate dehydrogenase (LDH) was prepared from Brevibacterium flavum MJ233AB-41 (FERM BP-1498) according to JP-A-11-206385. That is, a PCR reaction was performed on a partial fragment of the lactate dehydrogenase gene using two primers described as SEQ ID NOs: 5 and 6 in the patent using the total DNA extracted from MJ233AB-41 strain by a conventional method as a template. 3 μl of the obtained reaction solution and 1 μl of a cloning vector pGEM-T (PROMEGA) for PCR products were mixed, 50 mM Tris buffer (pH 7.6), 10 mM dithiothreitol, 1 mM ATP, 10 mM MgCl ₂ and T4 DNA Each component of ligase 1 units was added and reacted at 4 ° C. for 15 hours for binding. Using the resulting plasmid mixture, Escherichia coli JM109 (Takara Shuzo) was transformed by the calcium chloride method, and a medium containing 50 mg of ampicillin (10 g tryptone, 5 g yeast extract, 5 g NaCl, and 16 g agar dissolved in 1 L distilled water). It was applied to. The growing strain on this medium was subjected to liquid culture by a conventional method, and plasmid DNA was prepared from the culture solution. To 20 μl of this plasmid DNA, 50 mM Tris buffer (pH 7.5), 1 mM dithiothreitol, 10 mM MgCl ₂ , 100 mM NaCl, restriction enzymes SphI and SalI 1 unit were added and reacted at 37 ° C. for 1 hour. A 300 bp fragment was recovered from the obtained DNA solution using Gene Clean II (manufactured by Funakoshi), and 10 μl of the DNA solution and 1 μl of chloramphenicol resistant cloning vector pHSG396 (manufactured by Takara Shuzo Co., Ltd.) After mixing, components of 50 mM Tris buffer (pH 7.6), 10 mM dithiothreitol, 1 mM ATP, 10 mM MgCl ₂ and 1 unit of T4 DNA ligase were added and reacted at 4 ° C. for 15 hours for binding. Using the resulting plasmid mixture, Escherichia coli JM109 (Takara Shuzo) was transformed by the calcium chloride method, and a medium containing 50 mg of ampicillin (10 g tryptone, 5 g yeast extract, 5 g NaCl, and 16 g agar dissolved in 1 L distilled water). It was applied to. A growing strain on this medium was subjected to liquid culture by a conventional method, and a plasmid DNA into which a fragment of about 300 bp, which is a partial fragment of the lactate dehydrogenase gene, was introduced from the culture solution was prepared. The plasmid was introduced into Brevibacterium flavum MJ233AB-41 by electric pulse method, and a medium containing 5 mg of chloramphenicol (urea: 2 g, ammonium sulfate: 7 g, 1 potassium phosphate: 0.5 g, 2 potassium phosphate 0 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate heptahydrate: 20 mg, manganese sulfate hydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 1 g, 1 g of casamino acid, and 16 g of agar were dissolved in 1 L of distilled water). Among the strains grown on this medium, a strain having an LDH activity of 1/10 or less was selected as a strain in which the gene was disrupted by homologous recombination.
[0021]
Urea: 4 g, ammonium sulfate: 14 g, monopotassium phosphate: 0.5 g, dipotassium phosphate 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate heptahydrate: 20 mg, sulfuric acid Manganese hydrate: 20 mg, D-biotin: 200 μg, thiamine hydrochloride: 200 μg, yeast extract: 1 g, casamino acid: 1 g, and distilled water: 1000 ml of a medium is placed in a 500 ml Erlenmeyer flask at 120 ° C., 20 Sterilized by heat. This was cooled to room temperature, 4 ml of a pre-sterilized 50% glucose aqueous solution and 5 ml of a sterile filtered 0.1% chloramphenicol aqueous solution were added, and the aforementioned lactate dehydrogenase gene disruption strain was inoculated for 24 hours at 30 ° C. Seed culture.
[0022]
Urea: 8 g, ammonium sulfate: 28 g, 1 potassium phosphate: 1 g, 2 potassium phosphate 1 g, magnesium sulfate heptahydrate: 1 g, ferrous sulfate heptahydrate: 40 mg, manganese sulfate hydrate: 40 mg, D-biotin: 400 μg, thiamine hydrochloride: 400 μg, yeast extract 2 g, casamino acid 2 g, antifoaming agent (Adecanol LG294: manufactured by Asahi Denka): 1 ml and distilled water: 15000 ml of medium are placed in a 5 L fermentation trough. Sterilized at 20 ° C. for 20 minutes. After cooling this to room temperature, 50 ml of a 40% aqueous glucose solution sterilized in advance was added, and the whole amount of the above seed culture solution was added thereto, and the mixture was kept at 30 ° C. The pH was kept at 8.0 with 2M sodium carbonate, the reaction was carried out at 400 mL / min for aeration and 300 revolutions / min. Since the growth of the cells reached a stationary phase 15 hours after the start of the reaction, the reaction temperature was increased to 37 ° C. and the reaction was continued. As a result, glucose was almost consumed after 25 hours and succinic acid was accumulated at 33 g / L. Was.
[0023]
Example 2
The procedure was the same as in Example 1 except that the temperature after 15 hours after the start of the reaction was changed to 40 ° C. As a result, glucose was almost consumed after 26 hours and succinic acid was accumulated at 33 g / L.
Comparative Example 1
Except that the reaction temperature was kept constant at 30 ° C., the same procedure as in Example 1 was carried out. After 31 hours, glucose was almost consumed and succinic acid was accumulated at 31 g / L.
[0024]
Comparative Example 2
Except that the reaction temperature was kept constant at 37 ° C., the same procedure as in Example 1 was carried out. After 40 hours, glucose was almost consumed and succinic acid was accumulated at 29 g / L.
[0025]
【The invention's effect】
According to the method of the present invention, a target product can be obtained at a high reaction rate and yield in the production of an organic acid using bacteria.

Claims (1)

Brevibacterium flavum or Corynebacterium glutamicum in which the lactate dehydrogenase (LDH) gene of Brevibacterium flavum or Corynebacterium glutamicum is disrupted by homologous recombination and the LDH activity is reduced to 1/10 or less. In the production of succinic acid by the bacterial cell reaction of the bacterium, the growth of the bacterial cell is treated at the optimum temperature for growth of the bacteria, and the reaction temperature after reaching the stationary phase is 2 A method for producing succinic acid , characterized in that the temperature is higher by 10 ° C.