JPS5944039B2 - Method for producing L-glutamic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-glutamic acid from citric acid or ink citric acid using microorganisms.

More specifically, the present invention relates to (1) living microorganisms having the ability to produce L-glutamic acid when the ground material of the microorganisms is brought into contact with citric acid, isocitric acid or salts thereof, and ammonium ions; When L-glutamic acid is produced from citric acid, incitric acid, or their salts and ammonium ions using suspensions such as body culture fluids, the microorganisms are treated with antibiotics, treated with cell wall synthesis inhibitors, or dried. and (2) the ability of a microorganism in which a triturated product of its cells to produce L-glutamic acid from citric acid, ink citric acid or salts thereof and ammonium ions. Citric acid, using viable cells of microorganisms having
When producing L-glutamic acid from isocitric acid or salts thereof and ammonium ions, the microorganisms are treated with antibiotics, treated with cell wall synthesis inhibitors, or dried, or cultured in a citric acid-containing medium to become acclimated to citric acid. The present invention relates to a method for producing L-glutamic acid characterized by using L-glutamic acid that has been cultured (acclimated) and immobilized.

An object of the present invention is to stably produce glutamic acid over a long period of time from citric acid, isocitric acid or salts thereof and ammonium ions by simple operations.

Citric acid and ink citric acid are generally considered to have low permeability to microbial cell membranes.

Therefore, attempts have been made to convert citric acid and ammonium ions into L-glutamic acid using the physiological activity of microorganisms. There is a method in which bacterial autolysis fluid etc. are brought into contact with citric acid and ammonium ions (Japanese Patent Publication No. 36-3171).

However, this method requires special measures such as destroying the bacterial cells, and it also requires heat treatment after the reaction to stop the reaction and decomposes and removes the enzyme that coagulates and precipitates, making it impossible to use the enzyme repeatedly. there were.

Furthermore, it has been reported that the productivity of L-glutamic acid can be improved by treating microorganisms with a surfactant to improve the permeability of the small pleura.

C Special Publication No. 40-15952).

It is also known that L-glutamic acid can be produced by adding moldicidin and penicillin during mixed culture of citric acid-producing bacteria and L-glutamic acid-producing bacteria (
JP-A No. 51-35488).

Furthermore, L-glutamic acid-producing bacteria are cultured and acclimated to a seed medium containing citric acid, an organic acid other than citric acid, alcohol, or carbohydrate as a carbon source, and L-glutamic acid is produced using the acclimatized bacteria. A method is known (JP-A-51-35489).

The method disclosed in Japanese Patent Publication No. 40-15952 is excellent because it allows the use of viable microorganisms or a culture solution containing viable microorganisms for the production of L-glutamic acid. There is a problem in that the enzymes or coenzymes necessary for the production of microbial cells leak out of the bacterial cells over time, and the enzymes that leak out of the bacterial cells tend to deteriorate, such as inactivation.

Furthermore, especially in continuous fermentation, enzymes or coenzymes released from the bacterial cells are sent out of the system, resulting in the problem that L-glutamic acid production can only be sustained for a few hours to more than ten hours.

Therefore, the present inventors conducted various studies to impart to microorganisms the ability to stably convert citric acid, incic acid or salts thereof, and ammonium ions into L-glutamic acid over a long period of time. It has been found that microorganisms treated with a cell wall synthesis inhibitor or dried can stably produce L-glutamic acid from citric acid or isocitric acid over a long period of time, ie, over several days.

Furthermore, microorganisms treated as above or JP-A-51
By using L-glutamic acid producing bacteria cultured in a citric acid-containing medium and immobilized by various methods according to the method described in No. 35489, citric acid, isocitric acid or their salts, and It has been discovered that L-glutamic acid can be produced from ammonium ions.

Another advantage of the present invention is that the rate at which citric acid, ink citric acid, or salts thereof, and ammonium ion mustard monoglutamate are produced is faster than with other substrates such as carbohydrates, acetic acid, ethanol, etc. It is faster, the main component of the reaction is L-glutamic acid, there are almost no by-products, and the viscosity of the reaction solution is low.
Since there is no coloring, it is easy to isolate and purify L-glutamic acid from the reaction-completed liquid.

Next, the present invention will be explained in detail separately for cases in which the bacteria used are not immobilized and cases in which they are immobilized.

When the bacteria used are not immobilized Specifically, the present invention is carried out as follows.

That is, first, a microorganism whose cell pulverized product has the ability to convert citric acid, incic acid or salts thereof, and ammonium ions into L-glutamic acid is used as a carbon source, a nitrogen source, an inorganic substance, which is normally used in fermentation.
Cultivate in a medium containing moderate amounts of micronutrients, etc.

During culturing, the bacterial cells are treated with an antibiotic or a cell wall synthesis inhibitor, or the bacterial cells isolated from the culture solution are subjected to these treatments or a drying treatment.

Next, the bacterial suspension containing the treated bacterial cells is brought into contact with citric acid, incitric acid or salts thereof, and ammonium ions to produce L-glutamic acid.

The microorganism used in the present invention has the ability to produce L-glutamic acid from citric acid, ink citric acid, or salts thereof and ammonium ions, and
In addition, those treated with antibiotics, treated with cell wall synthesis inhibitors, or dried are used.

The microorganisms before these treatments include bacteria belonging to the genus Corynebacterium, Brevibacterium, Arthrobacterium, and Micrococcus, which are conventionally known as L-glutamic acid producing bacteria, or the aforementioned Tokko Sho 4.
Yeasts belonging to the genus Satucharomyces, Torula, etc. described in Publication No. 0-15952 are suitable.

Antibiotics used in the present invention include various antibiotics such as penicillin G1, cephalosporin, bacitracin, and cephalexin.

Furthermore, examples of cell wall synthesis inhibitors include deoxyglucone and the like.

As a method for bringing these antibiotics or cell wall synthesis inhibitors into contact with L-glutamic acid, these substances may be added to the culture solution of L-glutamic acid-producing bacteria;
Although the bacterial cells isolated from the culture solution may be brought into contact with these substances in water or the like, the former method is preferred.

The bacterial cells to be contacted are preferably in the logarithmic growth phase.

The amount of antibiotic added is 1 to 100 units/rrLl for penicillins such as penicillin G, 1 to 1000 units/ml for bacitracin, and 1 to 100 μF for cephalosporins such as cephalexin.
rn1° is preferred.

The amount of cell wall synthesis inhibitor added is 0.1 to 10 yny/r.
ail is preferred.

The drying treatment in the present invention is carried out by drying the collected and washed bacterial cells under ventilation at room temperature, or by drying them at 30° C. or lower after freezing.

In any case, the bacterial cells to be subjected to the drying treatment are preferably in the logarithmic growth phase.

Next, a culture solution containing L-glutamic acid producing bacteria subjected to such treatment or a suspension of the isolated bacteria in water, etc., and citric acid, incitric acid or a salt thereof, and ammonium ion are added. contact to produce L-glutamic acid.

This conversion reaction can be carried out using a conventional fermenter.

Examples of salts of citric acid or ink citric acid include ammonium salts and sodium salts.

There are various things that can provide ammonium ions, such as ammonia water, ammonium chloride, ammonium sulfate, and urea, but ammonium chloride is particularly preferred.

The reaction can be carried out using a conventional fermenter.

The reaction conditions are a temperature of 20 to 50°C, a pH of 5 to 9, and it is desirable to keep ventilation to a minimum.

It is necessary to supply ammonium ions in a sufficient amount to meet the production of L-glutamic acid.

The concentration of citric acid or isocitric acid is 40ml71r
Ll! It is preferable to supply as much as possible without exceeding.

When immobilizing the bacteria used To culture L-glutamic acid-producing bacteria in a citric acid-containing medium, use citric acid as the main carbon source, and use one or more of organic acids other than citric acid, alcohol, and carbohydrates as secondary carbon sources. L-glutamic acid producing bacteria may be cultured in a medium containing the L-glutamic acid.

Such a method may be carried out in accordance with the method described in JP-A-51-35489, Journal of General and Microbiology 10 23 (1964), and the like.

To immobilize the microbial cells, various known methods may be used, such as entrapping them in an acrylamide gel, entrapping them in a collagen gel, and entrapping them in cellulose acetate fibers.

That is, to enclose in an acrylamide gel, bacterial cells, acrylamide, and N are mixed in a dispersion medium such as cyclohexane and a dispersant such as Span 20.

Acrylamides such as N'-bisacrylamide, N,
Add diamines such as N,N',N'-tetramethylethylenediamine, phosphate buffer, ammonium persulfate, etc., and copolymerize acrylamide and diamines under conditions that do not expose to oxygen such as nitrogen gas atmosphere. Then, an acrylamide gel containing the bacterial cells may be produced.

The method of enclosing it in acrylamide gel is described in U.S. Patent Nos. 3 and 5.
42,662, etc.

To enclose in a collagen gel, bacterial cells are mixed with a solution in which collagen fibers are homogenized, the mixture is poured into a mold, and then dried.

This method is described in JP-A-49-133579 and other publications.

To enclose cellulose acetate fibers, cellulose triacetate is dissolved in a solvent such as methylene chloride, and bacterial cells are added and mixed.

The mixed liquid is coagulated by extruding it into a coagulation bath of toluene or the like through a nozzle or the like.

The solidified product is subjected to a vacuum drying treatment or the like to remove the solvent.

Thereby, it is possible to obtain bacterial cells enclosed in cellulose acetate fibers.

This method is used in process biochemistry 1(8), 9
(1972) and others.

The conversion reaction of citric acid, ink citric acid, or their salts to L-glutamic acid can be carried out using a continuous stirring reaction tank or a packed column. Bed flow is preferable.

Other conditions are the same as those for the case without immobilization.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Glucose broth medium 300mA with yeast extract 5g/l! A seed culture of Corynebacterium glutamate ATCC 13761, which had been cultured with shaking using
L K2HPO41.5g/l, NH4Cl12g
/l, MgSO4・7H200,5g/'L Mn50
4-4 H2O20mI? / 11F e 804 ・
7 H2020Tn9/i.

ZnSO4・7H2057n9/l, biotin 30tt
9Δ, Corn Nuchyplica 2g/L Thiamine/HCL
ITn9/l (separate steaming), urea 39/l (separate steaming) (pH 7
, 2) was transplanted into a 101 jar containing medium 31, and incubated at 30°C, 500 r, p, m, , ■1 alr.
/l/m1n (hereinafter referred to as V, V, m), and when the carbon source is consumed and the pH starts to rise rapidly, the potassium salt of penicillin G is added at 5 units/ml.
Add citric acid (210g/7 solution) to 100m4/
Continuously feed at 30°C while controlling the pH to 7.2 with 28% (wt/wt) aqueous ammonia.
, without stirring, at 0.2 v, v, m, for a further 60
As a result of culturing for a period of time, L-glutamic acid was produced during that time,
The production rate was maintained at an average of 3-19/l/hr.

Example 2 Brevibacterium flavum ATCC 13826 was used as the L-glutamic acid producing bacterium, 5 μg AfLl of cephalexin was added as the antibiotic, and culture was carried out in the same manner as in Example 1 except that isocitric acid (210 g/l solution) was used instead of citric acid. After adding cephalexin, 60
During this period, an average production of 33 g/13/hr of L-glutamic acid was observed.

Example 3 Glucose 209/l, (NH4)2SO42g/11
, yeast extract 4g/l, malt extract 10EI/l
A seed culture of Satucharomyces cerevisiae ATCC 20017 cultured with shaking in 300 ml of a medium consisting of (pH 5,5) was transplanted to a 101 jar fermenter containing the same medium 31, and incubated at 30°C and 450 r, p, m-11v, v.
, m, and when the carbon source is consumed and the pH starts to rise rapidly, 2-deoxy-D-glucose is added at 2 g/l, and citric acid (210 g/l solution) is added at 1 g/l.
Continuous feed at 00 mVhr, 28% (w/w)
While controlling the pH to 5.5 with ammonia water,
As a result of further culturing at 0° C. and 0.2 v, v, m without stirring for 60 hours, L-glutamic acid was produced during that time, and the production rate was maintained at an average of 2.8 g/13/hr.

Example 4 The cells of Cornebacterium and Glutamicum ATCC 13761, which were cultured in the same manner as in Example 1, were collected by centrifugation at the point when the carbon source consumption progressed and the pH started to rapidly rise, and after washing, Freeze (dry at 7.20°C).

The obtained bacterial cells were mixed with 1 ([
Adjust to jar fermenter and add citric acid (210g/
1 solution) was continuously fed at 100 ml/hr,
While controlling the pH to 7.2 with 8% (wt/wt) ammonia water, 0.2v at 30°C without stirring.
.

When cultured for an additional 60 hours at v and m, L
-Glutamic acid was produced, and the production rate was maintained at an average of 3.2"z/hr.

Example 5 East.

Glucose broth medium 300 with extract 59/l
A seed culture of Corynebacterium glutamic acid ATCC 13761 cultured with shaking using
0g/11KH2PO40.5g/l, 2 HP
04 1.5 g/11(NH4) 25O42g/l
, MgSO4.7 H2O0.5g/l, ■ 04.4
H2020■/11FeSO4・7H20201rIv
'l, ZnSO4-7H205mty/L biotin 3
01tEl/11 Corn Steve Liquor 2Vl, Thiamine HCl 1719/l (separate steaming), Urea 3g/l
(separately steamed) (pH 7.2), transferred to a 51 jar fermenter containing medium 31, heated at 30°C, 500 rpm,
1 v.

When the carbon source is consumed and the pH starts to rise rapidly, 5 nu i t/ml of potassium salt of penicillin G is added, and glucose is further added to 3 ml of glucose.
0g/I! After adding and culturing for 5 hours, the culture was terminated, and the bacteria were collected and washed by centrifugation.

On the other hand, 500 m of cyclohexane as a dispersion medium was placed in a stirred reaction tank with a baffle in No. 21.
s powder) and 10 g of nitrogen gas.
After aeration for minutes, 95 parts by weight of acrylamide and N,N'
- 60 parts of a mixture consisting of 5 parts by weight of bisacrylamide
0g/IJ aqueous solution 67TLl, the above bacterial cells 12g (as dry weight), 50g/lN, N,N',N'-tetramethylethylenediamine 10mL and 0.01 m
o 25 g/l in I/l phosphate buffer (pH 7,8)
Dissolve 9 liters of ammonium persulfate to make 200TL
Add 200ml of 100ml of 100ml and heat at 10°C for 30 minutes.
After continuing to stir in a nitrogen gas stream, 6 m/259/12 ammonium persulfate was further added and stirring was continued for 60 minutes, and about 400 ml of the resulting granular gel was separated from P and washed.

The obtained gel-immobilized bacterial cells (400 rnl) were
IL, packed in a column with a height of 200 crrL, sodium citrate 70 m mo I/1)s ammonium chloride 0.2 mol/II, Mn 804.4H205 m
A substrate solution with a composition of mol/A (pH 7,8) is given a space velocity S, V, -0,1 (l-solution/h r/11
- As a result of passing through the column using the ascending method (immobilized bacterial cells), L-glutamic acid in the passing solution increased to 66 m2 over 120 hours.
Example 6 Glucose with addition of yeast extract 5g/l while maintaining mol/ij.

Shaking culture was performed using 300 ml of broth medium.

Corynebacterium glutamate AT CC1376
The seed culture of 1 was prepared with 15 g/l of glucose, 15 g/l of sodium citrate, 1.5 g/l of K2HPO4 (NH
4) 2SO42g/l, MgSO4・7H200.5g
/l, MnSO4・4H4H2O20/11FeS04
・7H7H2O20/l, ZnS04-7H205mt
? /L Biotin 30 μg/l, Corn Nureve Liquor 2Vl.

Thiamine I (ClIVl (separately vaporized), urea 39/13
(Separate steaming) Transfer to a 101 jar fermentor containing medium 31 (pH 7.2) at 30°G, 50r, p.

After culturing for 20 hours at m, t, 1v, v, m,
Bacteria were collected and washed by centrifugation.

Meanwhile, the collagen fibers were homogenized and the pH
3, add 6 g of the above bacterial cells (
(as dry weight) was mixed, cast on a Teflon plate, air-dried overnight at 20°C, and then vacuum-dried.
0.3crnX0.3 (after shredding into 1771 pieces,
10f! /11 gel tar aldehyde solution (pH 8) 3
It was immersed for 0 seconds and then washed with water for 24 hours.

66 g of the gel-immobilized bacterial cells obtained were mixed with 70 mmol/A' of sodium ink citrate and 0.2 m of ammonium chloride.
o l /, 1! s Mn SO4・4H205mo
The mixture was placed in a 21 stirred reaction tank containing a reaction solution 11 with a composition of 1.1, # (pH 7, s), and heated at 38°G1 without ventilation.
The reaction was carried out at 0 rpm, and the reaction solution with the same composition was
After this reaction tank, second and third reaction tanks of the same volume containing the same amount of immobilized bacteria were connected in series, and each type was equipped with a glass filter. A partition wall was provided to prevent immobilized bacterial cells from moving out of the tank.

As a result, the concentration of L-glutamic acid in the liquid continuously discharged from the third tank was 63 mmol over 150 hours.
/l was maintained.

Example 7 Glucose 20g/4 (NH4)2SO42g, /
l, yeast extract 4 g/l and malt extract 10
g/l.

A seed culture of Satucharomyces cerevisiae ATCC 20017 cultured with shaking in 300 ml of a medium consisting of (pH 5.5) was transplanted to a 51-jar fermenter containing the same medium 31, and incubated at 30° C. and 450 rpffh 1 v, v.

When the carbon source is consumed and the pH starts to rise rapidly, 20 g/l of glucose is added and the culture is continued for 3 hours, then the culture is terminated and the bacteria are collected by centrifugation. , washed, frozen and dried at 20° C. in a conventional manner.

Using 60 g (dry weight) of this bacterial cell, the bacterial cell 9 was encapsulated in a polyacrylamide gel in the same manner as in Example 5.
00mA was prepared.

The obtained immobilized bacterial cells were placed in each reaction tank 3 times in the same manner as in Example 6.
Dispense 70 m of sodium citrate.
mo I/ls ammonium chloride 0.2 mo
l/l, Fe504H7H205mmol/13
．． MnSO4・4H205mmolA (pH7,8)
As a result, the concentration of L-glutamic acid in the effluent was 58 mm over 100 hours.
l/1. was maintained.

Claims (1)

[Scope of Claims] 1. The ability of a cell belonging to the genus Corynebacterium, Brevibacterium or Satucalomycenu to produce L-glutamic acid from citric acid, incitric acid or salts thereof and ammonium ions. A method for producing L-glutamic acid from citric acid, isocitric acid, or salts thereof and ammonium ions using viable microorganisms obtained by treating microorganisms with antibiotics, cell wall synthesis inhibitors, or drying. 2. The method according to claim 1, wherein the living microbial cells are immobilized microbial cells.