JPS5832595B2 - L-Glutamine Seizouhou - Google Patents

Description

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

Typical L-glutamic acid-producing bacteria, which require biotin for growth, have L-glutamic acid productivity that is extremely sensitive to the biotin concentration in the medium at normal culture temperatures, such as 30°C, and the amount of L-glutamic acid is the limiting amount for growth. L-glutamic acid can only be produced when the biotin concentration is .

In order to produce L-glutamic acid in a medium containing an excess of biotin, antibiotics such as penicillin,
Expensive physiologically active substances such as surfactants such as ``Twin 60'' must be added during culture, which is one of the drawbacks when using various carbohydrate raw materials rich in biotin economically. Ta.

The present inventors have demonstrated that when a temperature-sensitive mutant strain, which has been mutated from a conventionally known L-glutamic acid producing bacterium as a parent strain, is cultured aerobically, a physiologically active substance is added to suppress the action of biotin. I learned that it is possible to produce L-glutamic acid in high yield without any problems.

This invention has been completed based on these findings.

This will be explained in more detail.

The temperature-sensitive mutant strain of the present invention is a strain induced by mutation using a microorganism of the genus Brevibacterium having L-glutamic acid-producing ability as a parent strain.

The mutation induction method may be a conventional method, and the method for selecting a temperature-sensitive mutant strain from a mutated parent strain is to select a mutant strain that cannot grow at the high temperature at which the parent strain can grow, and You can choose a strain that can grow in the same way as the parent strain at low temperatures.

The temperature-sensitive mutant strains thus obtained include, for example, the following strains.

These strains are Brevibacterium flavum ATC
C14067 and Brevibacterium lactofermentum ATCC13869 were derived as parent strains, respectively.

Brevibacterium flavum AJ3957FERM-
P 3307 Brevibacterium lactofermentum AJ 3955 FERM-P3305 Brevibacterium lactofermentum AJ3956 FER
Table 1 shows the growth status of these mutant strains of M-P 330 under both high and low temperature conditions in comparison with the parent strain.

The temperature-sensitive mutant strain of the present invention exhibits particularly remarkable effects when L-glutamic acid is produced using a medium containing excess biotin.

That is, conventional L-
When culturing glutamic acid producing bacteria, penicillin,
A drug such as a surfactant that suppresses the action of biotin must be added to the medium.

However, when producing L-glutamic acid using the temperature-sensitive mutant strain of the present invention in a medium containing excess biotin, the temperature must be low enough to allow good growth during the time period 1 when the large mutant strain grows to a certain extent. By simply culturing at a high temperature and changing the temperature to a high temperature that inhibits growth once the desired growth amount is reached, L- can be grown in high yield without adding any drugs that inhibit the action of biotin. Glutamic acid can be obtained.

The medium for culturing these temperature-sensitive mutants is the usual L
- Any well-known culture medium for glutamic acid production can be used.

Carbon sources include glucose, sucrose, acetic acid,
Ordinary substances such as ethanol can be used.

In particular, when natural raw materials containing a large amount of biotin, such as molasses and fruit juice, are used as the carbon source, particularly remarkable effects can be obtained as described above.

The culture conditions are not particularly different from those used for conventional L-glutamic acid producing bacteria, but when using a medium containing excess biotin, as mentioned earlier, it is difficult to maintain a certain growth amount. L-glutamic acid can be obtained in a high yield by culturing at a low temperature suitable for growth until reaching this point, and then producing L-glutamic acid at a high temperature.

The low temperature suitable for growth can be determined by culturing the mutant strain to be cultured at various temperatures, but it is usually 29 to 35°C, preferably 30 to 34°C.

The high temperature at which L-glutamic acid is produced is preferably a temperature at which the mutant strain can hardly proliferate, but L-glutamic acid is actively produced.

Usually this temperature is between 34° and 43°C, particularly preferably between 35° and 42°C.

The amount of growth when changing from a low temperature to a high temperature is the same as, or slightly lower than, the amount of growth at which the parent strain most actively produces L-glutamic acid.

Generally, even when the temperature is shifted to a higher temperature, growth is not immediately suppressed and some growth is often allowed. Therefore, in order to know the desirable conditions for L-glutamic acid production, it is necessary to determine the amount of growth at the time of temperature shift and the temperature at which the temperature will shift. It is a good idea to investigate the optimal value of .

Thus, after ■ to 3 days, a significant amount of L-
Glutamic acid is produced and accumulated.

L-glutamic acid may be collected from the culture solution by any known method.

Example 1 Glucose 3.6 g/dl, KH2PO40.1
g/dl, MgSO4' 7H200, 1g/dl,
FeSO4” 7H201m9/cll, Mn SO
4.4 H201In97dl! , soybean decomposition concentrate (as total nitrogen) 24μ/di, biotin 30μ9/11,
Prepare a medium with the following composition: thiamine hydrochloride 100μg/13, urea (separately sterilized) 0.797di, and adjust the pH
After adjusting the temperature to 7.0, 30wLl each was placed in a 500r/l shaking flask and sterilized by heating at 115°C for 10 minutes.

This medium was inoculated with the bacterial strains shown in Table 2 A.
When shaking culture was performed at the temperature shown on the left side of the temperature column for the time shown in Table 2 A, the bacterial concentration (562 n of the 26-fold diluted culture solution)
The absorbance at m) was as shown in Table 2A.

At this point, the temperature was raised to the temperature shown on the right in the temperature column of Table 2 A, and the culture was further incubated. After the total culture time shown in Table 2 A, the final bacterial concentration and the accumulated amount of L-glutamic acid were measured. , respectively as shown in Table 2A.

Future Example 2 In the medium composition described in Example 1, the carbon source was changed from glucose to Cane molasses.

When an amount equivalent to 3.6 g/dl of glucose is added, the medium contains a high concentration of biotin of 200 μ9/13.

This medium (however, for AJ3955, the initial concentration of urea was 0.4597 dl, and an additional 0.4 dl was added at the 23rd hour of culture).
5 g/dl (for AJ3956, 0.997 di added from the beginning) was put into 500 mA shaking flasks in 20 ml portions and sterilized, then inoculated with the strains shown in Table 2 H. When shaking culture was carried out at the temperature shown for the time shown in Table 2B, the bacterial concentrations were as shown in Table 2B.

At this point, the temperature was raised to the temperature shown on the right side of the temperature column in Table 2 B, and the culture was further incubated. After the total culture time shown in Table 2 B, the final bacterial concentration and the accumulated amount of L-glutamic acid were measured. , as shown in Table 2B.

Claims (1)

[Claims] 1 L-glutamic acid is produced and accumulated in the culture solution by aerobically culturing a temperature-sensitive mutant strain of Brevibacterium that has the ability to produce glutamic acid, and the L-glutamic acid is collected. - A method for producing glutamic acid.