JPH0354550B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention belongs to the genus Corynebacterium or Brevibacterium, and is sensitive to lysozyme, and is caused by excess biotin present in the medium.
- A novel microorganism capable of producing L-glutamic acid whose production of glutamic acid is not suppressed. The L-glutamic acid productivity of L-glutamic acid-producing bacteria that requires biotin for growth is extremely closely related to the biotin concentration in the medium, and L-glutamic acid can only be produced when the biotin concentration is at a limiting amount for growth. On the other hand, blackstrap molasses, starch hydrolyzate, and the like, which are used as crude raw materials for inexpensive culture media, contain large amounts of bithione. Methods for culturing L-glutamic acid producing bacteria in a medium containing these crude materials are disclosed in Japanese Patent Publication No. 37-1695 and Japanese Patent Publication No. 38-1989.
Although the method described in Japanese Patent No. 25288 is known, an even better method is desired from an industrial perspective. The present inventors conducted research on a method for producing L-glutamic acid using inexpensive crude raw materials and avoiding the effects of excessive amounts of bithione. It has been found that by using a sensitive mutant strain, L-glutamic acid can be produced in high yield without being inhibited by biotin even if an excess biotin-containing medium is used. The present invention will be explained in more detail below. According to the present invention, a microorganism belonging to the genus Corynebacterium or Brevibacterium, sensitive to lysozyme, and having the property that the production of L-glutamic acid is not suppressed by excess biotin present in the medium is used in a medium. When cultured, L-glutamic acid accumulates in the medium, and by collecting this, L-glutamic acid can be obtained in high yield. The microorganism used in the present invention belongs to the genus Corynebacterium or Brevibacterium, is sensitive to lysozyme, and has the property that the production of L-glutamic acid is not inhibited by excess biotin present in the culture medium. Any bacterial strain can be used. In general, a strain belonging to the genus Corynebacterium or Brevibacterium and having the ability to produce L-glutamic acid is used as a parent strain, and a strain sensitive to lysozyme is selected from the mutant strains obtained by mutagenesis treatment. Use. As a method for inducing mutations, conventional methods such as ultraviolet irradiation, radiation irradiation, and treatment with a mutagenic agent are used. To select strains sensitive to lysozyme from mutagenized mutant strains, select strains that cannot grow in a medium containing lysozyme at a concentration that allows the parent strain to grow, but can grow in the same manner as the parent strain in a medium without lysozyme. All you have to do is choose. Therefore, being sensitive to lysozyme here means that the minimum inhibitory concentration for lysozyme is lower than that of the parent strain. Furthermore, the production of L-glutamic acid is not suppressed by excess biotin present in the medium.
This means that the inhibition of L-glutamic acid production by excess biotin present in the medium is substantially negligible. Specifically, this means that L-glutamic acid can be produced without being affected by excess biotin even when using the above-mentioned crude raw materials. As an example of a specifically suitable strain for use in the present invention, Corynebacterium glutamicum
Corynebacterium glutamicum KY9703 obtained using ATCC13032 as the parent strain
No. 4412, NRRL11271), Corynebacterium
Glutamicum KY9705 (Feikoken Bacillus No. 4413,
NRRL11272) and Brevibacterium flavum KY9733 obtained using Brevibacterium flavum ATCC14067 as the parent strain
No. 4414, NRRL11273). Corynebacterium glutamicum
A method for obtaining a lysozyme-sensitive mutant strain using ATCC13032 as a parent strain will be specifically described below. The parent strain was added to powdered broth (Kyokuto Pharmaceutical Co., Ltd.) 20
The cells were inoculated into a medium (PH 7.2 before sterilization, hereinafter referred to as C medium) having a composition of 5 g/g and yeast extract and cultured with shaking at 30°C. The culture was stopped at mid-logarithmic phase, the bacteria were harvested, and after washing with physiological saline, the cells were added to M/20 Tris-malate buffer (PH6.0) at 5 x 10 ⁸ cells/ml.
Suspend so that This suspension has a final concentration of
Add nitrosoguanidine to a concentration of 500 μg/ml, leave at 25°C for 30 minutes, collect bacterial cells by centrifugation, wash the cells with the same buffer, suspend in physiological saline, and add physiological saline as appropriate. Dilute and spread on a solid medium (hereinafter referred to as CA medium) containing C medium and 2 g/dl agar. Culture this at 30°C for 2 days, and spread the resulting colonies (approximately 6000) onto the following three types of solid media using the replica method. CA medium CLA medium: After heat sterilizing the CA medium and cooling it until the temperature of the medium drops to 45℃, 200mg/
A medium supplemented with lysozyme so that MA medium: glucose 10g/, NH ₄ Cl4
g/, urea 2g/, KH ₂ PO ₄ 1g/,
K ₂ HPO ₄ 3g/, FeSO ₄・7H ₂ O 10mg/,
MgSO ₄・7H ₂ O 400mg/, MnCl ₂・4H ₂ O 2
mg/, ZnSO ₄・7H ₂ O 0.9mg/, CuSO ₄・
5H ₂ O 0.4mg/, Na ₂ B ₄ O ₇・10H ₂ O 0.09mg/
, (NH ₄ ) ₆ Mo ₇ O ₂₄・4H ₂ O 0.04 mg/, biotin 30 μg/, thiamine hydrochloride 1 mg/,
Cysteine hydrochloride 20mg/and agar 20g/
A medium with the composition (PH7.0 before sterilization). After culturing at 30℃ for 2 days, CLA was grown on CA medium.
Bacteria that do not grow in the medium are obtained as lysozyme-sensitive mutants. Out of 6,000 colonies tested, 110 autotrophic, lysozyme-sensitive mutants that grew similarly to the parent strain on MA medium were obtained. this
17 out of 110 strains produced large amounts of L- even in biotin-rich medium.
It had the ability to grow glutamic acid. Corynebacterium glutamicum KY9703 and
KY9705 is an example of a mutant strain thus obtained. Mutation induction using Brevibacterium flavum ATCC14067 as the parent strain was performed in the same manner as above to obtain Brevibacterium flavum KY9733. The above-mentioned mutant strains, MA medium, CA medium, CLA
Table 1 shows the results of comparison with the parent strain regarding growth in the medium and sensitivity to lysozyme. Growth on three types of solid media was done using the replica method and grown at 30°C.
Judgment was made after culturing for 2 days. In the growth column in the table, + indicates that bacterial growth was observed, and - indicates that no growth was observed. In addition, lysozyme sensitivity in the table was tested as follows. That is, in C medium for 24 hours.
After collecting the bacteria cultured with liquid shaking at 30°C, dilute appropriately with physiological saline to create a suspension of the bacteria. This suspension, equivalent to 10 ⁴ cells, is inoculated dropwise into a CA medium containing lysozyme at multiple concentrations and cultured at 30° C. for 2 days. The minimum lysozyme concentration at which the growth of bacteria could not be stopped was defined as the lysozyme susceptibility value (minimum growth-inhibiting concentration) of bacteria.

[Table] The medium for culturing the microorganism of the present invention may be either a natural medium or a synthetic medium normally used for L-glutamic acid production, as long as it contains an appropriate amount of carbon sources, nitrogen sources, inorganic compounds, and other nutrients. can also be used. For example, carbon sources include sucrose, glucose,
Carbohydrates such as molasses and saccharified starch liquid are used as nitrogen sources, while organic substances such as ammonia, ammonium sulfate, ammonium hydrochloride, ammonium sulfate, ammonium phosphate, ammonium carbonate, ammonium hydroxide, ammonium citrate, ammonium tartrate, ammonium acetate, and urea are used as nitrogen sources. inorganic nitrogen compounds,
Natural nutritional sources such as peptone, meat extract, corn steep liquor, etc., and inorganic compounds such as potassium phosphate, potassium phosphate, potassium sulfate, magnesium sulfate, magnesium chloride, ferrous sulfate, ferric chloride, and sulfuric acid. Manganese, manganese chloride, etc. are used, and biotin, thiamine, etc. are used as other nutritional sources. The culture is carried out under aerobic conditions such as shaking culture and aerated agitation culture, and the culture temperature is preferably 24 to 37°C, particularly 28 to 33°C. During cultivation, it is preferable to adjust the pH to 6 to 9 using a suitable neutralizing agent. If the culture is continued for 1 to 3 days, a significant amount of L-glutamic acid will be produced and accumulated in the culture solution. L-glutamic acid can be collected from the culture solution using conventional methods for producing L-glutamic acid, such as adsorption/desorption using an ion exchange resin, concentration crystallization, and isoelectric focusing from the supernatant after removing bacterial cells. This can be carried out using various commonly used methods as appropriate. Examples of the present invention are shown below. Example 1 Glucose 40g/, (NH ₄ ) ₂ SO ₄ 2g/,
MgSO ₄・7H ₂ O 0.5g/, KH ₂ PO ₄ 0.5g/,
K ₂ HPO ₄ 1g/, FeSO ₄・7H ₂ O 2mg/,
MnSO ₄ 4H ₂ O 2mg/, thiamine hydrochloride
Prepare a medium with a composition of 1mg/1mg/phenol, 10mg/biotin, and 2μg/or 100μg/biotin, adjust the pH to 7.0, put 30ml into a 300ml side-end flask, and heat sterilize at 115℃ for 15 minutes. did. After cooling, a separately heat-sterilized urea solution was added to the solution at a concentration of 2 g. The bacteria shown in Table 2 were inoculated into this medium and cultured with shaking at 30°C. In order to maintain the pH of the culture solution during culture at 6.5 to 8.0, urea solution was added twice at 12 hours and 20 hours at a concentration of 4 g/hour, and the culture was completed after 32 hours. The amount of L-glutamic acid thus accumulated in the culture solution is as shown in Table 2. Remove the bacterial cells from culture solution 1, concentrate, adjust the pH to 3.2 with hydrochloric acid, cool, and
- Crude crystals of glutamic acid were obtained. The amount (g) of crude crystals is shown in parentheses.

[Table] Example 2 The glucose in the medium used in Example 1 was replaced with cane molasses (40 g/equivalent amount as glucose),
The same procedure as in Example 1 was carried out except that the medium after heat sterilization was readjusted to pH 7.0. L- accumulated in the culture solution
The amount of glutamic acid is shown in Table 3.

【table】

Claims (1)

[Scope of Claims] 1. A microorganism that belongs to Corynebacterium glitamicum or Brevibacterium flavum, is sensitive to lysozyme, and has the property that the production of L-glutamic acid is not inhibited by excess biotin present in the culture medium. . 2. The microorganism is Corynebacterium glutamicum KY9703 (Feikoken Bacteria No. 4412, NRRL11271),
Corynebacterium glutamicum KY9705 (FER 4413, NRRL11272) or Brevibacterium flavum KY9733 (FER)
4414, NRRL11273)
Microorganisms listed in section.