JPS6119236B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-valine by fermentation. A number of methods for producing L-valine have been proposed. The best results have been obtained using Brevibacterium and Corynebacterium mutants that require isoleucine for their development. More recently, L-
Ajinomoto Co., Inc. has announced a biological production method for valine. However, it has been discovered that the amount of L-valine produced by this method is small, and that it is often produced in the presence of large amounts of other amino acids (such as L-threonine), which increases extraction and purification costs. There is. L-valine is essential for humans and other animals and therefore has wide use in human and veterinary medicine. However, in order not to limit its use to the pharmaceutical field, L-valine must be obtained economically and with sufficient purity. It has now been discovered that significant amounts of L-valine can be obtained in the fermentation broth if the fermentation is carried out with the new microorganisms described below. Furthermore, it has also been discovered that in order to obtain significant amounts of L-valine it is of considerable importance to use ammonium in the form of acetate as the main nitrogen source in the fermentation medium. The features and advantages of the present invention described above will become clearer from the following description. The object of the present invention is that the growth of cysteic acid and L
- mixture of threonine or cysteic acid and L
- achieved by using a Corynebacterium glutamicum strain, which is characterized by being subject to a co-inhibitory effect by a mixture with valine. These strains are obtained from L-glutamic acid producing strains by conventional mutagenesis methods using known mutagenic agents. Co-inhibitory action means that each of the above compounds, namely cysteic acid, L-valine, and L-threonine, has only a slight negative effect on the growth of the mutant strain, and that this influence is greater than that of the parent strain. This means that the strain shows virtually no growth in the presence of a mixture of cysteic acid and L-valine, or a mixture of cysteic acid and L-threonine. The co-inhibition phenomenon is illustrated in Example 1. Another feature of the strain used in the present invention is that the production of L-valine is very favorably influenced if ammonium acetate is used as the main nitrogen source. The highly favorable influence of ammonium acetate on the production of L-valine is illustrated in Example 2. Example 2 shows that in order to increase the production of L-valine it is necessary to introduce at least 25 g/ammonium acetate into the medium. Ammonium acetate can be used alone or in combination with other common nitrogen sources (e.g. other ammonium salts, urea, molasses from the sugar industry, corn infusion, peptone, yeast extract, meat extract, various hydrolysates, etc.)
It can also be used in combination with More specifically, the object of the present invention is achieved by the use of Corynebacterium glutamicum I-026 strain (National Institute of Microbial Technology, Agency of Industrial Science and Technology, deposit number 4035) obtained by dissociation of a mutant strain exhibiting all of the above characteristics. Ru. The Corynebacterium glutamicum strain is obtained by mutating a glutamic acid producing strain with N-methyl, N'-nitro-N-nitrosoguanidine (NTG) using a nutrient medium + L-valine, using the following method. Separated.

[Table] ↓
Corynebacterium glutamicum I−026
Table 1 shows the characteristics of the I-026 strain compared to the characteristics of the original glutamic acid-producing strain. The characteristic difference between these two strains lies in their response to the acetoin production test, ie, the Hoges-Proskauer test. As the carbon source, carbohydrates, glucose, fructose, mannose, galactose, sucrose, molasses, etc. can be used alone or in combination. The inorganic compounds added are magnesium sulfate,
Potassium dihydrogen phosphate, potassium hydrogen phosphate,
Essential inorganic salts such as iron sulfate and other commonly used salts; manganese, calcium, etc. Addition of vitamins is also necessary, especially if the medium is completely synthetic. The fermentation according to the invention is carried out under normal aerobic conditions, for example with stirring and introduction of sterile air, at a temperature of 25-37°C, preferably 25-33°C. The PH is maintained between 5 and 9, preferably between 7 and 8.5. Since pH tends to decrease during culture, adjust it by introducing ammonia, for example. Cultivation is generally continued for 2 to 5 days. During this time, a considerable amount of L-valine accumulates in the fermentation liquid. Other amino acids are also found, but in negligible amounts compared to the amount of L-valine produced. The following examples are illustrative so that the manner in which the invention may be practiced may be more fully understood. Example 1 The mother strain and its mutant strain I-026 were subjected to an in vitro growth comparison test. Each tube contained 5 ml of medium. A suspension of bacterial strains washed once with a salt solution was inoculated. The medium was incubated at 30°C for 24 hours on a rotary stirrer. After dilution 10 times, its optical density (650n
m) was read.

【table】

【table】

[Table] Example 2 Laboratory production of L-valine Carbohydrate source used = molasses Strain I-026 cultured at 30°C for 24 hours on gradient gelose was used. After culturing this gelose, it was inoculated into a previously cultured medium having the following composition.

【table】

[Table] After culturing at 30°C for 8 hours on a rotary stirrer at 200 rpm, the following production medium was inoculated (inoculation rate: 6%).

【table】

Table: 18.5 g/L-valine was obtained after fermentation for 72 hours at 30° C. on a rotary stirrer at 200 rpm. Example 3 Effect of the nitrogen source used on the production of L-valine The I-026 strain was again used. The method was the same as that of Example 2 up to the pre-culture stage. The method at the biological production stage was also the same as in Example 2. The medium used was the same except for the nitrogen source, whose effect on L-valine production was investigated. The amount of L-valine obtained after 72 hours of cultivation was dependent on the nitrogen source used. The results are shown in the table below.

[Table] Example 4 Experimental conditions were similar to those described in Example 2. However, the nitrogen source used this time was a mixture of ammonium salts, namely acetate and sulfate. The following table shows the amount of L-valine obtained depending on the amount of acetate and sulfate used (the proportion of ammonia is constant).

[Table] Example 5 The same method as in Example 2 was applied up to the preculture stage. Biological production was also carried out in the same manner as in Example 2, but the medium used had the following composition.

[Table] After 72 hours, 13 g/L-valine was obtained. Example 6 Semi-industrial production of L-valine (1) Preparation of inoculum Corynebacterium I-026 strain was cultured at 30° C. in nutrient gelose gradient for 24 hours. 6 triangular kolben has the following composition per 1 demineralized water
Two bottles of the gelose were inoculated into 1.5 medium.

[Table] Cultured on a rotary stirrer for 18 hours at 30°C. (2) Preliminary culture The components shown in the following table were dissolved or suspended in 10 vats containing the demineralized water from Step 4.

[Table] The culture medium was sterilized by autoclaving at 125°C for 30 minutes. The pH after sterilization was 5.70, which was adjusted to 7.80 with ammonia. Then 320 ml of the above inoculum was inoculated. The PH became equal to 7. The culture was carried out for about 14 hours with stirring at 600 rpm and aeration at 1 v/v min. The pH gradually changed and reached 5.40 at the end of the culture. (3) Production The production medium had the following composition per 1 part demineralized water.

【table】

[Table] A small 14 fermenter was used in this process. The ingredients were dissolved in 7 parts of water. The pH was 7.20. Autoclave sterilization was performed at 123°C for 30 minutes. The pH after sterilization was 6.75. This was inoculated with 700 ml of preculture. PH rose to 7.30.
Cultivation was carried out at 30°C for 64 hours (stirring = 600 r.pm).
, ventilation = 1v/v・min). The PH rose naturally from 7.30 to 8 (ie the value to be adjusted to). Molecule was supplied during the culture to maintain a residual sugar percentage of 3%. The feed was stopped so that the percentage of residual sugar at the end of fermentation was less than 1%. A silicone-based antifoam agent was added as needed. After 64 hours, 373 g of L-valine, ie 36.4 g of L-valine per fermentation liquid, was obtained. The yield expressed as a percentage of the amount of sugar introduced was about 19%. Small amount (3.76g in total per fermentation liquid).
Leucine and isoleucine (about 50% of which was derived from glycosylation) were discovered.

【table】

TABLE Naturally, the invention is in no way limited to the embodiments thereof shown for illustrative purposes only.

Claims (1)

[Claims] 1. A method for producing L-valine by fermentation,
at least one mutant strain of Corynebacterium glutamicum, the growth of which is inhibited by a synergistic mixture of cysteic acid and L-valine or a synergistic mixture of cysteic acid and L-threonine; A method characterized by culturing in a fermentation medium containing a carbon assimilation source, at least one nitrogen assimilation source, an organic nutrient component, and an inorganic nutrient component. 2. The method according to claim 1, wherein the microorganism used is Corynebacterium glutamicum, which has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under deposit number 4035. 3. The method according to claim 1, which uses carbohydrates as a carbon assimilation source. 4. The method according to claim 1, which uses molasses as a carbon assimilation source. 5. The method according to claim 1, wherein ammonium acetate is used as a nitrogen assimilation source. 6 The amount of ammonium acetate used is 1 of the fermentation medium.
6. The method according to claim 5, comprising 20 to 35 g per serving. 7. Fermentation is carried out at a pH of 5 to 9 and a temperature of 25 to 37°C.
A method according to claim 1. 8. The method according to claim 1, wherein the fermentation is carried out at a pH of 7 to 8.5 and a temperature of 30 to 33°C.