JPS6234397B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-leucine using a fermentation method. Conventionally, methods using microorganisms such as Brevibacterium, Serratia, Salmonella, and Corynebacterium are known as methods for producing L-leucine by fermentation. In contrast, the present inventors have developed a β-2-thienylalanine-resistant strain from a microorganism belonging to the genus Escherichia through mutation, thereby producing a significant amount of L-
A strain capable of producing leucine was obtained. Specifically, the microorganisms used in the method of the invention include the following. Escherichia coli AJ 11478 (β-2-thienylalanine resistant, β-hydroxyleucine resistant) (FERM-P 5274) Escherichia coli AJ 11479 (β-2-thienylalanine resistant) (FERM-P 5275) These strains , Esierhia Cori K-12
(ATCC 10798),
All of these strains are resistant to β-2-thienylalanine. Table 1 shows the degree of resistance of these strains to β-2-thienylalanine. These results are
Each drug was dissolved in a medium with the following composition to the concentration shown in the table, and after inoculating each bacterial strain, it was incubated at 31°C for 24 hours.
The growth of the bacteria was investigated by culturing over time. Medium composition: glucose 0.5g/dl,
(NH ₄ ) ₂ SO ₄ 0.1g/dl, KH ₂ PO ₄ 0.846g/dl, sodium citrate 0.05g/dl, KOH 0.226g/dl
dl, MgSO ₄・4H ₂ O0.04g/dl, FeSO ₄・7H ₂ O1
Contains mg/dl, MnSO ₄ 4H ₂ O 1mg/dl, thiamine hydrochloride 1mg/ml.

[Table] Drug resistance as used in the present invention refers to a case where the specific growth rate in the presence of a drug is higher than that of the parent strain Escherichia coli K-12 under the above culture conditions. In addition, the specific growth rate is the amount of bacterial growth (minus the amount of inoculated bacteria) when no chemicals are added.
And so. Growth was measured by absorbance at 570 nm. The culture medium for producing L-leucine is not particularly limited, and a conventional medium containing a carbon source, a nitrogen source, an inorganic salt, and, if necessary, an organic micronutrient is used. Hydrous carbon (glucose, sucrose, fructose, lactose, and hydrolysates containing these such as starch and cellulose) as a carbon source;
molasses, whey, etc.), organic acids (acetic acid, citric acid, etc.),
Alcohol (glycerin, ethanol) can be used. Nitrogen sources include ammonium salts (ammonium sulfate, ammonium nitrate, ammonium phosphate, ammonium chloride, etc.), ammonia gas,
Ammonia water etc. can be used. Inorganic salts include phosphates, magnesium salts, calcium salts, iron salts,
Use manganese salt, trace metal salt, etc. as necessary. As organic micronutrients, if there is a nutritional requirement, appropriate amounts of corresponding amino acids, vitamins, fatty acids, organic basic substances, etc. are added, and if necessary, amino acids, vitamins, and these are added as growth-promoting substances. Soybean hydrolyzate, yeast extract, peptone, casamino acids, etc. are used. Culture conditions may be the usual methods, pH 5 to 9;
It may be cultured at a temperature of 20°C to 45°C under aerobic conditions for 20 to 96 hours. When the pH drops during culture,
Calcium carbonate is sterilized separately and added, or neutralized with alkali such as aqueous ammonia or ammonia gas. L-leucine can be collected from the culture solution by a conventional method. Example 1 Glucose 5 g/dl, (NH ₄ ) ₂ SO ₄ 2.5 g/dl,
KH ₂ PO ₄ 0.2g/dl, MgSO ₄・7H ₂ O 0.1g/dl, yeast extract 0.05g/dl, thiamine hydrochloride 1000γ/
, FeSO ₄・7H ₂ O1mg/dl, MnSO ₄・4H ₂ O1mg/dl
dl, calcium carbonate 2.5g/dl, PH
Dispense 20ml of 7.0 aqueous medium into a 500ml flask,
One platinum loop of each strain was inoculated into this, and cultured at 31°C for 72 hours. The amount of L-leucine accumulated at the end of fermentation was as shown in Table 2.

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Claims (1)

[Claims] 1 A microorganism that belongs to the genus Escherichia and is resistant to β-2-thienylalanine is aerobically cultured in a liquid medium to produce and accumulate L-leucine in the culture solution, which is then collected. L-
Method for producing leucine.