JPH0362394B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] L-isoleucine is an important component of amino acid infusions and comprehensive amino acid preparations. The present invention is based on this L-
This method improves the method of producing isoleucine by fermentation. [Prior Art] In order for microorganisms of the genus Brevibacterium and Corynebacterium to have the ability to produce L-isoleucine, they must be made resistant to α-amino-β-hydroxyvaleric acid (hereinafter abbreviated as AHV), etc. I know what I should do. Furthermore, in addition to the above-mentioned drug resistance, the ability to produce L-isoleucine can be improved by imparting O-methylthreonine resistance, β-hydroxyleucine resistance, or trichloroalanine resistance, and by imparting requirements for purine substances or lysine. is known to improve. [Problems to be Solved by the Invention] Improving the L-isoleucine fermentation yield and accumulation is an important problem in industrial production. [Means for Solving the Problems] The present invention has been made to solve the above problems, and has the ability to produce L-isoleucine belonging to the conventionally known genus Brevibacterium and Corynebacterium. As a result of research to improve microorganisms and find strains with even higher fermentation yields, we discovered α-ketomalonic acid (hereinafter abbreviated as α-KM).
It has been discovered that among the strains that have been given resistance to L-isoleucine, there are strains that produce L-isoleucine at a higher yield than conventional L-isoleucine-producing bacteria. That is, the present invention belongs to the genus Brevibacterium or Corynebacterium and has α-KM resistance,
The present invention also relates to a method for producing L-isoleucine, which comprises culturing a microorganism capable of producing L-isoleucine in a liquid medium, and collecting L-isoleucine produced and accumulated in the medium. The microorganism used in the present invention belongs to the genus Brevibacterium or Corynebacterium, and α
- It is a mutant strain that has KM resistance and the ability to produce L-isoleucine. To obtain the mutant strain of the present invention, the following wild strain is first given L-isoleucine producing ability, and then α-
KM resistance may be imparted, or α-KM resistance may be imparted first and then isoleucine producing ability may be imparted. The wild strain that serves as the parent strain of this mutant strain is one known as a coryneform L-glutamic acid producing bacterium such as the genus Brevibacterium or Corynebacterium, and includes, for example, the following. Brevibacterium lactofermentum
ATCC13869 Brevibacterium deivalicatum
ATCC14020 Brevibacterium satscaroliticum
ATCC14066 Brevibacterium flavum ATCC14067 Corynebacterium glutamium ATCC13032 Corynebacterium acedophyllum
ATCC13870 To obtain the mutant strains of the present invention from these parent strains, conventional mutagenesis methods such as treatment with N-methyl-N'-nitro-N-nitrosoguanidine can be applied. The method for isolating the mutant strain of the present invention from a mutant-treated bacterial fluid is carried out by collecting a strain that grows in a medium containing α-KM. The relationship between the specific mutation induction method of the mutant strain shown in the present invention and the growth rate of the strain against α-KM is shown below. [Mutation induction method] Brevibacterium flavum grown on broth agar strands at 30°C for 24 hours
AJ3686FERM P-2433, FERM BP-755
(AHV-resistant strain derived from ATCC14067) and Corynebacterium glutamicum AJ12150FERM
Cells of P-7674 and FERM BP-756 (AHV-resistant strain derived from ATCC13032) were suspended in M/30 phosphate buffer to make a cell suspension with a cell concentration of 10 ⁸ to 10 ⁹ /ml.
500 μg/ml of N-methyl-N'-nitro-N-nitrosoguanidine was added and kept at 30° C. for 20 minutes.
The cells were then collected by centrifugation, washed well with M/30 phosphate buffer, inoculated into a medium having the composition shown below, and cultured at 31.5°C for 2 to 10 days.

[Table] Among the strains grown on agar medium, Brevibacterium flavum AJ12152, FERM P-7676, and B.
FERM BP-758 (AHV resistant strain, α-KM resistant)
and Corynebacterium glutamicum
AJ12153, FERM P-7676, FERM BP-758
(AHV resistant strain, α-KM resistant) was obtained. The degree of α-KM resistance of the mutant strain thus obtained was compared with that of the parent strain. Glucose 0.5g/dl, urea 0.2g/dl, ammonium sulfate
0.15g/dl, KH ₂ PO ₄ 0.3g/dl, K ₂ HPO ₄ 0.1
g/dl, MgSO ₄・7H ₂ O0.01g/dl, CaCl ₂・
2H ₂ O 0.1mg/dl, biotin 100μg/, thiamine hydrochloride 100μg/, FeSO ₄・7H ₂ O 0.002g/
dl, MnSO ₄ 7H ₂ O 0.002 g/dl, and the amount of α-KM shown in the table, a natural medium (peptone 1 g/dl, yeast extract 1 g/dl) was added to the medium adjusted to pH 7.0.
dl, NaCl 0.5 g/dl, PH 7.0) strands for 24 hours were suspended in sterilized water and inoculated, cultured for 24 hours, and the degree of growth was measured by turbidity.

[Effect]

The medium used for culturing such mutant strains is a normal medium containing a carbon source, a nitrogen source, inorganic ions, substances that should satisfy the above-mentioned requirements, and other organic micronutrients such as vitamins as necessary. . Preferred carbon sources include carbohydrates such as glucose and sucrose, organic acids such as acetic acid, and preferred nitrogen sources include aqueous ammonia, ammonia gas, and ammonium salts. As inorganic ions, potassium ions, sodium ions, magnesium ions, phosphate ions, and others are appropriately added to the medium as necessary. It is preferable to culture under aerobic conditions.
More favorable results can be obtained by controlling the pH to 4 to 8 and the temperature to 25 to 37°C. Thus, if the culture is continued for 1 to 7 days, a significant amount of L will be present in the medium.
-Isoleucine is produced and accumulated. L from the culture solution
-Isoleucine can be collected by a conventional method such as a method using an ion exchange resin. This will be explained below using examples. Example 1 Glucose 10g/dl, (NH ₄ ) ₂ SO ₄ 7g/dl,
KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O0.04g/dl,
FeSO ₄・7H ₂ O1mg/dl, MnSO ₄・4H ₂ O1mg/dl,
Thiamine/HCl 100μg/, Bithion 100μg/
A medium containing 60 mg/dl of soybean protein acid hydrolyzate (as total nitrogen) and 5 g/dl of calcium carbonate (separately sterilized) was adjusted to pH 7.0, and 20 ml of it was placed in a 500 ml shoulder flask and sterilized by heating. A loopful of the bacterial strains shown in Table 1 was inoculated into this, and the mixture was shaken for 4 days while being kept at 31.5°C. The amount of L-isoleucine shown in Table 2 was accumulated in the culture solution of each strain. AJ12152 was cultured using the above method to obtain culture solution 1, from which the bacterial cells were removed by centrifugation, and the supernatant was passed through a strongly acidic ion exchange resin "Diaion" SK-IB (NH ₄ ⁺ type). I let it happen. After washing the resin with water, it was eluted with 2N aqueous ammonia, and the eluate was concentrated to obtain 16.0 g of crude crystals of L-isoleucine.

【table】

Claims (1)

[Claims] 1 Belongs to the genus Brevibacterium or Corynebacterium and has resistance to α-ketomalonic acid, and
- A method for producing L-isoleucine, which comprises culturing a microorganism capable of producing isoleucine in a liquid medium, and collecting L-isoleucine produced and accumulated in the medium.