JPS6234392B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel mutant strains of the genus Brevibacterium. The present inventors derived a mutant strain with acetic acid auxotrophy from a microorganism belonging to the genus Brevibacterium.
succeeded in separating. This new mutant strain has high efficiency in producing amino acids from carbon sources, including L-arginine, L-glutamine, L-glutamic acid,
L-histidine, L-isoleucine, L-leucine, L-methionine, L-ornithine, L-phenylalanine, L-threonine, L-valine, L
- Useful as a bacterium that produces amino acids such as tryptophan. That is, the present invention is a mutant strain that belongs to the genus Brevibacterium and has acetic acid auxotrophy. Representative examples of the mutant strains of the present invention include the following. Brevibacterium flavum AJ11277
FERM-P4551 (AEC〓, AcOH ^- ; L-lysine producing bacterium) Brevibacterium flavum AJ11278
FERM―P4552 (AEC〓, Hse ^- , AcOH ^- ; L―
Lysine producing bacteria) Brevibacterium lactofermentum
AJ11280 FERM―P4554 (AEC〓, CCL〓,
Ala ^- , AcOH ^- ; L-lysine producing bacteria) Brevibacterium lactofermentum
AJ11515 FERM―P5335 (AcOH ^- ) Brevibacterium lactofermentum
AJ11516 FERM-P5336 (AcOH ^- , isocitrate lyase reduction; L-glutamic acid producing bacteria) Brevibacterium flavum AJ11517
FERM-P5337 (AcOH ^- , L-glutamic acid producing bacteria) AEC〓: S-(2-aminoethyl)-cysteine resistant. Hse ^- : L-homoserine requirement. CCL: α-chlorocaprolactam resistance. Ala ^- : L-alanine requirement. AcOH ^- : Acetic acid requirement. The parent strains of the above-exemplified mutants are Brevibacterium lactofermentum ATCC13869 and Brevibacterium flavum ATCC14067.
In addition, Brevibacterium immariophyllum ATCC14068, Brevibacterium satucaroliticum ATCC14066, Brevibacterium
A group of microorganisms of the genus Brevibacterium, particularly known as L-glutamic acid-producing bacteria, such as Brevibacterium devaricata ATCC 14020 and Brevibacterium roseum ATCC 13825, are suitable as parent strains. Furthermore, in addition to the wild strains exemplified above, amino acid-requiring,
A mutant strain imparted with drug resistance or the like may be used as a parent strain. Any of the usual methods such as ultraviolet irradiation, X-ray irradiation, radiation irradiation, mutagenic agent treatment, etc. can be used as the mutation induction method. The medium for culturing such mutant strains contains a carbon source,
It is a conventional medium containing a nitrogen source, inorganic ions and, if necessary, organic micronutrients. Carbon sources include carbohydrates (glucose, fructose, sucrose, etc. and molasses containing these, starch hydrolysates, fruit juices, cellulose decomposition products, etc.), alcohols (ethanol, glycerin, etc.), and organic acids (acetic acid, propionic acid, gluconic acid, etc.). , higher fatty acids, etc.) can be used. As the nitrogen source, urea, ammonium salt, aqueous ammonia, ammonia gas, and other common nitrogen sources are used. As inorganic ions, phosphate ions, magnesium ions, potassium ions, etc. are added to the medium as appropriate and necessary. Cultivation is best carried out under aerobic conditions, with temperatures between 24 and
The most favorable results will be obtained if carried out at 39°C and pH 5.0 to 9.0. To adjust the pH, inorganic or organic acids or alkalis, as well as urea, calcium carbonate, ammonia gas, etc. can be used. Example 1 Brevibacterium flavum ATCC21475
(AEL〓) was treated with N-methyl-N'-nitro-N-nitrosoguanidine (250μg/ml, at 30℃).
30 min), then add 0.2 acetic acid to the minimal medium shown below.
Colonies that grew in the medium with % addition and could not grow in the medium without addition were collected by the replica method. Minimum medium composition Glucose 2g/dl Urea 0.25g/dl Ammonium sulfate 1g/dl KH ₂ PO ₄ 0.1g/dl MgSO ₄・7H ₂ O 0.04g/dl FeSO ₄・7H ₂ O 1mg/dl MnSO ₄・4H ₂ O 1mg /dl Biotin 50μg/ Thiamine hydrochloride 100μg/ NaCl 5mg/dl L-alanine 50mg/dl Nicotinamide 0.5mg/dl L-homoserine 50mg/dl (PH) (7.2) Add acetic acid to the growth thus obtained. AJ11277 (FERM-
P4551) (AEC〓・AcOH ^- ), and AJ11278
(FERM-P4552) (AEC〓・Hse ⁻・AcOH ⁻ ) was collected. Furthermore, using the same method, Brevibacterium lactofamentum AJ11082 (FERM-P3840)
(AEC〓, CCL (α-chlorocaprolactam))
Mutant strain ^AJ11280 (FERM-P4554) (AEC〓,
CCL〓, Ala ^- , AcOH ^- ) were collected. 20 ml of a medium having the composition shown below was dispensed into a 500 ml shaking flask and steam sterilized at 110°C for 5 minutes. Medium composition Glucose 10g/dl Ammonium sulfate 4.5g/dl KH ₂ PO ₄ 0.1g/dl MgSO ₄・7H ₂ O 0.04g/dl FeSO ₄・7H ₂ O 1.0mg/dl MnSO ₄・4H ₂ O 1.0mg/dl Biotin 500μg/ Thiamine hydrochloride 200μg/ Soy protein hydrolysis solution concentrate
1.5 mg/dl (total nitrogen 7%) Calcium carbonate (separate sterilization added) 5.0 g/dl (PH7.0) Table 1 was prepared by growing on glucose bouillon slant in advance in the medium in the flask prepared as above. Inoculate one platinum loopful of the bacterial strain shown in
They were cultured with shaking at 31°C for 72 hours. The amount of L-lysine produced in the medium after 72 hours of culture was determined by a colorimetric method using an acidic-copper ninhydrin reaction.
The results are shown in Table 1.

[Table] Supernatant liquid 1 after collecting the cultured liquid of AJ11277 strain and removing bacterial cells and calcium salts by centrifugation.
, a strongly acidic ion exchange resin (“Amberlite”)
IR-120OH type) to adsorb L-lysine. Then, L adsorbed with 3% ammonia water
-Lysine was eluted, and the eluate was concentrated under reduced pressure. After adding hydrochloric acid to the concentrate, it was cooled and L-lysine was added to L-lysine.
-Precipitated as lysine hydrochloride secondary hydrate, crystallized
22.5g was obtained. Example 2 Brevibacterium lactofermentum
ATCC13869 and Brevibacterium flavum
ATCC14067 was treated with 200 μg/ml of N-methyl-N′-nitro-N-nitrosoguanidine.
acetic acid auxotrophic mutant AJ11515, treated for 20 min at °C.
AJ11516 and AJ11517 were obtained. Table 2 shows the growth rates of these strains relative to the concentration of acetic acid added. The growth rate of each strain was detected as follows. Glucose 0.5g/dl, Urea 0.15g/dl, Ammonium sulfate
0.15g/dl, KH ₂ PO ₄ 0.3g/dl, K ₂ HPO ₄ 0.1g/
dl, MgSO ₄・7H ₂ O0.01g/dl, CaCl ₂・2H ₂ O0.1
mg/dl, thiamine hydrochloride 10 μg/dl, biotin 3 μg/dl, Na ₂ B ₄ O ₇・10H _{2 O} 0.44 mg/dl,
FeCl ₂・6H ₂ O4.85mg/dl, CuSO ₄・5H ₂ O1.95
mg/dl, (NH ₄ ) ₆ Mo ₇ O ₂₄・4H ₂ O0.185 mg/dl,
ZnSO ₄・7H ₂ O44mg/dl, MnCl ₂・4H ₂ O0.36mg/
Contains dl and the amount of acetic acid shown in the table and has a pH of 7.0.

【table】

[Table] Natural medium (peptone 1g/dl,
(Yeast extract 1g/dl, NaCl 0.5g/dl, PH7.0) Bacteria cultured on a slant for 24 hours were suspended in sterile water and inoculated, cultured for 24 hours, and the growth value was measured by turbidity. In addition, the above-mentioned isocitrate lyase (hereinafter referred to as
Abbreviated as ICL. ) The ICL activity of the reduced strains is shown in Table 3. ICL activity was assayed as follows.

[Table] Glucose 2.5g/dl, ammonium acetate 0.8
g/dl, KH ₂ PO ₄ 0.1g/dl, MgSO ₄・7H ₂ O0.1
g/dl, FeSO ₄・7H ₂ O1mg/dl, MnSO ₄・4H ₂ O1
mg/dl, urea 0.4g/dl, biotin 0.3μg/dl,
Prepare a medium containing 20 μg/dl of thiamine hydrochloride and 48 mg/dl (PH7.0) of soybean decomposition concentrate (as TN), put 20 ml of each into a 500 ml shaking flask, and incubate at 115°C for 10 minutes. Heat sterilized. The test strain was inoculated into this medium and cultured at 31.5°C in a reciprocating shaking incubator until the early stage of logarithmic growth (10 to 16 hours). Bacterial cells were isolated from the culture solution, washed, disrupted by ultrasonication, and treated with Sephadex G-10, and used as an enzyme preparation. Enzyme activity was measured according to the method of Shiio et al. (Journal of Biochemistry 49
Vol. 262, 1961) Glucose 2.3g/dl, Ammonium acetate 1
g/dl, sodium acetate 1g/dl, KH ₂ PO ₄ 0.1
g/dl, MgSO ₄・7H ₂ O0.1g/dl, Thiamine・
Hydrochloride 20μg/dl, urea 0.6g/dl, FeSO ₄ .
_7H2O1mg /dl, _MnSO4・_4H2O1mg /dl, soybean decomposition concentrate (as T-N) 36mg/dl and biotin 2
Prepare a medium containing μg/(PH7.0) and place 20 ml of it into a 500 ml shake flask at 115°C.
Sterilized by heating for 10 minutes. Each of the bacteria shown in the table below was inoculated into this medium and cultured at 31.5°C using a reciprocating shaking culture solution. During cultivation, the pH of the culture solution was adjusted to 6.5 to 8.0 using 45 g/dl urea or 2NH ₂ SO ₄ . The culture was terminated after 36 hours, and the substrate yield of L-glutamic acid accumulated in the culture solution was determined. The results are shown in Table 4 below.

【table】

Claims (1)

[Claims] 1 Brevibacterium lactofermentum or Brevibacterium flavum
flavum) and has acetic acid auxotrophy.