JPS6224074B2 - - Google Patents

Description

[Detailed description of the invention]

This invention relates to a method for producing L-lysine. As a result of research to induce more efficient L-lysine producing bacteria, the present inventors found that citrate synthase derived from conventional L-lysine producing bacteria belonging to the genus Brevibacterium or Corynebacterium (4.1.3.7Citrate oxaloacetate-lyase) activity is lower than that of the parent strain, and the mutant strain, which does not have L-glutamic acid requirement, has a higher yield than the conventional strain.
discovered that it produces lysine. This invention was completed based on this knowledge. The mutant strain used in the present invention belongs to the genus Brevibacterium or Corynebacterium,
Characteristics that are conventionally known to be necessary for having L-lysine production ability, that is, homoserine requirement;
In addition to having properties such as resistance to S-(2-aminoethyl)-cysteine, in other words, the ability to produce L-lysine, it is a mutant strain with reduced citrate synthase activity. Deletion of citrate synthase activity or
Strains with significantly reduced levels require glutamic acid for growth, and cannot grow unless glutamic acid is added to the medium. As described above, the L-lysine production ability of a strain whose citrate synthase activity has been reduced to the extent that it exhibits glutamate auxotrophy is lower than that of a strain whose citrate synthase activity has been reduced to the extent that it does not have glutamate auxotrophy, which is undesirable. The parent strains of these mutant strains are microorganisms of the genus Brevibacterium or Corynebacterium, which are conventionally known as glutamic acid bacteria. Specifically, for example, there are the following strains. Brevibacterium deivaricata ATCC
14020, Brevibacterium flavum ATCC
14067, Brevibacterium lactofamentum
ATCC 13869, Brevibacterium roseum ATCC
13825, Corynebacterium acetoacidophyllum
ATCC 13870, Corynebacterium Lilium ATCC
15990, Corynebacterium glutamicum ATCC
13032. As a method for inducing the mutant strain of the present invention from these parent strains, conventional methods such as ultraviolet irradiation, N-methyl-N-nitro-N-nitrosoguanidine treatment, etc. can be applied. An example of a specific method for inducing the mutant strain of the present invention will be shown below. (1) Brevibacterium flavum ATCC
21475 (AEC〓) was treated with N-methyl-N'-nitro-N-nitrosoguanidine (250μ
g/ml for 30 minutes at 30°C), colonies that grew in the minimal medium shown below to which 0.5% citric acid or glutamic acid was added, and which could not grow in a medium without the addition, were collected using the replica method. Minimum medium composition Glucose 2.0g/dl Urea 0.25g/dl Ammonium sulfate 1.0g/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 50μg/ Thiamine hydrochloride 100μg/ NaC 5.0mg/dl L-alanine 50mg/dl Nicotinamide 0.5mg/dl PH 7.2 Citric acid or glutamic acid is required for the growth thus obtained. Collect the mutant strain and use the PASrere method (Biochim.Biophys.
Acta, 77, P693 (1963)), a citrate synthase-deficient strain was selected. These citrate synthase-deficient strains were treated with N-methyl-N'-nitro-N-nitrosoguanidine (250 μg/ml, 30°C, 30°C,
After 5 minutes), colonies that could grow were collected in the above minimal medium. (Thus, none of these strains has glutamate auxotrophy.) This is a revertant strain with reduced citrate synthase and is one of the strains that is resistant to lysine analogs. , AJ11507 (FERM-P5312) as a mutant strain with excellent L-lysine production ability.
(AEC〓.CS ^Weak ) was collected. In addition, by the same method, Corynebacterium
Acetoglutamicum AJ 11094 (FERM−
P3856) AEC〓, Ala ^- ) as parent stock AJ 11508
(FERM-P 5313) (AEC〓, Ala ^- , CS ^Weak )
was collected. Furthermore, by the same method, Brevibacterium.
Lactofermentum AJ11082 (FERM−
P3840) AEC〓, CCL〓, Ala ^- ) as parent stock AJ
11509 (FERM-P 5314) (AEC〓, CCL〓,
^Ala- , CS ^Weak ), Brevibacterium. Lactofermentum AJ 11273 (FERM-P
AJ 11510 (FERM-P ^{5315 )} (AEC〓,
CCL〓, Ala ^- , FP ^s , CS ^Weak ) were collected. AEC〓: S-(2-aminoethyl)-cysteine resistance Ala ^- : L-alanine requirement CCL〓: α-chlorocaprolactam resistance FP ^s : Fluoropyruvate sensitivity CS ^Weak : Decreased citrate synthase activity These mutants Table 1 shows the results of measuring the citrate synthase activity of this strain and its parent strain.

【table】

[Table] Experimental method: Biochim.Biophys, Acta, 77, 693
This was carried out according to the method described in . Enzyme preparations were prepared as follows. Glucose 10g/dl, ammonium 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 1.0 mg/dl, MnSO ₄・4H ₂ O
1.0mg/dl, biotin 500μg/, cyacin.
Using a medium adjusted to pH 8.0 containing 200 μg/dl of hydrochloride, 1.5 mg/dl of soy protein hydrolyzate concentrate (7% total nitrogen), and 5 g/dl of calcium carbonate (added separately for sterilization). After shaking culture at ℃ for 48 hours, bacteria were collected. Tris-hydrochloride buffer (PH
7.5 and 0.56M) twice, and after ultrasonic disruption (10 kc, 5 minutes), centrifugation was performed at 12,000 rpm for 30 minutes to remove the crushed materials. The supernatant was gel-filtered using "Sephadex G-10" to remove low-molecular substances, and the liquid was used as an enzyme preparation. There is no particular difficulty in producing L-lysine using these microorganisms, and it is carried out in a conventional manner using a conventional nutrient medium containing a carbon source, a nitrogen source, inorganic salts, and, if necessary, organic trace nutrients. Examples of carbon sources that can be used include sugars such as glucose, sucrose, molasses, and starch hydrolyzate; organic acids such as acetic acid, propionic acid, and benzoic acid; and alcohols such as ethanol and propanol. As the nitrogen source, ammonium salts, urea (ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphorus, etc.), aqueous ammonia, ammonia gas, and others can be used. In addition, if necessary, by using mutant strains that exhibit nutritional auxotrophy, vitamins, amino acids, etc., and organic micronutrients such as soybean protein hydrolyzate, corn staple liquor, yeast extract, or peptone containing these can be added. Add to medium. Cultivation is best done under aerobic conditions, 24-37
The most favorable results will be obtained if the reaction is carried out at ℃ 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. Thus, if the culture is continued for 2 to 7 days, a significant amount of L-lysine will be produced and accumulated in the medium. Collection of L-lysine from the fermentation broth is usually carried out by a combination of the ion exchange resin method and other known methods, and depending on the type of medium, it can also be carried out by direct crystallization. Example 1 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 2.0g/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/sodium glutamate 0.25g/dl Soy protein hydrolysis solution concentrate 1.5mg/dl (total nitrogen 7%) Calcium carbonate (separate sterilization added) 5.0g/dl PH 7.0 Prepared as above Inoculate the culture medium in a flask with one platinum loopful of the strains shown in Table 2, which have been grown on glucose bouillon slant in advance, and
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,
It was carried out by a colorimetric method using an acidic-copper ninhydrin reaction. The results are shown in Table 2.

【table】

[Table] 1.5 hours of the cultured solution of AJ11510 strain was prepared in the same manner as above, and the bacterial cells and calcium salts were removed by centrifugation. The supernatant liquid 1 was passed through a strongly acidic ion exchange resin (Amberlite IR-1200H type) to adsorb L-lysine.
Elute the adsorbed L-lysine with aqueous ammonia,
The eluate was concentrated under reduced pressure. After adding hydrochloric acid to the concentrate, it was cooled, and L-lysine was converted into L-lysine hydrochloride No. 2.
It was precipitated as a hydrate to obtain 4.1 g of crystals. Example 2 One platinum loop of Brevibacterium flavum AJ 11507 and its parent strain ATCC 21475 were each scraped off from the top of the slant, inoculated into 50 ml of the following seed culture medium, and cultured with aeration and stirring at 31°C for 18 hours. A culture solution was prepared. Seed culture medium composition: Glucose 1.5g/dl Ammonium sulfate 0.3g/dl Urea 0.1g/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 50μg/ Thiamine hydrochloride 200μg/ Sodium glutamate 0.25g/dl Soy protein hydrolysis solution concentrate 2mg/dl (Total nitrogen 7%) PH 7.5 Meanwhile, 1 volume small glass jar - Dispense 300 ml of the main fermentation medium with the following composition into a fermentor and sterilize it using a conventional method. Each of these was inoculated with 15 ml of the above seed culture solution, and culture with aeration and stirring was started at 31°C. Main fermentation medium composition: Glucose 2g/dl Ammonium sulfate 0.5g/dl Urea 0.2g/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 50μg/ Nicotinamide 1mg/ Soy protein hydrolyzate hydrolyzate concentrate 3mg/dl (Total nitrogen 7%) PH 7.2 A mixture of acetic acid and ammonium acetate in the culture medium was added so as to maintain the pH of the medium between 7.2 and 8.0, and culture was carried out at 31 to 33°C for 55 hours. The results are shown in Table 3.

[Table] Example 3 One loopful each of Brevibacterium flavum AJ 11507 and its parent strain ATCC 21475 were grown in the seed culture medium shown in Example 2 (however, 0.5 g/ml of ethyl alcohol was added instead of ammonium acetate).
ml and further changed urea to 0.3g/dl) 50
ml and cultured with aeration at 31°C for 18 hours. On the other hand, in a 1-volume small glass jar fermenter, the main fermentation medium shown in Example 2 (however, the glucose concentration was adjusted to 1 g/dl, 1 g/dl of ethyl alcohol instead of ammonium acetate, and 1 g/dl of ammonium sulfate) 0.5g/dl) was dispensed into 300ml volumes and sterilized. Each of these was inoculated with 15 ml of the above seed culture solution, and culture with aeration and stirring was started at 31°C. During culture, when the pH decreased, the pH was maintained between 7.2 and 8.2 with ammonia gas. Ethyl alcohol was added when its consumption in the medium was determined by gas chromatography and its concentration in the medium decreased to around 0.3%. When cultured at 31-33°C for 48 hours, L-lysine was accumulated in each culture solution in the amount shown in Table 4.

【table】

Claims (1)

[Claims] 1 Belongs to the genus Brevibacterium or Corynebacterium, has 50% to 95% lower activity of citrate synthase (4.1.3.7Citrate oxaloacetate-lyase) than the parent strain, and has L-
1. A method for producing L-lysine, which comprises culturing a mutant strain having the ability to produce L-lysine without requiring glutamate, and collecting L-lysine produced and accumulated.