JPH047196B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-proline by fermentation, and more particularly to a method for obtaining L-proline in high yield using a mutant strain resistant to purine analogs or pyrimidine analogs. Traditionally Corynebacterium, Arthrobacter,
Methods for producing L-proline using microorganisms belonging to Microbacterium, Brevibacterium, Bacillus, Kurtia, Satucharomyces, etc. are known. Also known is a method for producing L-proline using bacteria that require proline and the like, and bacteria that are resistant to sulfur drugs and the like. Microorganisms that produce L-proline in high yields are always in demand, and as a result of studies for this purpose, we have developed mutant strains of microorganisms belonging to the genus Corynebacterium or Brevibacterium that confer purine analog resistance, or The present invention has been completed based on the discovery that a mutant strain of a microorganism belonging to the genus Bacterium, Arthrobacter, or Brevibacterium that is endowed with pyrimidine analog resistance produces L-proline in good yield. Furthermore, it was also found that L-proline can be obtained in good yield by culturing the mutant strain with the addition of a specific amount of NH ₄ ⁺ ions. According to the present invention, a microorganism belonging to the genus Corynebacterium or the genus Brevibacterium, having resistance to purine analogs and having an ability to produce L-proline, or a microorganism belonging to the genus Corynebacterium, the genus Arthrobacter, or the genus Brevibacterium L-proline can be produced in high yield by culturing microorganisms belonging to the genus pyrimidine analogs that are resistant to pyrimidine analogs and capable of producing L-proline in a medium containing carbon sources, nitrogen sources, and other nutrients. Obtainable. In addition, by adding 0.05 to 0.5 M of NH ₄ ⁺ to the culture medium during culture, L
- The yield of proline can be improved. The microorganisms used in the present invention include strains belonging to the genus Corynebacterium or Brevibacterium that are resistant to purine analogs and have the ability to produce L-proline, or strains of the genus Corynebacterium, Arthrobacter, or Any strain that belongs to the genus Brevibacterium, is resistant to pyrimidine analogs, and has the ability to produce L-proline can be used, but it also has any other properties that contribute to L-proline production, such as nutritional requirements for proline, etc. Bacteria that are resistant to drugs such as anti-inflammatory drugs, sulfur drugs, and proline analogs can also be used. Examples of purine analogs include 6-mercaptoguanine, 8-azaguanine, 2-fluoroadenine, libersidin, 6-methylpurine (hereinafter referred to as 6MP), 8-azaxanthin, 8-azaadenine, 8-mercaptoguanosine, 6-mercapto Guanosine (hereinafter referred to as 6MG), 2-aminopurine, 2-amino-6-mercaptopurine,
Examples include decoinin and psychofuranin.
Examples of pyrimidine analogs include 5-bromouracil (hereinafter referred to as 6BU), 6-azauracil (hereinafter referred to as 6AU), 5-fluorouracil,
Examples include 5-bromo-2-deoxyuridine, 2-thiouracil, 6-methyl-2-thiouracil, and amicetine. Mutation induction methods for obtaining purine and pyrimidine analog resistant strains include ultraviolet irradiation, X-ray irradiation, drug treatment (eg, nitrosoguanidine, ethyl methanesulfonate, etc.), and natural mutation. The mutation manipulation of the bacterial strain used in the Examples of the present invention will be described in more detail. After culturing the parent strain overnight with shaking in bouillon medium, collect the bacteria.
Trisumarate buffer (ammonium sulfate 1
g/, magnesium sulfate heptahydrate 0.1 g/,
Calcium chloride/dihydrate 5mg/, ferrous sulfate/
After washing with PH6.0, 0.05M Tris-maleic acid buffer containing 0.25 mg/ml of heptahydrate, suspend in the same buffer containing 1 mg/ml of nitrosoguanidine, leave at 28°C for 30 minutes, and then immediately Wash twice with the same buffer, then inoculate the suspension into bouillon medium for 60 minutes.
After culturing with shaking at ℃, after collecting the bacteria and washing once with Tris-malate buffer, an appropriate amount is spread on an agar medium containing purine or pyrimidine analogs with the composition listed in Table 1. It was obtained as a pyrimidine analog resistant strain. At this time, glucose is used as the carbon source for the agar plate medium, but lactic acid, succinic acid, glycerol, etc. can also be used instead of glucose. The thus obtained strain having purine or pyrimidine analog resistance and L-proline producing ability is H-3334, which is mutated from Corynebacterium glutamicum ATCC-13032.
(6MG resistant) (NRRL B-15511), Corynebacterium acetophilum (FERM-P 4045)
H-3336 (6MP resistance) mutated from
(NRRL B-15512), H-3337 (5BU resistance)
(NRRL B-15513), H-3338 (6AU resistant) mutated from Corynebacterium acetoacidophyllum ATCC-13870 (NRRL B-
15514), H-3356 mutated from Brevibacterium lactofermentum ATCC-13869
(5BU resistance) (NRRL B-15515), H-3357
(6MP resistant) (NRRL B-15516) and H-3358 (6AU resistant) mutated from Arthrobacter citreus FERM-P 4963 (Feikoken Bacterial Serial No. 412). Experimental results regarding the degree of purine or pyrimidine analog resistance of these strains are shown in Table 1.

[Table] Experimental method Medium: glucose 10g/, monopotassium phosphate 1
g/, dipotassium phosphate 3 g/, ammonium chloride 4 g/, urea 2 g/, magnesium sulfate heptahydrate 0.4 g/, ferrous sulfate heptahydrate 0.99 mg/, zinc sulfate heptahydrate 0.88 mg/ ,
Copper sulfate pentahydrate 0.39mg/, manganese chloride 4
Water salt 0.072mg/Sodium borate decahydrate
0.88mg/, agar 20g/, PH7.2 Culture: Spread the above strain on agar medium and culture at 28℃ for 30 hours Judgment: Sufficient growth + Growth ± Slight growth - No growth Note that the above purine and pyrimidine analog resistant strains Both have the same susceptibility to sulfur drugs as the parent strain, and have no resistance to sulfur drugs. The culture medium used in the present invention includes a carbon source, a nitrogen source,
Both synthetic and natural media can be used as long as they contain appropriate amounts of inorganic substances and other trace nutrients required by the strain used. Any carbon source or nitrogen source may be used in the culture medium as long as it is available to the strain used. That is, carbon sources include carbohydrates such as glucose, fructose, sucrose, maltose, mannose, and sorbitol, sugar alcohols, glycerol, starch,
Starch hydrolyzate, molasses, etc. can be used, and organic acids such as acetic acid, pyruvic acid, lactic acid, fumaric acid, and gluconic acid, amino acids such as glutamic acid and aspartic acid, and lower alcohols such as ethanol can also be used. Nitrogen sources include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or amino acids such as glutamic acid and aspartic acid, urea, and nitrogen-containing substances such as peptone and meat. Various nitrogen-containing organic substances such as extract, corn stave liquor, casein hydrolyzate, and soybean meal hydrolyzate can be used. L-glutamate and D-, L- and
Addition of DL-pyrrolidonecarboxylic acid is effective in improving the yield of L-proline. As the inorganic substance, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, etc. are used. Furthermore, vitamins necessary for the growth of microorganisms, such as biotin, nicotinamide, pantothenic acid, and thiamine, are also used as necessary, but especially if they are provided in the medium along with the other medium compositions mentioned above. It's fine even if you don't have it. Cultivation is performed under aerobic conditions such as shaking culture or aerated agitation culture. Cultivation is performed at 20 to 40°C and at a pH around neutrality (PH5 to 9). Usually, a significant amount of L-proline accumulates in the medium after 1 to 5 days of culture. After the cultivation is completed, the bacterial cells are removed and L-proline is recovered from the culture solution by a known method such as treatment with an ion exchange resin or activated carbon as shown in the Examples. Aspects of the present invention will be explained below with reference to Examples. Example 1 Glucose 10g/, meat extract 5g/, peptone 10g/, yeast extract 3g/, salt 3g/
250ml of seed culture medium with the composition of (PH7.2)
ml Erlenmeyer flask, sterilized, inoculated with the strains shown in Table 2, and cultured with shaking at 30°C and 220 rpm for 24 hours. As the main fermentation medium, 300 ml of the medium with the following composition was dispensed into 2-volume Erlenmeyer flasks with a baffle, and after sterilization, 30 ml of the above seed culture solution was inoculated, and the temperature was 32°C and 220 rpm.
Culture for 4 days. The amount of L-proline accumulated in the fermentation broth is shown in Table 2. Main fermentation medium glucose 100g/, KH ₂ PO ₄ 3g/,
MgSO ₄・7H ₂ O 0.5g/, ammonium sulfate 10g/,
FeSO ₄・7H ₂ O 10mg/, nicotinic acid 10mg/,
Thiamine hydrochloride 100μg/, biotin 100μg/
, Corn Steep Liquor 20g/, Sodium L-Glutamate 20g/ and CaCO ₃ 30g/
(PH7.4)

[Table] Example 2 The strains shown in Table 3 were cultured in the same seed culture medium as in Example 1, and then cultured in the same manner as in Example 1 in a medium with the same composition as in Example 1 except for the ammonium sulfate concentration shown in Table 3. do. The amount of L-proline accumulated in the fermentation liquid under each condition is shown in Table 3.

[Table] Example 3 200 ml of a seed culture medium having the composition shown below was placed in a 2-volume Erlenmeyer flask with a baffle, and after sterilization, H
-3336 or H-3338 inoculated, 30℃, 220rpm
Incubate with shaking for 24 hours. Seed culture medium Sucrose 60g/, KH ₂ PO ₄ 2g/,
MgSO ₄・7H ₂ O 0.5g/, FeSO ₄・7H ₂ O 10
mg/, MnSO ₄ 4H ₂ O 10mg/, Corn Steep Liquor 10g/, Thiamine Hydrochloride 100μ
g/, biotin 100μg/, urea 3g/
(PH7.4) As the main fermentation medium, use one having the following composition, dispense 700ml each into a 2-volume jar fermenter, and after sterilization, inoculate 100ml of the above-mentioned seed medium.
Culture with aeration and agitation at 32°C. To control the pH during culture, acetic acid solution is automatically added to maintain the pH at 7.0. Main fermentation medium Sucrose 20g/, ammonium acetate 7
g/, MgSO ₄・7H ₂ O 0.6 g/, KH ₂ PO ₄ 4
g/, ammonium sulfate 5g/, FeSO ₄・7H ₂ O 10mg/
, MnSO ₄・4H ₂ O 10mg/, CuSO ₄・5H ₂ O
1mg/, thiamine hydrochloride 100μg/, biotin 100μg/, (PH7.4) After 72 hours of culture, 15% of acetic acid was consumed based on the initial solution volume, and the proline accumulation amounts of H-3336 and H-3338 were as follows: 45g/, 43g/. Each parent strain FERM-P-4045 was cultured in the same manner,
ATCC-13870 is 28.5g/, 0.7g/.

Claims (1)

[Claims] 1. A microorganism belonging to the genus Corynebacterium or Brevibacterium that is resistant to purine analogs and has the ability to produce L-proline, or a microorganism that belongs to the genus Corynebacterium, Arthrobacter, or Brevibacterium and that contains pyrimidine L-proline is characterized by culturing a microorganism that is resistant to analogs and capable of producing L-proline in a medium, producing and accumulating L-proline in the culture solution, and collecting L-proline from the culture solution. -Process for producing proline.