JPS6141553B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-serine using a fermentation method. More specifically, a mutant strain belonging to the genus Pseudomonas that has the ability to produce L-serine from glycine and is resistant to chloroacetylserine, carbobenzooxyserine, or glycine hydroxamate is grown in a nutrient medium containing glycine. This is a method for producing L-serine using a fermentation method, which is characterized by culturing L-serine in a culture medium and collecting L-serine produced and accumulated in a medium. As a method for producing L-serine by fermentation method,
A method of producing glycine using microorganisms such as Corynebacterium and Pseudomonas is known. As a result of extensive research into the production method of L-serine, the present inventors found that many mutant strains belonging to the genus Pseudomonas that are resistant to chloroacetylserine, carbobenzyloxyserine, and glycine hydroxamate are more resistant to glycine than glycine. L- in high yield
We discovered the fact that serine is produced and accumulated. The microorganism used in the present invention is a mutant strain belonging to the genus Pseudomonas that is resistant to chloroacetylserine, carbobenzyloxyserine, and glycine hydroxamate. A resistant mutant strain refers to a strain that exhibits a reduced degree of growth inhibition against these drugs compared to the parent strain. To collect these mutant strains, the parent strain is subjected to known mutation treatment methods such as ultraviolet rays, drug treatment, and γ-ray irradiation. A strain that can grow on an agar medium or liquid medium containing benzyloxyserine, glycine hydroxamate, etc. may be selected. Specifically, the mutant strain used in the present invention is Pseudomonas rhodomethylofaciens.
AJ 11503FERM-P5308 (carbobenzyloxyserine resistant), Pseudomonas rhodomethylofaciens AJ 11504 FERM-P5309 (chloroacetylserine resistant), Pseudomonas rhodomethylofaciens AJ 11505 FERM-P5310 (D-
serine resistance, glycine hydroxamate resistance),
Pseudomonas rhodomethylofaciens AJ
11506 FERM-P5311 (glycine hydroxamate resistant). These mutant strains are Pseudomonas rhodomethylofaciens
This strain was obtained by further mutating AJ 11261 and FERM-P4531 as parent strains. Parent stock AJ 11261
The method for inducing mutations from is as follows. In broth liquid medium containing 2 ml/dl of methanol.
Pseudomonas rhodomethylofaciens AJ 11261 cells cultured at 30°C for 24 hours were suspended in 10ml of 50mM phosphate buffer (PH7.0) containing 250μg/ml of nitrosoguanidine, and this suspension was incubated at 30°C for 24 hours.
Shake for 15 minutes. After shaking, the bacterial cells were washed and diluted appropriately, and 0.1 to 10 mg/ml of D-serine, chloroacetylserine, carbobenzoxyserine, or glycine hydroxamate was added. Minimal growth medium containing methanol 1.5ml/dl, KH ₂ PO ₄ 0.06
%, (NH ₄ ) ₂ HPO ₄ 0.2%, MgSO ₄ 7H ₂ O 0.02%,
NaCl 0.05%, _FeSO4・_7H2O0.001 %, _MnSO4・
Bacterial cells were inoculated in 0.0005% nH ₂ O and cultured at 30°C for 7 to 15 days. The culture solution was applied to a bouillon agar plate containing 2 ml/dl of methanol and cultured at 30°C for 2 to 4 days to grow. Among the colonies, those with excellent L-serine productivity were collected. The specific method for collecting these mutant strains is as follows: minimum growth medium KH ₂ PO ₄ 0.06%, (NH ₄ ) ₂ HPO ₄ 0.2%,
_MgSO4・_7H2O0.02 %, NaCl 0.05%, _FeSO4・
Put 5 ml of a medium with a composition of 0.001% 7H ₂ O, 0.0005% MnSO ₄ nH ₂ O, and 1.5% methanol into a test tube, and add D-serine, chloroacetylserine, carbobenzooxyserine, and glycine hydroxamate. Each drug was added to a test tube at a concentration of 0 to 1000 mg/dl. In test tubes containing each of these drugs,
The parent strain AJ 11261, which was cultured for 48 hours at 30°C in a broth slant containing 2% methanol, was suspended in 10ml of 50mM phosphate buffer (PH7.0), and 0.1ml of the suspension was inoculated and cultured with shaking at 30°C for 2 to 4 days. I went to The bacterial growth rate of the cultured solution containing each concentration of drug was measured at a wavelength of 562n.
By measuring in m, it was possible to know the growth-inhibiting concentration for each drug. Therefore, to collect resistant mutant strains, the parent strain after mutation treatment with nitrosoguanidine is treated with the minimum growth inhibitory concentration of each drug (D-serine, chloroacetylserine, carbobenzoxyserine, glycine hydroxamate) added to the growth-inhibiting concentration. Inoculated into a medium and cultured at 30°C for 7 to 15 days.
The resistant mutant strain that had grown was removed to a broth plate containing 2% methanol, and the growing colonies were isolated. Table 1 shows the growth-inhibiting concentrations and concentrations of resistant mutants collected for each drug.

[Table] Pseudomonas rhodomethylofaciens AJ 11261
FERM-P4531 is classified as a new bacterial species, and its mycological properties are as follows. (a) Morphology (1) Cell shape and size: Large rod, 1.0-1.5 x 2-5 microns (2) Presence or absence of cell pleomorphism: Yes, poly-β-hydroxybutyric acid granules Formed in the body (3) Motile status Flagellum epiphytic status: Yes Polar flagella (4) Spore presence, shape, size, and location: None (5) Gram staining: Negative (6) Acid-fast: Negative ( b) Growth status in each culture medium (1) Broth agar plate culture: Moderate growth, circular, whole-rimmed, semi-lenticular, sparse, yellow-orange, opaque (2) Broth agar slant culture: Moderate growth, filamentous, red Orange, opaque, no water-soluble pigments (3) Meat juice liquid culture: moderate growth, ring-shaped (4) Meat juice gelatin puncture culture: growth on top, aerobic, no liquefaction (5) Litmus milk: no change ( c) Physiological properties (1) Nitrate reduction: Positive (2) Denitrification reaction: Negative (3) MR test: Negative (4) VP test: Negative (5) Indole production: Negative (6) Hydrogen sulfide production : Negative (7) Hydrolysis of starch: Negative (8) Use of citric acid: Koser medium Use slightly Christensen medium Use slightly (9) Use of inorganic nitrogen sources: (Methanol medium) Nitrate Use ammonium Use (10) Pigment production: No production (11) Urease: Negative (12) Oxidase: Positive (13) Catalase: Positive (14) Growth range temperature 10-38℃ Cannot grow at 41℃ PH 6-8 (15 ) Attitude towards oxygen: aerobic

【table】

【table】 (17) Accumulation of poly-β-hydroxybutyric acid: + (18) Auxotrophy: None

【table】

[Table] (20) Other properties L-serine hydroxymethyltransferase activity: Positive Hydroxypyruvate reductase activity:
Positive As mentioned above, this bacterial species is a Gram-negative bacillus, has a polar monoflagellate, is motile, and is aerobic. It also has the property of producing acids rather than sugars, and according to the 8th edition of Barge's Manual, it belongs to the genus Pseudomonas. In addition, new bacteria differ from known bacterial species due to characteristics such as lack of auxotrophy, accumulation of poly-β-hydroxybutyric acid within the bacterial body, lack of ability to assimilate arginine, and red pigmentation. It was determined to be a species and was named Pseudomonas rhodomethylofaciens. The medium for culturing the mutant strain of the present invention may be either a synthetic medium or a natural medium as long as it contains organic micronutrients such as carbon sources, nitrogen sources, inorganic ions, vitamins, and amino acids in addition to glycine. can do. Glycine may be added to the medium in advance or during the culture. Alternatively, the glycine may be added in small portions so that the concentration of glycine in the medium is lowered to reduce inhibition of growth of the mutant strain. Methanol is most suitable as a carbon source, but alcohols such as ethanol and propanol, organic acids such as formic acid and acetic acid, and carbohydrates such as glucose can also be used if an appropriate strain is selected. As the nitrogen source, aqueous ammonia, ammonium salts, nitrates, amino acids, and any other commonly used nitrogen sources can be used. Inorganic ions include potassium, magnesium, iron,
Ions such as manganese, calcium, chloride, sulfuric acid, and phosphoric acid are used as necessary. In addition, organic micronutrients such as vitamins, amino acids, corn staple liquor, yeast extract,
Meat extract, soybean protein hydrolyzate, etc. may be added. Cultivation is best carried out under aerobic conditions, with a pH of usually 5.
-9, preferably around neutrality. It is desirable to culture at a temperature in the range of 25 to 37°C. The pH of the medium is adjusted using CaCO ₃ , acid, alkaline solution, ammonia gas, etc. By culturing under aerobic conditions for 2 to 8 days, a significant amount of L-serine is accumulated in the fermented liquid. L- from the culture finished solution
Serine can be recovered by commonly known recovery methods such as a method using an ion exchange resin method. Hereinafter, the present invention will be specifically explained with reference to Examples. Example 1 KH ₂ PO ₄ 0.05%, (NH ₄ ) ₂ SO ₄ 0.5%, MgSO ₄ .
_7H2O0.04 %, NCl 0.05%, _FeSO4・_7H2O0.001
%, _MnSO4・_nH2O0.0005 %, yeast extract 0.2%,
Methanol 1.5% (separate sterilization), _CaCO3 5%, (PH
7.0) was placed in a 500 ml Sakaguchi flask and sterilized. One platinum loopful of each of the strains shown in Table 1, which had been cultured in a broth slant containing 2% methanol at 30°C for 48 hours, was inoculated into each strain, and cultured with shaking at 31.5°C. After 24 hours of culture, 1.5% methanol was added, and after 24 hours of culture, 1.5% glycine was added.
4% methanol was added, followed by further culturing for 48 hours. The amount of L-serine produced was as shown in Table 2.

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【table】

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Claims (1)

[Claims] 1 Belongs to the genus Pseudomonas, and from glycine to L-
A mutant strain that has the ability to produce serine and is resistant to chloroacetylserine, carbobenzyloxyserine, or glycine hydroxamate was cultured in a medium containing glycine, and L was produced and accumulated in the culture solution. - A method for producing L-serine, which comprises collecting serine.