JPS5933357B2 - Method for producing L-methionine - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing L-methionine.

L- has a wide range of uses as amino acid infusions, feed additives, etc.
Regarding methionine, the present inventors conducted research to find a more efficient production method, and found that homocysteine has a higher L- We have discovered several microorganisms that have the ability to efficiently produce methionine.

That is, the present invention provides for the production of (1) methanol or betaine and (2) homocysteine by the action of microorganisms of the genus Pseudomonas, Microticulus, or Methylomonas that have the ability to assimilate methanol and produce L-methionine from homocystine. This is a method for producing L-methionine, which is characterized by producing L-methionine and collecting it.

Pseudomonas, Microticulus, and Methylomonas assimilate methanol, and homocysteine is L-
Specifically, microorganisms that have the ability to produce methionine include the following.

Methylomonas thiomedogenes 0M33 (FE RM-P 5719) (Methyl
omonast h i om'edogenes
) Pseudomonas sp FM518 (FERM
-P 5841 ) (Pseudomonas s
p) Pseudomonas sp FE244 (FERM-P
5840) (ethionine-resistant strain mutated from FM518) Microticulus evaneus ATCC21373 (Mi
crocyclus eburneus) Methylomonas thiomedogenes 0M33 and Pseudomonas s
pFM518 was isolated by the present inventors and has the following mycological properties.

Mycological properties of 0M33 (a) Morphology ■) Cell shape and size: Bacillus 0.3-0.5 x 0.
6-2.5 μm0 2) Presence or absence of cell pleomorphism: None.

3) Motility: Yes, polar flagella.

4) Presence or absence of spores: None.

5) Durham staining: negative.

6) Acid-fast dinegative (b) Growth status in each medium■) Broth agar plate culture (methanol 1.0% human): 1-
2 mm, circular, convex circular, fully bound, smooth, translucent to opaque, mixed light, buttery uniform gloss, homogeneous, yellowish white to pale yellow.

2) Broth agar slant culture (methanol 1.0%): Moderate growth, thin film-like, filamentous, light yellow to light brown.

3) Meat juice liquid culture (methanol 1.0%): Uniformly cloudy, with a slight powdery precipitate, no film formed on the surface.

4) Meat juice gelatin puncture culture: Does not liquefy.

5) Lidmus milk: No change.

6) Others: Does not grow in broth medium that does not contain methanol.

(c) Physiological properties ■) Reduction of nitrates 2) Weakly reduced.

2) Denitrification reaction: Negative.

3) MR test: negative.

4) VP test: Negative.

5) Indole production: negative.

6) Generation of hydrogen sulfide: negative.

7) Starch hydrolysis: Negative.

8) Utilization of citric acid: Not utilized in Koset medium.

Not used in Christensen medium. 9) Use of inorganic nitrogen source: Use nitrate.

Use ammonium salts.

10) Formation of pigment: Not formed.

11) Urease: Negative.

12) Oxidase: Positive.

13) Catalase: Positive.

Rare growth range: Grows at a temperature of 30°C, but not at 37°C.

pH 5-9° 15) Attitude towards oxygen: aerobic.

16) 0-F test (according to Hugh & Leifson method): No acid is produced.

■7) Presence or absence of acid and gas generation from sugars: Acid generation
Gas production L-arabinose - -D-xylose - -D-glucose
- - D-mannose - -D-fructose - - Maltose - - Sucrose - - Lactose - - Acid production Gas production Tren-10-su - -D-Sorvit - D-Mannit - -Inosit
− − Glycerin − − Starch − − Roughinose
− −Adonito
-- Sacilin -- Dulcit -- Rhamnose -- 18) Utilization of malonic acid: Negative (Ewing et al.
, method) 19) Phenylalanine deaminase reaction: Negative (E
Wing et al.'s method) 20) Decarboxylase reaction: (MΦller's method) Lysine: Negative Ornithine: Negative Arginine: Negative 2υArginine dihydrolase reaction: Negative (5tani
er et al. method) 22) Poly-β-hy
Accumulation of droxybutyrate: No accumulation.

23) Assimilation of the following compounds (according to Stanier's medium) D-glucose - mesaconic acid - trehalose - erythritol - 2-keto-glue -
2,3-butylene glyconic acid iorumeso-inositide - meta-benzoic acid - L-valine - para-benzoic acid - β-alanine -
Tryptamine -DL-Arginine-α-
Amylamine - Betaine - DL-lactic acid - L-arabinose - D-fructose -
Satucarose - Malonic acid - Propionic acid - Testosterone Acid - Cellobiose - Ah, yup, DL-β-
・Miya Doro Oxifate Propylene Griyo 2. - L-histidine - ethanol - pantothenic acid - D-xylose -
Acetic acid - D-Lipose - Succinic acid - L-Rhamnose - Citric acid -
Porisorbate 80- L-ornithine -L/j
+) 7-acid -5-ketose) leconic acid - citraconic acid - L-lysine - meso-tartaric acid
-L-Alanine -D(-)-Tartaric Acid -Dulcit -Sorbitol Methanol-''& Methylamine + Dimethylamine, ethanolamine, formic acid, trimethylamine, diethylamine, ethylamine, formamide and formaldehyde, glycerol, pyruvic acid,
Lactose is not assimilated.

24) GC content of DNA: 51.6% (Tm method).

25) Has 3-hekusulose phosphate synthase activity.

26) Does not have hydroxypyruvate reductase activity.

0M33 is a Gram-negative motile bacillus with polar flagella that can only utilize methanol and monomethylamine as carbon sources, but cannot assimilate other carbon sources.

The ribulose monophosphate pathway is used as the methanol assimilation route, and the serine pathway is not used.

“BERGEYS MANUAL OFDETERMI
According to the 8th edition of NATIVE BACTERIOLOGYJ, this bacterial strain belongs to the genus Methylomonas.

All of the strains belonging to the genus Methylomonas described in the same document can utilize methane as a carbon source, but this strain differs greatly in that it cannot.

On the other hand, although it is not described in the same document, Methylomonas is a microorganism recently identified as belonging to the genus Methylomonas.
Methanorovolance (Ohno, Hamada, Takada, Terui, J, F
erment, Technol, 55.

295 (1977)), Methylomonas aminofascens (clamping force, Izumi, Kawamori, Asano, Tani, J.

Ferment, Technol, 55.
444 (1977)), Methylomonas methanlophylla (precious wood, Kuhn, Berglund, Unden, Beden, J., Ferment.

TechnoL55, 459 (1977)), Methylomonas methylopora (Kasho, Oki, Sealer, Ozaki, J, G
en, Appl, Microbiol, 19
, 11 (1973)), Methylomonas thalassica (Dashi et al. J, Ferment, Technol, 58,
99 (1980)), Methylomonas clara (Hornrosel et al., European J, Applied
Microbiol and Biotech
6.167 (1978)), Methylomonas M15 (Sahm, Wagner-1 European J,
Microbiology 2y147 (1975)
), but this strain has different properties from any of these strains.

That is, Methylomonas methanoloporans differs from this strain in that it produces water-soluble pigments and does not have the ability to assimilate methylamine.

Methylomonas aminofascens is identified as this strain because it produces acetoin (VP test), grows at 37°C, accumulates polybeta-hydroxybutyric acid, and does not assimilate methylamine. Unusual,
This strain differs from Methylomonas methanolophila in that it produces acetoin, grows at 37°C, and does not have the ability to assimilate methylamine.

Furthermore, Methylomonas Methylopora does not have the ability to assimilate methylamine, Methylomonas Salasica requires NaCV and vitamin B12 for growth, and 37℃
They differ in that they grow in water, have a low GC content of 43.8-47.3%, and have the ability to assimilate formaldehyde.

In addition, Methylomonas clara has the ability to assimilate methane and does not have the ability to assimilate monomethylamine.37
Methylomonas M15 differs from this strain in that it grows at 42°C and has a low GC content, and that it grows at 42°C and does not have the ability to assimilate methylamine.

Based on the above results, the vehicle was identified as a new bacterial species belonging to the genus Methylomonas, and was named Methylomonas thiomedogenes.

Mycological properties of FM518 (a) Morphology 1) Cell shape and size: Bacillus, 0.8-1.0×0
．． 8-3.0 μm 2) Presence or absence of cell pleomorphism: None 3) Presence or absence of motility, epiphytic state of flagella: Yes, polar flagella.

4) Presence, shape, size, and location of spores: None.

5) Durham staining: negative.

6) Anti-acidity: Negative.

(b) Growth status in each culture medium 1) Broth agar plate culture: moderate growth, circular, convex to rounded
Cushion-like, golden-edged, smooth, translucent to opaque, buttery gloss, homogeneous, orange to pink.

2) Broth agar slant culture: Moderate growth, thin film-like, filamentous, mixed light, orange to brick-colored.

3) Meat juice liquid culture: Moderate growth, not uniformly cloudy, with flaky precipitate.

4) Meat juice gelatin puncture culture: Does not liquefy.

5) Lydmus mill polysaccharide: No change.

(c) Physiological properties 1) Nitrate reduction: Yes.

2) Denitrification reaction: None.

3) MR test: None.

4) Do the VP test.

5) Production of indole: f, K L/.

6) Hydrogen sulfide generation: None.

7) Hydrolysis of starch: None.

8) Use of citric acid: Do not use in Koser medium.

Not available in C11ristensen medium.

9) Utilization of inorganic nitrogen sources: Utilize nitrates.

Use ammonium salts.

■ Formation of pigment: Not formed.

11) Urease: Yes.

12) Oxidase: Yes.

13) Catalase: Yes.

14) Range of growth: Temperature: Grows at 35°C and does not grow at 40°C.

pH5~9° 15) Attitude towards oxygen: aerobic.

16) 0-F test (according to Hugh & Leifson method) 20.

17) Production of acids and gases from sugars
Gas production L-arabinose - - D-xylose ± - D-glucose - D-mannose - - D-fructose - D-galactose - - Maltose - Yo sugar - Acid production Gas production Milk Sugar -- Tren 10-su -- D-Sorvit -- D-Mannit -- Inosit -- Glycerin + -- Starch -- 18) Decarboxylase reaction (MΦler's method): Lysine; None Ornithine; None Arginine; None 19) Arginine, dihydrolase reaction (Sta-ni
(according to the method of Er et al.): None.

20) Degradability of casein: Does not decompose.

21) Accumulation of poly-β-hydroxybutyric acid: Accumulates.

22) Auxotrophic requirement: None.

23) Assimilation of the following compounds (based on 5tanier medium) D-glucose + mesaconic acid - trehalose - erythritol - 2-keto-2,3-butylegluconic acid glycol - meso-inodite - meta-benzoic acid -L- Valine −
Para-benzoic acid - β-alanine - DL-arginine - Betaine + L-arabinose - Sucrose - Tryptamine - Propionic acid - α-amylamine Monoacid
-DL-lactic acid 10 attonits - D-fructose + malonic acid 10 ethanol + testosterone -D-xylose - cellobiose -D-lipose
- DL-β-''hydrooxyfidylate + L-rhamnose - L-histidine - formic acid - pantothenic acid - levulinic acid - acetic acid decacitraconic acid - succinic acid
Decafumaric acid + citric acid -D(-
) -Tartaric acid -L-ornithine -So, Yu, To-/
l/5-keto-gluconic acid mesaconic acid-L-lysine-top 2-p0''
10L-alane, di-7゜dulcit - methylamine + methanol + dimethylamine - glycerin
+ Ethanolamine -25) Hydroxypyruvate reductase: Positive.

26) 3-Hekyusulose phosphate synthase activity: negative FM518 is 1) a Durham-negative bacillus, 11) has a polar flagellum, grows only under aerobic conditions, 1v)
It is oxidase positive and therefore belongs to the genus Pseudomonas.

When searching for species, FM518 is M anua 1
of Determinative Bacteria
According to the 8th edition of Pseudomonas, it accumulates poly-β-hydrobutyrate, is not auxotrophic, and cannot grow at 41°C, so it belongs to the Pseudomonas genus section (2).

When comparing FM518 with the known bacterial species belonging to section ①, there are no known bacterial species that match in terms of carbon source assimilation pattern and colony color tone.

Therefore, according to the 8th edition, FM518 can be said to be a new bacterium, but in literature after the 8th edition was published, bacteria similar to FM518 in terms of the color tone of the colonies are known.

(P, rhodomethylo faciens,
P. On the other hand, there is controversy over the taxonomic treatment of methanol-assimilating bacteria.

Considering the above, we believe that it is appropriate to refrain from naming the species as a new species at this stage and hold off on naming the FM518.
was designated as Pseudomonas sp.

The method of causing the above-mentioned microorganisms to act on methanol or betaine and homocystine is to combine these raw materials and the cells of the above-mentioned microorganisms in an aqueous solution containing (1) methanol or betaine and (2) homocysteine. The contact may be carried out at a pH range of 4 to 10 and a temperature range of 15°C to 60°C.

If methanol, betaine, or homocystine as a raw material has a toxic effect on the microorganisms mentioned above, the concentration is adjusted to a low level that does not cause a toxic effect, by supplementary addition if necessary.

Further, more favorable results are often obtained by adding an antioxidant to the aqueous solution.

The above microorganisms are generally cultured using methanol as a carbon source, since they can assimilate methanol well, but when using a strain that can also assimilate other carbon sources such as glucose, may be used as a carbon source.

The medium used is a conventional medium containing, in addition to the above-mentioned carbon source, a nitrogen source, inorganic ions, and, if necessary, organic micronutrients such as amino acids and vitamins.

As the nitrogen source, ammonium ions, nitrate ions, etc. are preferable.

If the methanol concentration in the medium becomes too high, the growth of microorganisms will be inhibited, so supplementary addition may be made as appropriate.

The culture medium is preferably carried out under aerobic conditions, and during cultivation, p
More favorable results can be obtained by adjusting H5rj to 8 and keeping the temperature between 25 and 40°C.

To collect the L-methionine thus produced and accumulated in the aqueous solution, conventional methods such as methods using ion exchange resins can be used.

Example 1 (NH4)2HPO43,0,9/#, to 2HP0,2
．． 0g/111NaCl 109/13z M
g5o4+ 7 H200, 29/11 yeast extract 0.
59/l! 1 Methanol 109/111 FeCI3/6
H2O101n9. #.

MnCl2 ・4H2027Q, #, CuSO4・5
H5H2O1/l, Na2B407-10H200,
4 drops, # and (NH4)a Mo7024-4 H2
20 ml of culture medium containing 0.27 rvl and adjusted to pH 7.0 was placed in a 500 Tll shoulder flask and sterilized.

Pseudomonas 5pFE244 was inoculated into this, and 2
The cells were cultured at 8°C for 40 hours.

Bacterial cells were obtained by centrifugation and washed once with 0.1M potassium phosphate (pH 7.4).

KH2PO46,8m97m1. DL-homocystine 3,65 m9/ru11 methano-”32 μll
/rul in an aqueous solution (in a test tube) containing 3
Zero drops of the washed bacterial cells were suspended and adjusted to pH 7.4.

When this was shaken at 28℃ for 24 hours, 285μ
9/rnl! of L-methionine was produced.

Example 2 In the method shown in Example 1, instead of 32 p, 11/13 methanol in the aqueous solution, 23.41179/
When shaking for 8 hours using 189 μfl/ml of L-methionine was produced in the aqueous solution.

Example 3 In the method shown in Example 1, when washed bacterial cells obtained using betaine 109/l instead of methanol 109/l in the culture medium were used, 150 μg was obtained after 8 hours of reaction.
/ml of L-methionine was produced.

Example 4 In the method shown in Example 1, the microorganisms shown in Table 1 were cultured as washed bacterial cells, and 40η of this was used to incubate L-
When methionine was produced, the results shown in Table 1 were obtained.

However, the aqueous solution of DL-homocystine is 65~/ml.
And so.

Claims (1)

[Claims] 1 Pseudomonas, Microticulus or Methylomonas assimilate methanol and produce L- from homocysteine.
1. A method for producing L-methionine, which comprises producing L-methionine from (1) methanol or betaine and (2) homocysteine by the action of a microorganism capable of producing methionine, and collecting the L-methionine.