JPS5816878B2 - Hatsukouhou Niyor L- Serin no Seizouhou - Google Patents

Description

[Detailed Description of the Invention] The present invention involves culturing microorganisms capable of assimilating methanol using methanol as a carbon source in a glycine-containing medium, accumulating L-serine in the culture solution, and recovering this. The present invention relates to a method for producing L-serine characterized by the following.

Conventionally, L was cultured in a medium containing glycine and
- Methods for producing and accumulating serine include Brevibacterium (Japanese Patent Publication No. 45-11114), Corynebacterium (Japanese Patent Publication No. 47-38994), Arthrobacterium, Visia, or Candeida (Japanese Patent Publication No. 46-1972)
Although methods for culturing microorganisms such as those described in Japanese Patent Publication No. 32793 are known, the production of L-serine from glycine by microorganisms that assimilate methanol is completely unknown.

The present inventors have previously isolated a large number of methanol-assimilating microorganisms that assimilate methanol and exhibit vigorous growth, and have conducted various fermentation studies on strains that were recognized as new bacterial species. Focusing on the fact that many of these bacterial strains have serine hydroxymethyltransferase activity, which converts glycine to L-serine in methanol metabolism, we are searching for microorganisms that have the ability to produce and accumulate L-serine from glycine. I did it.

Among them, the genus Aeromonas
s), Achromobacter spp.
The present invention was completed by discovering a new fact that microorganisms belonging to category r) have such abilities and are excellent.

The microorganisms used in the present invention belong to the genus Aeromonas and Achromobacter, and have the ability to produce L-serine from glycine.
s me thanoph i luminov, sp
, ) R-1014 (Feikoken Bibori No. 2808), Aeromonas meth
a-nocola nov, sp, ) R-1332
(Feikoken Bibori No. 2809), Achromobacter methanoloxidans
ethanolo-xidans nov, sp,)
There is R-1013 (Feikoken Bibori No. 2693).

All of these strains are entrusted to the Microbial Technology Research Institute (Japanese Patent Application No. 138534-1983,
-112554, patent application No. 49-112555).

The mycological properties of these bacteria are as follows.

Aeromonas methanophyllum R-1014 Morphological properties (1.5% methanol broth agar plate 32℃, 24
Time culture) Pattern: Short bacilli, single or 2-3 linked, with single pole flagella.

Size 20, 5 to 0.7 x 0.8 x 1.3 microns Motility: Motile Durham staining: Negative Acid-fast staining: Negative Spores: Not formed Cell pleomorphism: None 03) Culture properties 1.5% methanol gravy agar Colony grows well, round, smooth surface, ridges, gold edges, glossy, cream color, buttery, no pigment formation.

Good colony growth on gravy agar, circular, smooth surface, ridges, gold edges, glossy, pale skin color, somewhat viscous, no pigment formation 61.5% methanol synthetic agar Colony growth is good, round, smooth surface, ridges, gold edges, glossy , white cream color, buttery, non-pigment forming.

1.5% methanol gravy agar Slope growth is good, filamentous growth, glossy, pale skin color, no pigment formation.

Good growth on gravy agar slant, filamentous growth, glossy, pale skin color, no pigment formation.

Good growth on 1.5% methanol synthetic agar slope, filamentous growth, glossy, pale skin color, no pigment formation.

1.5% methanol synthetic liquid medium Growth is good, white film formation, white precipitate, liquid is cloudy, no pigment formation.

Good growth of meat juice, white film formation, white closed ring, sediment, liquid is cloudy, no pigment formation.

Flesh gelatin puncture culture Upper surface ash cream, pale skin-colored growth at the puncture site, gas generation at the puncture site, no liquefaction.

(Q Physiological properties (test medium contains 1.5% methanol).

Grows at growth temperature = 19°C to 42°C.

It does not grow at 45°C.

Growth pH: 5-9 Oxygen requirement: Aerobic when methanol is used as a carbon source.

Facultatively anaerobic when using glucose as the carbon source.

OF test (Pureifson medium): Acid is also produced anaerobically through fermentation.

Gas evolution (glucose medium): Gas evolution occurs.

IJ l-mass milk: Good growth, forming white closed rings.

Peptonize.

Lidmus is bleached.

No milk curdling. Gelatin liquefaction: Does not liquefy.

Formation of hydrogen sulfide: Produces.

Decomposition of starch 2. Decompose.

Indole generation: Not generated.

Reduction of nitrate: produces nitrite.

Catalase activity: Positive oxidase activity: Positive urease activity: Positive VP reaction: Negative MR test: Negative Ammonia production: Positive Nitrogen availability: Uses ammonium salts, nitrates, urea.

Denitrification reaction: Yes Use of citric acid: Use Koser medium.

Christensen's medium is utilized.

Salt tolerance: Grows up to 6%.

Carbon source availability, acid, and gas production Note: Although acid production is observed, the pH indicator is decolored as the culture progresses.

Isolation source: Seawater Aeromonas meta/': 1-Ra R-1332 (a) Morphological properties (1,5% methanol broth agar plate 32℃, 2
Cultured for 4 hours) Bacteria Form: Short rod, single or 2-3 chains, with 2-4 pole flagella.

Size: 20.4-0.7 x 0.8-1.2 microns Mobility: Motile.

Durham stain: negative Acid-fast staining: negative Spores: Not formed.

Cell pleomorphism: None.

(B) Culture properties: 1.5% methanol broth agar Colony grows well, round, smooth surface, raised center, golden rim, glossy, cream color, buttery, no pigment formation.

Good growth of gravy agar colonies, round shape, smooth surface, raised center, golden edges, glossy, cream color, buttery texture, no pigment formation.

1.5% methanol Synthetic agar Colony grows well, round, surface smooth, flat with no bulges, slightly wavy around, gray cream color, glossy, buttery, no pigment formation.

1.5% methanol meat juice Good growth on agar surface, filamentous growth, glossy, gray cream color, no pigment formation.

Good growth on gravy agar slope, filamentous growth, glossy, off-white color, no pigment formation.

Good growth on 1.5% methanol synthetic agar slope, filamentous growth, glossy, gray-cream color, no pigment formation.

Good growth in 1.5% methanol synthetic liquid medium, white film formation, white precipitate, cloudy liquid, no pigment formation.

Good growth of meat juice, slightly white closed ring, formation of film, cloudy liquid,
No pigment formation.

Meat juice gelatin puncture culture At the puncture site, gray-cream colored cotton-like growth with liquefaction.

(C) Physiological properties (methanol 1.5% in test medium)
) Growth temperature: Grows at 13°C to 39°C.

It does not grow at 43°C.

Growth p) (:5-9 Oxygen requirement: aerobic when methanol is used as a carbon source,
Facultatively anaerobic when using glucose as the carbon source.

OF test (Huleifson medium): Acid is also produced anaerobically through fermentation.

Gas generation (glucose medium): No gas generation.

Lidomus milk: Good growth, forms white closed ring, peptonizes.

Lidmus is bleached.

Milk curdles. Gelatin liquefaction: liquefy.

Formation of hydrogen sulfide: Produces.

Starch decomposition and two decomposition.

Indole generation: Not generated.

Nitrate reduction: produces nitrite.

Catalase activity: Positive oxyguse activity: Positive urease activity: Positive VP reaction: Positive MR test: Negative Ammonia production: Positive Nitrogen availability: Utilizes ammonium salts, nitrates, and urea.

Denitrification reaction: Yes.

Use of citric acid: Use Koser medium.

Christensen's medium is used.

Grows up to 8% salt tolerance.

Carbon source availability, acid, and gas production Note: Although acid production is observed, the pH indicator is decolored as the culture progresses.

Isolation source: Seawater Achromobacter methanol oxidans-1013 (5) Morphological properties (cultivated on 1.5% methanol broth agar plate at 32°C for 24 hours) Bacterial shape: Short rod, no long cells observed. ).

Single or in chains of two. No pleomorphism.

No flagella. Size 2 0.5-0.8 micron x 0.9
~1.2 microns Motile: non-motile Duram staining Nigeram negative Acid-fast staining: negative spores: none B) Cultural properties 1.5% methanol broth agar Colony grows well, round, surface smooth, flat ridges, whole Spelled, greyish-yellow, glossy, buttery.

1.5% methanol Synthetic agar Colony growth is good, round, surface smooth, raised, all bound, slightly skin-colored gray cream color, glossy, buttery.

1.5% methanol Meat juice Good growth on agar surface, filamentous, glossy, grayish-brown. Growth on 1.5% methanol synthetic agar slope, filamentous, glossy, slightly skin-colored, gray-cream color.

1.5% methanol synthetic liquid medium No film formation was observed.

The liquid becomes cloudy. There is a slightly skin-colored sediment.

gravy No film is formed.

The liquid is not cloudy. There is a gray cream colored sediment.

Gravy agar slope Good growth, grayish-brown cream color, filamentous growth.

(C) Physiological properties (methanol 1.5% in test medium)
) Growth temperature: Good growth at 20°C to 40°C.

It does not grow at 43°C.

Growth pH: Grows at pH 6-9.

Oxygen requirement: aerobic IJ Thomas Milk: Forms a slightly white pedestal.

No peptonization occurs, but milk coagulation is observed.

The liquid is purple, but it is not alkaline. stomach.

Gelatin liquefaction: Does not liquefy.

Formation of hydrogen sulfide: Produces.

Does not decompose or decompose starch.

Indole generation: Not generated.

Nitrate reduction: No nitrite formation.

Catalase activity: positive Urease activity: negative vP reaction: negative MR test: negative Ammonia production: negative Availability of nitrogen sources: Use ammonium salts and nitrates.

Usability of citric acid: Not used.

Salt tolerance: Grows up to 4%.

Utilization of carbon source, acid production Growth Acid production methanol 10 - Ethanol - The fermentation medium used in the present invention contains methanol as the main carbon source, and further contains a conventional nitrogen source, inorganic salts, etc. and if necessary. A medium containing trace organic nutrients.

Methanol is used as a carbon source, but if the concentration of methanol is too high, it will inhibit the growth of microorganisms, so the concentration during culture should not be too high.

Therefore, although it is possible to terminate the culture using only the initially added methanol, better results can be obtained by starting with a low concentration and replenishing methanol as it is consumed.

Good results are usually obtained by keeping the concentration at 1-3% (V/V).

As the nitrogen source, ammonium sulfate, ammonium chloride, ammonia, ammonium phosphate, urea, ammonium nitrate, sodium nitrate, etc. used in ordinary fermentation are used.

Inorganic salts include potassium phosphate, magnesium sulfate, phosphoric acid,
In addition to potassium chloride and sodium chloride, iron, manganese,
Addition of salts such as zinc and calcium is effective.

As trace organic nutrients, it is effective to add vitamins such as vitamin B1 and biotin, or soybean protein hydrolyzate containing them, cane molasses, cornstarch liquor, yeast extract, etc., especially if the bacteria used If the substance is auxotrophic, it is necessary to add an appropriate amount of the auxotrophic substance.

Glycine may be added to the medium in advance, or may be added at the beginning of fermentation, or during fermentation.

Furthermore, if necessary, adding a small amount of antibiotics such as tetracyclines during the culture will increase the production of L-serine.

The conditions for main fermentation are good aeration and stirring, and the fermentation temperature is 25-3.
Fermentation time is usually 2 to 5 days at 8°C.

The pH at the start of fermentation and during culture is preferably 5.0 to 9.0. To adjust the pH, use inorganic and organic acidic and alkaline substances, pH buffers such as phosphate buffer, calcium carbonate, etc. can be used.

The monoserine produced in the culture solution may be recovered by a combination of the usual ion exchange resin method and other known methods.

The obtained L-serine was confirmed and quantified by conventional paper chromatography method and microbial quantification method.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but these Examples are merely illustrative and do not limit the present invention in any way.

Example 1 Ammonium sulfate 03%, urea 0.1%, monopotassium phosphate 0.2%
, potassium phosphate 0.7%, magnesium sulfate heptahydrate 0.05%, ferrous sulfate heptahydrate 0.003%, manganese sulfate tetrahydrate 0.004%, sodium chloride 0.01
%, yeast extract 0.1%, calcium carbonate 20%, pH
30 ml of an aqueous medium consisting of 7,0 was dispensed into a 500 ml Sakalo flask, sterilized at 110°C for 10 minutes, and 3.0% (v/v) of methanol was added to incubate each of the strains shown in Table 1. I made platinum ear fluid seeds.

Glycine was added at two points: at the start of culture and at 24 hours after culture, and the amount added was fixed at 0.5%.

When these were cultured with shaking at 32°C for 3 days, accumulation of L-serine as shown in Table 1 was observed.

Example 2 Achromobacter methanoloxidans R-101
3 strains were used, and 0.5 glycine was added to the same medium as in Example 1.
% was added at the start of the culture.

In addition, various antibiotics were added at the start of culture or 24 hours after culture, and culture was carried out under the same conditions and method as in Example 1. Significant accumulation of L-serine was observed in the lacyclines shown in Table 1.

In flasks without added glycine, L-serine is 0.
03 f? /l l.

Or did not accumulate.

Example 3 Ammonium phosphate 1.2%, magnesium sulfate heptahydrate 0.05%, ferrous sulfate heptahydrate Q, 003%, manganese sulfate tetrahydrate 0.004%, calcium chloride 2 Water salt 0.005%, sodium chloride 0.01%, cornstarch liquor 0.05%, yeast extract 0.01%, °
Pour 30 ml of an aqueous culture medium containing 1.0% glycine and pH 7.0 into a 500 ml Sakalo flask and heat at 110°C.
After sterilization for 10 minutes, 2.0% (V/v) of methanol was added to Achromobacter methanoloxidans R-1.
One platinum loop of 013 was inoculated.

After culturing with shaking at 32°C for 24 hours, methanol was further added for 2.
0% (v/v) and at the same time 10μ of tetracycline hydrochloride? /ml was added, and shaking culture was continued for 3 days from the start of culture.

The pH during the culture was adjusted to a range of 6.5 to 8.0 by adding ammonia water, and the pH was 2.6 Vt at the end of fermentation.
of L-serine was produced and accumulated.

1 ton of the fermentation-finished liquid was centrifuged to remove tooth bodies, and the supernatant liquid was passed through a column filled with DOWEX 50 (H type 10) resin to adsorb L-serine to the resin. After washing with water, it was washed with ammonia water. By eluting and concentrating the L-serine fraction, 2.12 crude crystals of L-serine were obtained.

Claims (1)

[Claims] 1. Genus Aeromonas
), Achromobacter genus (genus Achromo
bacter), which has the ability to assimilate methanol, is cultured in a glycine-containing medium using methanol as a carbon source, and L-serine is produced and accumulated in the culture solution,
A method for producing shiichiserine, which is characterized by recovering this.