JP3390072B2 - Method for producing and purifying L-threo-hydroxyaspartic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antibacterial L-sulfate.
For production and purification of leohydroxyaspartic acid
Related. [0002] 2. Description of the Related Art L-Threoxyhydroxyaspartic acid
Is Earthrinium Phaeospam Speech (Art
hrinium phaeospermum  sp. )
T-53 and Streptomyces sp.Stre
ptomyces  sp. ) Raw by 7540-MC
To be performed [J. Antibiotics 23, 8
21 (1975)].
And [Bull. Chem. Soc. Japan 40,
2154 (1967)]. [0003] L-threo-hydroxyas from culture
The following are known as methods for purifying paraginic acid.
You. First, the culture filtrate was subjected to activated carbon and weak acid cation exchange.
Pretreatment with resin (Amberlite IRC-50)
You. Next, the eluate was washed with a strongly basic anion exchange resin (Do
Wex1 × 8 or IRA410)
Elute with HCl. This eluate is further subjected to an adsorption resin (Amb
erlite XAD-2). on the other hand,
Separately from this method, the eluate from IRC-50 is applied to a strongly acidic solution.
Ion exchange resin (Amberlite 1R-120)
0.3N-NH_FourEven if eluted with OH solution
It is said that it can be manufactured. [0004] SUMMARY OF THE INVENTION L-Threo-hydroxy
Siaspartic acid has an antibacterial effect and is a drug.
And what is expected as a pharmaceutical intermediate
Therefore, there is a demand for the development of a new and superior method for producing this substance.
You. As for the purification method, the conventional method requires a large number of steps.
And a long purification time, a simple method is desired.
ing. In addition, L-threo-hydroxyaspartic acid is used.
Activated carbon and weakly acidic cation exchange as described above
Pre-treated with resin and adsorbed on strongly acidic cation exchange resin 0.3
N-NH_FourWe repeated the method of elution with OH solution,
Many minutes were not purified. Also, aspartic acid and hide
If roxyaspartic acid is contained in the solution
Is 0.3N-NH after adsorption on a strongly acidic cation exchange resin._Four
Elution with OH also elutes aspartic acid,
Sheaspartic acid is not purified. [0005] [Means for Solving the Problems]Papull
aria) Genus, L-threo-hydroxyaspara
Microorganisms capable of producing formic acid, L-aspartic acid
Culture in a medium containing L-aspartic acid
In the form of L-threo-hydroxyaspartic acid.
It is found that it is generated conveniently. Further, L-threo-
How to purify hydroxyaspartic acid from culture solution
As a result of the examination, using a strongly acidic cation exchange resin, pH 3
Purification can be easily performed by elution with aqueous solutions of ~ 8.
I came out. That is, the first invention of the present application isP
apuliaL-threo-hydroxya belonging to the genus
Microorganisms capable of producing spargic acid are
By culturing in a medium containing paraginic acid, L-thread
Produce o-hydroxyasparagine and collect it
L-threo-hydroxyaspara
The present invention relates to a method for producing formic acid. Further, the second invention of the present application is a hydroxyas
Converting the culture filtrate containing paraginic acid into a strongly acidic cation exchange resin
Adsorbed and eluted with an aqueous solution of pH 3-8
To a method for purifying hydroxyaspartic acid. Than
More specifically, a solution containing hydroxyaspartic acid is strongly acidic
Contact with cation exchange resin, hydroxy asparagine
The acid is adsorbed on the resin and then eluted with neutral water,
It specializes in isolating hydroxyaspartic acid from effluent.
The present invention relates to the purification method according to claim 4. [0008] Papulia used in the present invention (Papu)
Laria) belonging to the genus L-threo-hydroxyaspa
One example of a bacterium producing laginic acid is Papraria Arun.
Dinis (Papulia arundines)
NF00779. Papallia Arundini below
Su (Papulia arundines) NF0
This shows the bacteriological properties of the 0779 strain. 1. Potato dextrose agar (2
(5 ° C), very good growth in 3 days,
Reaches 2-54 mm. The surface of the settlement is wool to fibrous
Aerial mycelium grows and becomes white to light brown depending on the number of days of culture.
You. Spread a black conidium mass on the aerial mycelium, and then
Form conidia. The back surface has a light brown color. Good growth on 2.2% malt extract agar medium (25 ° C)
In three days, the diameter of the settlement reaches 43 to 44 mm. Collection
A wool-like aerial mycelium grows on the surface of the drop and depends on the number of culture days.
It becomes white to white-grey / brown. Conidium formation is good
Good. The back has a light brown color. 3. Growth on Tuapec agar medium (25 ° C) is slightly bad
In three days, the diameter of the settlement reaches 15-27 mm. Collection
The surface of the fall becomes white by the formation of wool-like aerial hyphae. Separation
Good child formation. 4. It grows well on cornmeal agar (25 ° C)
In three days, the diameter of the settlement reaches 34-36 mm. Of the village
The aerial mycelium spreads thinly on the surface, and conidia formation is good. [0010] 5. The fungus is potato dextrose agar
It grows quickly on the medium and has a large conidial growth on the entire surface
You. The mycelium is colorless to brownish, the wall has a smooth surface and septum.
1.6-4.0 μm. Conidium cells are colorless to brownish,
Flask type to ampule type, 4.5 to 8.0 x 3.2 to
5.2 μm. Conidia are formed from the top. Conidium pattern
Is colorless, slender cylindrical, smooth, partition walls are irregular, length 6
Up to about 0 μm, diameter is about 0.8 μm. The conidium is Vasoji
Type conidia, lens shape, surface is smooth, light brown, both sides
The joint is colorless, the diameter of the front circle is 4.8-7.2 μm,
The minor axis of the lateral spindle is 2.8 to 4.8 μm
Along the entire surface. Optimal growth temperature
The temperature is around 25 ° C and does not grow at 37 ° C. Optimal growth p
The range of H is 6.0 to 8.0. 6. Based on the above mycological properties, this bacterium is "Ai
nsworth and Bisby's Dicti
onary of the Fungi "(by D.E.
L. Hawksworth, B .; C. Sutton a
nd G. C. Ainsworth, 7th ed. ,
C. M. I. , Kew, 1983), according to the fungal phylum
Complete subphylum, Paplaria of incomplete filamentous fungi (Papull
aria) A genus was identified. Further species search results
As a result, paprialia Arundinis (Papulia
arundinis)
Fungus on paparia arundinis (Papulia
arundinis) And identification papriaria arundini
Su (Papulia arundinis) NF0
0779. The strain is manufactured by the National Institute of Advanced Industrial Science and Technology
With the accession number FERM P-13773 to the National Institute of Advanced Industrial Science and Technology
And was deposited. In the present invention, a strongly acidic cation exchange resin is used.
Therefore, a sulfonic acid-based cation exchange resin is preferable.
Anything that has a sulfonic acid group as an on-exchange group
Resin is polystyrene, phenol, etc.
Although not limited, polystyrene resin is generally used.
Preferably, the degree of crosslinking is about 5 to 15%. Ingredient
Physically Dowex 50W (trade name, Dow), Amba
-Light 120B (trade name; ROHM and Haas) etc.
It is. In the present invention, an aqueous solution having a pH of 3 to 8 is used.
Is pH adjusted with neutral or hydrochloric acid, etc.
Is about 0.01 to 1 M NaCl, Na_TwoSO_FourEtc. salt
An aqueous solution containing In the present application, neutral water is preferably used.
Good. According to the present invention, L-aspartic acid is hydroxylated.
For producing L-threo-hydroxyaspartic acid
Describes a microorganism capable of producing this substance as L-aspara
Culture in a medium containing formic acid to produce and store this substance in the culture.
You can pile it and then collect it. Culture method is original
In principle, follow the culture method for filamentous fungi, but usually use liquid culture.
A submerged culture method is advantageous. Medium used for culture
In addition to L-aspartic acid,
Ndinnis (Papulia arundini
s) In the medium containing the nutrient sources utilized by NF00779
I just need. As a nutrient source, it has traditionally been useful for culturing filamentous fungi.
Well-known materials used can be used.
Glucose, mannitol, dextrin, starch, water
Candy (starch malt saccharified product), soybean oil, etc., alone or in combination
Can be used. As a source of inorganic and organic nitrogen
Is ammonium chloride, ammonium sulfate, urea, nitric acid,
Ammonium, sodium nitrate, peptone, meat extract, yeast
Extract, dried yeast, corn steak liquor, soybean oil
Cass, soybean meal, oatmeal, casamino acid,
Bacto soyton, solvable vegetable protein, etc.
It can be used alone or in combination. Other required
Salt, magnesium sulfate, copper sulfate, zinc sulfate,
Inorganic salts such as manganese chloride, calcium carbonate and phosphate
Organic substances that can be added and promote the growth of the fungus
Substances such as nucleic acids, vitamins and inorganic substances
Can be In the present invention, the addition of L-aspartic acid
The amount is not particularly limited as long as it does not inhibit bacterial growth.
About 0.01 to 10 w / w% of the total amount of the medium,
Practically, about 0.05 to 5 w / w%, preferably 0.1 to 5 w / w%.
It is about 1-3 w / w%. Addition of L-aspartic acid
May be stored in the initial medium, or may be
May be added in portions during the culture. A in the medium
The concentration of sparagic acid is preferably 0.01 to 2 w / w%, preferably
Is controlled to be about 0.02-1.2w / w%
Preferably. The culture temperature is 20 to 35 ° C., preferably 25 to 35 ° C.
Cultivation is performed at 30 ° C. and a pH of 5 to 9, preferably 6 to 8.
It is desirable to do. In liquid culture, culture for 1-3 days
Sea urchin L-threo-hydroxyaspartic acid in the culture
Is accumulated in When the amount of production in the culture reaches the maximum
Stop the culture and filter the cells from the culture broth.
The target is purified and isolated. For purification and isolation of this substance from culture filtrate
Is the adsorption / desorption method using adsorption resin or activated carbon, ion exchange
Resin, Sephatex, silica gel column chromatography
Use a suitable combination of methods such as photography
Can be. In the present invention, the purification step and the purification
For the purpose of shortening the time required, strongly acidic cation exchange resin
Is preferred. Hydroxyasparagine
Contacting a solution containing an acid with a strongly acidic cation exchange resin,
For example, the solution is passed through a column filled with the resin,
Adsorb spargic acid, then aqueous solution of pH 3-8
Preferably near neutral water, for example, pH 5.5 to 7.5
(E.g., distilled water).
Concentration of sparaginic acid or addition of insoluble solvent
Method, the purified hydroxyaspara
Formic acid can be obtained. Hydroxyasparagi treated with the resin
If the solution containing the acid is colored,
Chromatography for decolorization and decolorization, activated carbon treatment, etc.
, And then the resin. For example
More specifically, first, the culture filtrate is treated with activated carbon or the like.
Decolorization, highly unsaturated substances and fat-soluble
Substances were excluded. Next, this eluate is used as a sulfonic acid-based resin.
Cation exchange resins such as Dowex 50
W (trade name; Dow) at 10 to 40 ° C, preferably at room temperature
At this temperature, an aqueous solution having a pH of 3 to 8, preferably medium
It can be easily purified simply by eluting with water. Then get
The resulting aqueous solution is concentrated under reduced pressure to methanol or ethanol.
The precipitated crystals may be collected by filtration. Activated carbon
Even if the order of treatment and strongly acidic cation exchange resin is reversed
The purification effect is achieved, but the activated carbon treatment is performed first.
Is preferred. An embodiment of the present invention will be described below. 【Example】 Example 1 Glue into a 500 ml Erlenmeyer flask for rotary shaking.
Course 1.0%, shoe close 2.0%, soybean
Meal 2.0%, potassium dihydrogen phosphate 0.1%, sulfuric acid
0.025% of magnesium and 0.6mg of iron sulfate, sulfuric acid
4.1 mg of copper, 0.8 mg of zinc sulfate, manganese chloride 5.
100 ml of a medium containing 0 mg (pH 6.3) was dispensed.
Autoclaved at 120 ° C for 20 minutes. To this
Pralia Arundinis (Papulia aru
ndinis) NF00779 strain (Institute of Industrial Science and Technology, Biotechnology)
Industrial Technology Research Institute accession number FERM P-13773)
Inoculate one platinum loop and shake at 27 ° C, 220 rpm for 3 days.
I was sorry. Separately, in an Erlenmeyer flask similar to the above,
Dispense 100 ml of the same medium (pH 6.3),
L-aspartic acid is 0.1, 0.2, 0.4, 0.8
%, And autoclaved at 120 ° C for 20 minutes.
Was sterilized. 2 ml of the culture was transplanted into the
The cells were cultured for 48 hours at 220 ° C. at 220 ° C. culture
The supernatant obtained by centrifugation from the solution was Nimura's method
[J. Chromatogr. , 352, 169-17
7 (1986)] with the OPTA-AcCys reagent.
L-threo-hydroxyasparagine produced and processed
After introducing the acid to the fluorescent substance, HPLC fluorescence analysis was performed. Attachment
The amount of L-aspartic acid added and the L-threo-
Table 1 summarizes the amounts of hydroxyaspartic acid. HP
The LC conditions are shown below. [0020] Column: Unisil Pack 5C18, 6.0 × 250
mm, 40 ° C Solvent: 50 mM sodium acetate, 50 mM phosphoric acid
-Sodium hydrogen / tetrahydrofuran / methanol
(96/2/2) Flow rate: 0.8 ml / min. Detection: excitation wavelength 350 nm, fluorescence wavelength 405
nm [0021]     Table 1 L-threo-hydroxya formed relative to amount of added L-aspartic acid           Spartic acid content             L-aspartic acid L-threo-hydroxyaspartic acid                   (%) (Ppm)                   0.0440                   0.1 570                   0.2 740                   0.4 1010                   0.8 1000 Embodiment 2 Glue into a 500 ml Erlenmeyer flask for rotary shaking.
Course 1.0%, shoe close 2.0%, soybean
Meal 2.0%, potassium dihydrogen phosphate 0.1%, sulfuric acid
0.025% magnesium, and 0.6 mg iron sulfate,
4.1 mg of copper sulfate, 0.8 mg of zinc sulfate, manganese chloride
100 ml of a medium (pH 6.3) containing 5.0 mg
The mixture was poured and autoclaved at 120 ° C. for 20 minutes. this
In the papriaria Arundinis (Papulia
arundinis) NF0779 strain (Graduate School of Industrial Technology)
Life Science Industrial Technology Research Institute accession number FERM P-1377
3) One platinum loop was inoculated and 27 ° C, 220 rpm, 3 days
Shake. Separately, 10L jar fermen
6 L of the same medium as above (pH 6.3)
Was dispensed. Furthermore, L-aspartic acid is added to each.
0.4% at 120 ° C for 20 minutes
It was sterilized in a Toclave. To this was added 30 ml of the above culture solution,
° C, 350 rotations / minute, 2/3 VVM (4 L / min.)
For 48 hours. Filtrate the culture solution and filtrate 1
4.5 L were obtained. Pass the filtrate through 1.2 L of activated carbon
The excess liquid was added to Dowex 50W (H⁺) Adsorb to 3.2L
After that, elution was performed with 20 L of distilled water. Concentrate the eluate under reduced pressure
Dry and then use 50 ml of water-ethanol (1: 1)
The powder was collected to obtain 17.4 g of a white powder. (Purity 82
%). From the IR spectrum, the substance was identified as L-threo-hydro
It was identified as xyaspartic acid. In addition, it was added to the medium.
L-aspartic acid was not eluted. [0024] According to the present invention, as a pharmaceutical and a pharmaceutical
L-threo-hydroxyaspara useful as an intermediate material
Formic acid was obtained in good yield. Activated carbon and strong acid yang
Purification process by simple purification method combined with ion exchange resin
In addition, the purification time was significantly reduced. In addition, L
-Purification was good even with the addition of aspartic acid.
Was.

──────────────────────────────────────────────────続 き Continuation of front page (56) References ATCC CATALOGUE OF FUNGI / YEASTS, 16th edition (1984), p. 199 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12P 13/00-13/24 BIOSIS / WPI (DIALOG)

Claims (1)

(57) Claims: 1. A microorganism belonging to the genus Papulia and having the ability to produce L-threo-hydroxyaspartic acid is cultured in a medium containing L-aspartic acid. -Threo-hydroxyaspartic acid is produced and collected.
-A process for producing threo-hydroxyaspartic acid.