JPS6363377A - Bacterium ns303 of genus pseudomonas and production of l-amino acid - Google Patents

Abstract

PURPOSE:To separate and collect L-amino acid not to be influenced by bacterium NS303 (FERM P-8878) of genus Pseudomonas of novel strain in high purity and in high yield, by reacting DL-amino acid (salt) with the bacterium cell or a treated material of the cell. CONSTITUTION:Bacterium NS303 of genus Pseudomonas acting on a wide range of D-amino acids such as methionine, alanine, valine, phenylalanine, phenylgycine, lysine, etc., has the following mycological properties. (A) Morphological properties: having 0.6 - 0.7 X 1.5 - 2.0mum cell size, being a bacillus free from polymorphism, having mobility by flagellum, containing no spore and having negative Gram'stain free from acid-fastness. (B) Growth states in each medium as shown in the table. (C) Physiological properties: positive in reduction of nitrate, denitrification reaction, use of citric acid, use of inorganic nitrogen source, urease, oxidase and catalase, negative in MR test, VP test, formation of indole, formation of H2S and hydrolysis of starch and aerobic properties.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for enzymatically producing L-amino acids by allowing microorganisms to act on DL-amino acids or their salts.

(Prior Art) Conventionally, as a method for obtaining L-amino acids by reacting DL-amino acids with Pseudomonas aeruginosa, Biochem-Biphysica Acta has been used.
phys-Acta) Volume 78 136 Page 19
A method described in 1963 is known.

However, this method has problems in that the substrate specificity of the enzyme for DL-amino acids is narrow and the conversion rate for D-amino acids is slow.

[Means for solving problems]

The present inventors searched for microorganisms that have the ability to specifically act on D-amino acids to convert them into keto acids and do not act on L-amino acids, and found that they have excellent D-amino acid oxidase activity and We succeeded in isolating a new bacterium that has an enzyme with a wide range of substrate specificity, and after further investigation, we completed the present invention.

The present invention provides a method for producing L-amino acids by contact-reacting the microorganisms or their processed products with DL-amino acids or salts thereof, and then separating L-amino acids that are not affected by the microorganisms from the reaction product. It is.

Examples of the D-amino acids that the microorganism of the present invention specifically acts on include methionine, alanine, valine, phenylalanine,
A wide range of amino acids include phenylglycine and lysine.

The microorganism used in the present invention is Sheudomonas NS303 (AIST
No. 78), which is a new bacterial species newly isolated by the present inventors. The mycological properties of the NS303 bacterium are as follows.

(al form 1) Cell shape and size: 0.6-0.7 x 1.
5-2. θ μm Bacillus 2) Presence or absence of cell pleomorphism: None 3) Presence or absence of motility: Yes Flagella epiphytic state Polar flagella 4) Presence or absence of spores: None 5) Durham staining: Negative 6) Acid-fastness: None bl Growth status in each medium 1) Broth agar plate culture: Good growth, circular, convex circular, gold-rimmed, opaque, smooth, white 2) Broth agar slant culture: Good growth, does not produce filamentous soluble pigments 3) Flesh juice Liquid culture: Good growth, filmy 4) Meat juice gelatin puncture culture: Does not liquefy 5) Lidomus milk
: No change [C1 Physiological properties 1) Nitrate reduction +2) Denitrification reaction +3) MR test
-4) VP test
-5) Production of indole -6) Production of hydrogen sulfide
-7) Hydrolysis of starch
-8) Utilization of citric acid; Christensen medium: +
(Christensen) Simmons medium: + Koser medium: + (Xoser) 9) Utilization of inorganic nitrogen source; Nitrate: +7 Nitrium salt: +10) Production of pigment
Not produced 11) Urease
+12) Oxidase
+13) Catalase
+14) Growth range: Temperature: Grows at 38°C, but not at 42°C, pH: 6-9 15) Attitude towards oxygen: Aerobic 16) O-
F test oxidation 17) Formation of acid and presence of acid and gas from WilI Formation of gas fl) L-arabinose 10 - (2) D
-Xylose + (weak) -(3) D-glucose + -(4) D-mannose 10 -(5) D-fructose 10
-(6) D-galactose 10 -(7)
Maltose 10 - (8) Shi! FwM
10-(9) Lactose
+<'a weak) -01) Lebarose 10
-(11) D-Sorvit + (weak)
-(12) D-mannit + (weak) =
(13) Inonsoto 10 - (1
4) Glycerol + (weak) - (15) Starch - 18) Oxidation of gluconic acid - 19) Decarboxylase reaction Møller's method Lysine - Ornithine - Arginine + 20) Degradability of DNA -
21) (Presence or absence of accumulation of !I-β-hiFIll xybutyric acid:
Yes22) Auxotrophy No23) Assimilation of carbon compounds (Stanier et al.
al method) 1) L-arabinose
+2) D-xylose +3)
D-glucose +4) D-
Fructose +5) Shig Tong
+6) Trehalose
+7) D-Repose +
8) L-rhamnose +9) Atonite +10) Erythritol -11) Sorbitol
+12) Ethanol
+13) β-alanine +1
4) L-Arginine +15) L-
Valine-16) Butyric acid
-IT)DL-lactic acid
+18) Propionic acid 119) Mesotartaric acid -20)
D (-)Tartaric acid -21) m-hydroxybenzoic acid -22) p-hydroxybenzoic acid 123) Glycolic acid
-24) Malonic acid =
25) DL-β-hydroxybutyric acid 126) Levulinic acid -27) Citraconic acid -28) Mesaconic acid
-29) Triple min
-30) Propylene glycol +31) 2.3-butylene glycol +32) n-amylamine
-33) Betaine Bergey's Autumn Equinox H (Bergei')
sManual of Determinative
Bacteriology (8th edition), it is found that it belongs to the genus Pseudomonas, and when searching for species, it is found that known bacterial species and in the home country are consistent in terms of properties such as the presence or absence of decarboxylase reaction and the ability to assimilate carbon compounds. It was determined that it was a new bacterial species.

The above microorganisms are usually cultured using shaking culture or aeration.
Perform under aerobic conditions such as agitated deep culture.

Culture 11 degrees is 10-35℃, culture pH is 6-9, 1-
Incubate for 3 days. The medium contains carbon sources, nitrogen sources, inorganic salts, and trace organic nutrient sources that can be used by the bacteria used. That is, as a carbon source, carbohydrates such as glucose, fructose, starch hydrolyzate, and molasses, as well as ethanol, etc. can be used. Various inorganic and organic ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, or natural organic nitrogen sources such as meat extract, yeast extract, corn steep liquor, casein hydrolyzate, etc. can also be used as nitrogen sources. . As the inorganic salt, salts of magnesium, iron, manganese, potassium, sodium, etc. are used as appropriate.

There is no particular restriction on the concentration of the DL-amino acid that is the substrate used in the reaction, but a concentration of 1 to 69 (concentration) is usually used, the reaction temperature is 10 to 50 °C, preferably 30 to 45 °C, the reaction pH is 3 to 10, Preferably, the reaction time is in the range of 6 to 9 for 1 to 3 days.

To separate L-amino acids from the reaction solution, for example, concentration,
This can be carried out by a known method such as a direct crystallization method using isoelectric point precipitation or treatment with an ion exchange resin.

Quantification and quantification of the amino acids produced can be done using thin layer chromatography (TLC) or high performance liquid chromatography (HPL).
C), the method by Paio Fusei can be used. Optical isomers can also be determined by optical rotation analysis, using an optical isomer separation plate, and a column.

(Example) Example 1 Nutrient medium (pH 7) containing glucose 0.5χ, yeast extract 0.5χ, polypeptone 0.5χ
) Pseudomonas NS303 m was inoculated into 10 ml, and precultured by shaking at 30°C for 24 hours. Main culture was performed by adding 5 ml of the preculture solution to 500 ml of the above-mentioned nutrient medium and shaking at 30° C. for 24 hours. This medium 4!8 solution was centrifuged and seedlings were collected. To the obtained bacterial cells,
3% concentration of DL- adjusted to pH 8 with ION soda
Methionine (CLSCHzCLC)! (NHZ) COO
H] After adding 100ml and performing Qli, 160ml for 1 minute.
The reaction was carried out at 42°C for 2 days with rotational shaking. After the reaction was completed, bacteria were sterilized by centrifugation, and the supernatant was analyzed using HPLC and an optical isomer separation column (Chiral Pafuku H manufactured by Daicel Chemical Industries, Ltd.). analyzed.

輔CHsSC）IzCHzCOCOOII” Cl
After supplying the above supernatant adjusted to pH 3 to a Clost column packed with 5 g of 1i SCHzCl (zcOOH/ion exchange resin (Amberlite + R12OB)), the keto acid and propionic acid were filtered out by washing the column with water. Then, methionine was extracted with a 0.4 M?Ei aqueous ammonia solution. After that, the eluate? By condensation and drying, L-methionine 1,
2g was obtained.

[α]. ”-+23.6° (C-1,04,5N-
MCI) Example 2 Pseudomonas NS3 cultured in the same manner as in Example 1
After suspending the bacterial cells obtained from 5 ml of culture solution of 0.03 bacteria in 1 ml of 0.1 M phosphate buffer (pH 8), DL
-valine ((CH3)IC)IcH(NL)COO)I
) 0.01 g was added. After the 0 reaction, which was carried out by shaking at 42°C for 2 days, the supernatant obtained by centrifugal sterilization was
HPLC, chiral Bafuku H and optical isomer separation plate (chiral plate: Mack
As a result of analysis using a method (manufactured by Erey-Nagel-Duen), it was confirmed that the remaining valine was L-integrated, and D-integrated was not detected at all. The remaining amount of L-valine was 5 .mu./ml.

Example 3 Pseudomonas i NS cultured in the same manner as in Example 1
The bacterial cells obtained from 5+ml of culture solution of 303 bacteria were 0.1
After suspending in 1 ml of M concentration phosphate buffer (pH 8), DL-phenylalanine CC,H5CH,C)I(
0.01 g of NHffi)COOH) was added. 42℃
After the reaction was carried out by shaking for 2 days, the supernatant obtained by centrifugal sterilization was analyzed using IF'L11:, Chiral Pasok WH and Chiral Plate. As a result, the remaining phenylalanine was L-integrated and D It was confirmed that nothing was detected at all. The remaining amount of L-phenylalanine was 4.6 ml/m+.

〔Effect of the invention〕

According to the method of the present invention, by using Pseudomonas NS303 bacteria, L-amino acids can be obtained with high purity and high yield from various DL-amino acids.

Claims (2)

[Claims] (1) Pseudomonas NS303 bacterium that has the ability to convert D-amino acids to keto acids (2) Pseudomonas NS303 bacteria having the ability to convert D-amino acids into keto acids or a processed product thereof,
1. A method for producing L-amino acids, which comprises carrying out a contact reaction with DL-amino acids or salts thereof, and then separating L-amino acids that have not been affected by the above-mentioned microorganisms from the reaction product.