JPH0342879B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-serine. L-serine is a substance useful as a raw material for pharmaceuticals, foods, or chemicals. It has been known that enzyme preparations extracted from bacteria belonging to Alcaligenes faecalis produce trace amounts of L-serine from β-hydroxyaspartic acid [J. of Biological Chemistry,
Vol244.P353-358 (1969)]. There is always a need for new methods for producing L-serine. As a result of various studies on methods for efficiently producing L-serine from β-hydroxyaspartic acid, the present inventors found that a culture, bacterial cells, or bacteria of a microorganism that has the ability to convert β-hydroxyaspartic acid to L-serine was developed. It has been discovered that L-serine can be efficiently produced by allowing a body-treated product to act on β-hydroxyaspartic acid. The present invention will be explained in detail below. The microorganisms used in the present invention belong to the genus Achromobacter, Aeromonas, Bacillus, Brevibacterium, Titrobacter, Corynebacterium, Erwinia, Pseudomonas, or Vibrio, and contain β-hydroxyaspartic acid. Any microorganism that has the ability to convert L-serine can be used. A specific example is Achromobacter Cycloclastus
ATCC21921, Aeromonas punctata
ATCC11163, Bacillus subtilis IFO3022,
Brevibacterium imariophylium
ATCC14068, Brevibacterium sterolicum ATCC21387, Titrobacter freundei
ATCC6750, Corynebacterium glutamicum
FERM−P3295, Erwinia herbicola
ATCC21434, Pseudomonas pseudoalcaligenes ATCC17440, Pseudomonas chlorolahuis ATCC9446, Vibrio tyrosinaticus
ATCC19378 is mentioned. As a method for allowing these microorganisms or their processed products to act on β-hydroxyaspartic acid, these microorganisms may be cultured in a fermentation medium containing β-hydroxyaspartic acid, or the bacterial cells of these microorganisms may be cultured in a fermentation medium containing β-hydroxyaspartic acid. Alternatively, the treated bacterial cells may be brought into contact with β-hydroxyaspartic acid in an aqueous solution. Among the above methods, β-hydroxyaspartic acid may be added to the medium from the beginning of the culture head, or it may be added all at once or in portions during the culture to produce L-serine in the fermentation liquid. . As a medium used for culturing these microorganisms, a medium containing a usual carbon source, nitrogen source, and inorganic ions is used. Furthermore, if necessary, vitamins, amino acids (alanine, aspartic acid, glutamic acid, etc.), nucleic acid-related substances, and substances containing these are added to the medium. If the bacteria used exhibit nutritional requirements, an appropriate amount of the required substance is added. Carbon sources include glucose, sucrose,
Fructose, mannose, molasses, starch, starch hydrolyzate, acetic acid, propionic acid, formic acid, methanol, ethanol, etc., which can be assimilated by the bacteria used, can be used alone or in combination. As the nitrogen source, ammonia gas, aqueous ammonia, urea, various ammonia salts, various nitrates, and amino acids (alanine, aspartic acid, glutamic acid, glutamine, proline, methionine, glycine, lysine, etc.) are used. As the inorganic ion, salts of magnesium, potassium, iron, manganese, zinc, nickel, cobalt, etc., and salts of phosphoric acid, sulfuric acid, and nitric acid can be used. Cultivation is carried out under aerobic conditions. The culture temperature is 20
The culture is carried out at ~40°C and the pH of the culture medium is adjusted to 4-10. β-hydroxyaspartic acid is converted to L-serine after 5 to 70 hours of culture. In addition, in the case of a method in which microbial cells or a treated product thereof is brought into contact with β-hydroxyaspartic acid in an aqueous solution as an enzyme source, a reaction solution containing β-hydroxyaspartic acid and bacterial cells or a treated product of bacterial cells is mixed for 10 to 10 minutes. Allow to react by standing or stirring while adjusting the temperature within the 70°C range. At this time, as the bacterial cells, a culture solution containing the bacterial cells may be used as is. Examples of processed bacterial cells include mechanically ground bacterial cells, ultrasonicated bacterial cells, freeze-dried bacterial cells, acetone and other solvent-treated bacterial cells, oxygen-treated bacterial cells, freeze-dried bacterial cells, and surfactant-treated bacterial cells. For example, immobilized products of microbial cells, protein fractions of microbial cells, microbial cells and bacterial cell extracts, etc. are used. In order to obtain bacterial cells with high activity of exchanging β-hydroxyaspartic acid to L-serine, β
-hydroxyaspartic acid, aspartic acid,
Fumaric acid, malic acid, etc. are often added. As for the basic composition of the medium and the culture method, the usual methods described above are employed. Antioxidants, surfactants, coenzymes, solvents, and amines may be added to the reaction solution for converting β-hydroxyaspartic acid to L-serine as necessary. In addition, as the raw material β-hydroxyaspartic acid, L-erythro form, L-
There are four possible stereoisomers: threo, D-erythro, and D-threo, and they can be used alone or in combination depending on the bacteria used. After 5 to 100 hours of culture or reaction, a large amount of L-serine is produced in the reaction solution. As a method for collecting L-serine thus obtained from the culture solution or reaction solution, known methods such as a method using an ion exchange resin are used. Example 1 Ammonium fumarate 0.25 g/dl, sodium fumarate 0.5 g/dl, peptone 1.8 g/dl, corn steep liquor 1 g/dl, KH ₂ PO ₄ 0.05
Bacillus grown on bouillon agar medium was placed in a 300 ml Erlenmeyer flask containing 40 ml of a medium with the following compositions: g/dl, MgSO ₄ 7H ₂ O 0.01 g/dl, and L-aspartic acid 0.1 g/dl (PH 7.0). Plumis
Contact one platinum loop of ATCC14884 bacterial cells and heat at 30°C.
Shaking culture was performed for 24 hours under shaking conditions of 210 rpm. The obtained bacterial cells were centrifuged and an additional 0.9%
After centrifugation washing twice with NaCl solution, add 50mM
Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (PH6.3) and 1% DL
- after reaction involving threo-hydroxyapartic acid
10 ml was added and shaken for 20 hours in the same manner as above. An average of 1.6 mg/ml of L-serine was produced in the reaction solution. After the reaction is complete, reaction solution 1 is collected, the bacterial cells are removed by centrifugation, and the supernatant is filtered using Dowex 50.
(H ⁺ ) type resin was passed through the column. After washing with water, L-serine was eluted with 1.5M ammonium water, and the eluate was concentrated under reduced pressure. After concentration, add cold alcohol and
- As a result of serine precipitation, crude crystals of L-serine
0.62g was obtained. Example 2 Various microorganisms shown in Table 1 were used as bacteria, and the growth of the bacteria was measured using a test tube containing 4 ml of culture medium.
The same procedure as in Example 1 was carried out except that the amount of reaction liquid was changed to 1 ml. As a result, L-serine shown in Table 1 was produced. In addition, confirmation of L-serine is n-butanol:acetic acid:water=5:2:2V/V, n-butanol:acetone:diethylamine:water=180:50:20:
20V/V, ethanol: ammonia: H ₂ O = 18:
Quantification of L-serine was carried out by a bioassay method using a serine-auxotrophic mutant of Corynebacterium glutamicum using thin layer chromatography using a 1:1 V/V solvent system and an automatic amino acid analyzer. 【table】

Claims (1)

[Claims] 1 Belongs to the genus Achromobacter, Aeromonas, Bacillus, Brevibacterium, Titrobacter, Corynebacterium, Erwinia, Pseudomonas or Vibrio and has the ability to convert β-hydroxyaspartic acid to L-serine. 1. A method for producing L-serine, which comprises reacting a culture, cells, or treated product of a microorganism with β-hydroxyaspartic acid to produce L-serine, and collecting the L-serine.