JPS6296092A - Production of l-serine - Google Patents

Abstract

PURPOSE:To produce L-serine useful as a component of cosmetics such as hair tonic, etc., at a low cost, by reacting glycine with 5,10-methylenetetrahyrofolic acid in the presence of a living cell, etc., of a specific microorganism. CONSTITUTION:A microbial strain belonging to Pseudomonas genus and having methanol assimilating capability and L-serine transhydroxymethylase productivity, e.g. Pseudomonas rhodomethylofaciens AJ12225 (FERM P-8316) (a methionine requiring strain) is cultured aerobically in a medium composed of carbon source, nitrogen source, etc., at 5-9pH and 25-37 deg.C. The cultured liquid is treated e.g. by centrifugal separation to obtain an enzyme specimen composed of living microbial cell, etc. A reaction system containing the enzyme specimen, 0.1-10% glycine and 5,10-methylenetetrahydrofolic acid is subjected to enzymatic reaction at 5-9pH and 20-50 deg.C under agitation to produce L-serine in the reaction system. The product is separated from the system e.g. by ion exchange resin treatment.

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] <Industrial Application Field> The present invention relates to a method for producing L-serine. L-serine is an important substance as a component of cosmetics such as hair tonics.

<Prior art> L-serine transhydroxymethylase (Ec, 2.
1.2.1) As a method for producing glycine, 5.10-methylenetetrahydrofolic acid, and mustard monoserine using Proteus sp.
Methods using Sarcina genus, Pseudomonas genus, Microbacterium genus (Ibid. 59-23796), etc. are known. In addition, a method using Escherichia coli, Salmonella tyhumurium, Klebsiella aeronoens, etc. in which the enzyme has been enhanced by genetic manipulation (Japanese Patent Application Laid-Open No. 59-1091
87) is known.

However, there is no known method for producing monoserine using microorganisms capable of assimilating methanol.

<Problems to be solved by the present invention> The problems to be solved by the present invention are as follows: Using a microorganism belonging to the genus Pseudomonas and having the ability to assimilate methanol,
Another object of the present invention is to establish a method for producing L-serine using an enzymatic method at a lower cost.

[Structure of the invention]

<Means to solve the problem> As a result of research to develop a more efficient method for producing monoserine, the present inventors found that microorganisms belonging to the genus Pseudomonas and capable of assimilating methanol were It has an excellent ability to produce serine transhydroxymethylase, and produces high, 10-methylenetetrahydrofolic acid in the culture solution, live bacterial cells, or processed bacterial cells of the microorganism and glycine and 5,10-methylenetetrahydrofolic acid or in the reaction solution. The present invention has been completed based on the discovery that a significant amount of L-serine can be accumulated by interacting with a substance that causes L-serine. Any strain belonging to the genus and having the ability to assimilate methanol via the serine pathway can be used.

For example, Pseudomonas rhodomethylofaciens and various mutant strains derived therefrom (eg, auxotrophic mutants, analog-resistant mutants, and other drug-resistant mutants) are used. A specific example of a suitable strain is Pseudomonas rhodomethylo7 asciens AJ122.
25 (FERM P-8316) (methionine requirement).

In addition, the production method of L-serine by fermentation method (% Showan 60-
138147), as shown by the present inventors, it can be expected that strains with methionine auxotrophy have high L-serine transhydroxymethylase activity.

The medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic ions, and, if necessary, organic micronutrients such as vitamins and amino acids.

Methanol is most suitable as a carbon source, but other alcohols such as ethanol and gulo-2-nor, organic acids such as formic acid and acetic acid, and carbohydrates such as glucose can also be used if an appropriate strain is selected.

As the nitrogen source, ammonia water, ammonia gas, ammonium salts, nitrates, amino acids, and other common nitrogen sources can be used.

Inorganic ions include potassium ion, magnesium ion,
Iron ions, manganese ions, calcium ions, chloride ions, sulfate ions, phosphate ions, and others are added to the medium as necessary.

Corn staple liquor, yeast extract, etc. that contain organic micronutrients such as vitamins and amino acids,
Meat extract etc. may be added.

Incidentally, if methanol is used as a carbon source, it is possible to promote the production of L-serine transhydroxymethylase, and a more favorable effect can be expected during the enzymatic reaction.

Cultivation is preferably carried out under aerobic conditions, and the most favorable results can be obtained by adjusting the pH to an appropriate level of -4 within the range of 5 to 9. Further, it is desirable that the culture be carried out while adjusting the culture temperature to an appropriate temperature in the range of 25 to 37°C.

The culture fluid thus obtained, live bacterial cells collected from the culture fluid by centrifugation, or processed bacterial cells (e.g., crushed bacterial cells, sonicated bacterial cells, bacterial extracts, and bacterial cell extracts obtained from the extracts). enzyme classification) can be used as enzyme preparations.

L-serine is produced by allowing such an enzyme preparation to react with glycine and 5,10-methylenetetrahydrofolic acid or a substance capable of producing 5,10-methylenetetrahydrofolic acid in the reaction solution. To carry out this enzyme reaction, the substances added to the reaction system are appropriately selected depending on the type of enzyme preparation.

For example, when using a culture solution or live bacteria isolated from the culture solution as an enzyme preparation, these enzyme preparations usually produce 5,10-methylenetetrahydrofolate from formaldehyde in addition to L-serine transhydroxymethylase activity. The substance added to the reaction system is a combination of glycine and 5.10-methylenetetrahydrofolic acid, or a combination of glycine and formaldehyde. Instead of using a combination of glycine and tetrahydrofolic acid, a combination of glycine and formaldehyde may be used, or glycine alone may be used alone. On the other hand, when using a bacterial cell-treated product as an enzyme preparation, the enzyme preparation usually has the ability to produce 5,10-methylenetetrahydrofolic acid from formaldehyde or 5,10-methylenetetrahydrofolate from glycine via formaldehyde.
Since it does not have the ability to produce 0-methylenetetrahydrofolic acid, or even if it does have it, it is extremely weak, the substances added to the reaction system are a combination of glycine and 5,10-methylenetetrahydrofolic acid, or a combination of glycine and formaldehyde. It is necessary to use a combination of and tetrahydrofolic acid.

The concentration of glycine used is not particularly limited, but is between 0.1 and 1.
Approximately 0% is preferable.

The enzyme reaction of the present invention is P) (at about 5 to 9, at a temperature of 2
It is preferable to leave it still, shake it, or press it down at about 0 to 50°C.

In addition, about 0.01 to 1% formaldehyde was added to the reaction system.
Alternatively, the reaction may be enhanced by adding about 0.1 to 10 mM of tetrahydrofolic acid or about 0.001 to 1 mM of pyridoxal phosphoric acid. Also,
If these are used in combination, the reaction may be further enhanced.

L-serine produced in the reaction system can be isolated by a conventional method such as a method using an ion exchange resin.

Example 1 Twenty liquid media having the following composition were placed in a 500 d shake flask and sterilized. To this, methanol 2, OTrLl
The first cultured at 30°C for 48 hours in ldi-containing meat juice slant.
One platinum loopful of each strain shown in the table was inoculated and cultured by shaking at 34°C. 24 hours later methanol 1.0ml1ldi
was added and cultured for an additional 24 hours.

Kn2PO40,59/1 (NH4)HPO45,Og/l MgSO4-7H200,4g/1l NaC10,5g/1 FeSO4・7 H2O10"!i'/l!MnSO
4.4H4H2O5/l Yeast extract 2.0 g/IL -
Met 0.15 g/l methanol (separate sterilization) 2. Om1ldlCaCos
(〃) 5 g/diPH6,8 Bacterial cells were collected from the culture solution and washed twice with 50 mM') potassium phosphate buffer (pH 7,0). The cells were suspended in the same buffer and subjected to ultrasonic treatment at 0°C for 10 minutes.

This treated solution was centrifuged (10,000X, !i', 1
5 minutes.

0°C), and the supernatant was used as an enzyme preparation.
a r *M, and Et-Obeid, H,A
, method (Biochem, Biophys.

See Acta 258.791-799 (1972)]
L-serine transhydroxymethylase activity was measured according to . Assuming that the activity to reduce l n mole of substrate (formaldehyde) per minute is 1 unit, the amount of extractable protein after sonication of cells/m
The activity per gram was as shown in Table 1 below.

Table 1 Pseudomonas rhodomethylofaciens AJ1126
1 (FERM-P4531) 9.
9 Pseudomonas rhodomethylofaciens AJ112
25 (FERM-P8316) 64
．． 0 Example 2 Pseudomonas rhodomethylofaciens AJ1122
5 (FERM-P8316) was cultured in Example 1 using 3% 7-lactose instead of methanol, and the L-serine transhydroxymethylase activity was measured in the same manner as in Example 1. Specific activity was 21.3.

Example 3 Pseudomonas rhodomethylofaciens AJ1122
5 (FERM-P8316) in the same manner as in Example 1, the cells were cultured in 40 mM glycine, 0.2 mM pyridoxal phosphate, and 1 mM tetrahydrofolic acid.

COCl2.6 0.1M salt/potassium buffer containing 0.02g H2O and 1d toluene (pi(7,0)2ON/
When formaldehyde was added to the mixture and formaldehyde was added at a rate of 2 mM/hour while the reaction was allowed to stand at 34° C. for 7 hours, 1.1 g/l of L-serine was accumulated in the reaction solution.

Example 4 A reaction was carried out in the same manner as in Example 3 in a reaction solution in which pyridoxal phosphoric acid and tetrahydrofolic acid were not added. The amount of serine accumulated in the reaction solution was 0.1 g/
It was l.

Patent issuer: Ajinomoto Co., Ltd. Procedural amendment October 29, 1985 Patent application dated October 22, 1985 (2) 2. Name of the invention: Process for producing L-serine 3. Relationship with the amended person case Patent Applicant address: 5-8-6, Kyobashi-chome, Chuo-ku, Tokyo Subject of amendment Column 7 of detailed explanation of the invention in the specification, Contents of amendment (1) Line 5 from the bottom of the second specification r (EC, 2121]
Correct 1 to r(EC2, 1, 2, 1) J.

(2) Add a separate text between lines 9 and 10 on page 4 of the detailed description.

<3) Specification page 11 line 8 “...Glycine 40 m
Correct "M Viridoki..." to "...glycine 40mM, Viridoki...".

[Attachment] Microorganisms that assimilate compounds containing one carbon such as methanol are
s-Manual or [)terminati
ve3 acteriology 7thed,), it is classified as Brotaminobacter genus or Methanomonas, and AJ 11261 and AJ 11262 of the present invention.
According to the Purge Aids Manual, 7th edition, it should belong to the genus Protaminobacter.

However, in the 8th edition of the Purge Aids Manual, the genus Protaminobacter is not recognized as a genus, and its representative species, Protaminobacter ruber, is related to methanol-assimilating microorganisms belonging to the genus Bacillus, Vibrio or Pseudomonas. microorganisms.

In addition, recent research by Yukami and Komagata et al.
・It has been revealed that louvers have cell morphology, coenzyme Q type, and bacterial cell fatty acid composition similar to those of methanol-assimilating bacteria that form red colonies of the genus Pseudomonas. Abstract m2
Page F-2070) In the current classification system, it is considered appropriate that Protaminobacter ruber and its closely related microorganisms belong to the genus Pseudomonas.

Therefore, the microorganisms belonging to the genus Pseudomonas used in the present invention include Protaminobacter looper 43 described in Purge Aids Manual, 7th edition, and microorganisms closely related thereto.

Claims (1)

[Claims] 1. Glycine and microorganisms belonging to the genus Pseudomonas and having the ability to assimilate methanol and produce L-serine transhydroxymethylase in the presence of a culture solution, live bacterial cells, or processed bacterial cells. A method for producing L-serine, which comprises reacting with 5,10-methylenetetrahydrofolic acid. 2. The method according to claim 1, wherein 2,5,10-methylenetetrahydrofolic acid is produced in a reaction solution from formaldehyde using a culture solution or viable cells of the microorganism. 2. The method according to claim 1, wherein 3,5,10-methylenetetrahydrofolic acid is produced from glycine in a reaction solution using a culture solution or viable cells of the microorganism. The method according to claim 1, wherein 4,5,10-methylenetetrahydrofolic acid is produced in the reaction solution by a reaction between formaldehyde and tetrahydrofolic acid.