JPS60160890A - Preparation of l-phenylalanine - Google Patents

Abstract

PURPOSE:To prepare L-phenylalanine efficiently without using an expensive amino group donor, by treating phenylpyruvic acid with a bacterium in the presence of an energy source, an inorganic ammonium compound etc. CONSTITUTION:A bacterium belonging to the genus Brevibacterium, Bacillus, etc., capable of converting phenylpyruvic acid into L-phenylalanine, is cultivated in a medium containing an energy source such as saccharide, alcohol, or organic acid, an inorganic ammonium compound such as ammonia water, ammonium salt, etc. or urea and oxygen in the presence of phenylpyruvic acid, or phenylpyruvic acid is treated with a culture of the bacterium or a treated material of it in the presence of the energy, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-phenylalanine. More specifically, A Brevibacterium, Bacillus, Citrobacter, Corynebacterium, Escherichia, Flavobacterium, Microbacterium, Serratia,
Alkaline earth metals, microorganisms belonging to the genus Salmonella, Enterobacter, Erwinia, or Klebsiella.
By culturing the microorganism as an energy source, ■ an inorganic ammonium compound or urea, and ■ phenylpyruvic acid in the presence of oxygen in the medium or at any time during the cultivation, or B. A culture of the above microorganism or a treated product thereof. The above,
By acting on phenylpyruvic acid in an aqueous solution in the presence of components ① and ②, L is added to a culture solution or an aqueous solution.
- A method for producing L-phenylalanine, which comprises producing phenylalanine and collecting the produced phenylalanine.

L-phenylalanine is useful as a raw material for sweeteners and the like.

Conventionally, a method for producing phenylalanine using microorganisms involves culturing L-phenylalanine-producing bacteria in a fermentation medium containing carbohydrates and other carbon sources and inorganic nitrogen sources such as ammonia as main raw materials. - So-called direct fermentation methods for accumulating phenylalanine and various reaction substrates (eg phenylpyruvate, cinnamic acid, DL-phenylalanine).

An enzymatic method is known in which microbial cells or enzymes derived therefrom are made to act on microorganisms (such as acyl-DL-phenylalanine).

Among these, the method for producing L-phenylalanine using phenylpyruvic acid as a raw material is to combine phenylpyruvic acid and amino acids as amino group donors with alkaline earth metals, Pseudomonas spp., Anirobaku spp., Escherichia spp., Achromobacter spp. , Sarcina spp., Taruihuella spp. or Micrococticus spp. (Special Publication No. 37-10672)
Or Serratia marcescens (Amino Ac
1ds, Vol. 5, p. 61, 1962), phenylpyruvic acid and various amino group donors [e.g., nylon 6 hydrolyzate (Japanese Patent Publication No. 44-17992>), nylon cyclic oligomer (Special Publication No. 44-17991), Lactam (Special Publication No. 44-1799)
0)) A method of making phenylpyruvic acid and various amino acids act on the bacterial cells of the Corynebacterium genus.

Absidia spp., Chaetmium spp., Fusarium spp.

A method of acting on microbial cells of any of the genus Penicillium, genus Mucor, genus Monascus, and genus Rhizobus (Japanese Patent Publication No. 45-20556) is known.

A better method for producing phenylalanine is always being sought.

As a result of consideration for this purpose, ■Energy sources.

It has been discovered that a spinning top can be obtained in significant amounts by using microorganisms having the ability to convert phenylpyruvic acid into L-phenylalanine in the presence of (1) an inorganic ammonium compound or urea and (2) oxygen. According to the present invention, the desired product can be produced without using expensive amino group donors such as amino acids, so it is an economically advantageous method.

As the microorganism used in the present invention, any microorganism can be used as long as it has the ability to convert phenylpyruvic acid into L-phenylalanine in the presence of (1) an energy source, (2) an inorganic ammonium compound or urea, and (2) oxygen. Preferred microorganisms include Brevibacterium, Bacillus, Citrobacter, and Corynebacterium.

Escherichia, Flavobacterium, Microbacterium, Serratia, alkaline earth metals.

Any microorganism belonging to the genus Salmonella, Enterobacter, Erwinia, or Talebsiella may be used, including wild strains, mutant strains, and recombinant strains induced by cell fusion methods or genetic manipulation methods. Um lactofermentum ATCC13869, ha f x
・SeL/Us I F 03131゜Citrobacter・
Freundi AT[:C8750, Corynebacterium glutamicum AT[:C13032, Escherichia coli ATCC11303, Flavobacterium ribeoleus ΔTCC958, Microbacterium ammoniaphilum ATCC15354.

Serratia marcescens ATCC19180, Alcaligenes faecalis IAM1015, Salmonella
ATE: [:19585, Enterobacter cloacae-IAM1528, Tcc.21434 for Erwinia herbicolae, and TCC8724 for Talebsiella pneumoniae. When culturing these microorganisms in a medium to obtain L-phenylalanine, the medium may be either a natural medium or an artificial medium as long as it contains at least (1) an energy source and (2) inorganic ammonium or urea.

Energy sources include sugars such as glucose, fructose, sucrose, maltose, galactose, xylose, molasses, il powder, carbohydrate raw materials such as 11 powder hydrolyzate, sugar alcohols such as glycerol and mannitol, ethanol, propatool, etc. Alcohols, formic acid, lactic acid,
Organic acids such as acetic acid, acetic acid, acetic acid, and fumaric acid are used.

Examples of inorganic ammonium include aqueous ammonia, ammonia gas, ammonium salts (for example, ammonium chloride,
Ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium acetate and other 1m ammonium, ammonium carbonate) are used.

Phenylpyruvic acid may be added to the medium from the beginning, or may be added during the cultivation of the microorganism.

Furthermore, inorganic substances (phosphoric acid-potassium,
phosphates such as dipotassium phosphate, sodium phosphate, disodium phosphate, and ammonium phosphate, sulfates, hydrochlorides, and nitrates of magnesium, manganese, calcium, iron, zinc, etc.), and when the microorganism is an auxotrophic strain. adds compounds necessary for growth.

Culture conditions include 20-50°C, preferably 30-40°C.
Under aerobic conditions such as shaking and aeration stirring at a temperature of 0°C, pH
3-10. Preferably, the culture is carried out for 1 to 3 days in the range of 6 to 9. In this way, L-phenylalanine is produced in the culture solution.

Next, as a medium for obtaining a culture of the microorganism,
Either a natural medium or an artificial medium can be used as long as the medium contains a suitable amount of carbon source, nitrogen source, and inorganic substances.

The above energy source is used as the carbon source. As the nitrogen source, in addition to the inorganic ammonium compound and urea, peptone, meat extract, protein hydrolyzate, fish meal, etc. are used.

As the inorganic substance, the above-mentioned inorganic substances are used. The conditions described above are used as the conditions for culturing the microorganism.

The microorganism culture thus obtained can be used as it is for the reaction, or furthermore, the culture can be subjected to various treatments and the processed products obtained can be used for the reaction. Processed products include concentrated or dried culture products, filtrate or bacterial cells obtained by treating the culture with a centrifuge, dried bacterial cells, acetone-treated products, surfactant-treated products, and lytic enzyme treatment. Examples include microorganisms, immobilized bacterial cells, etc.

The reaction is carried out in an aqueous solution by reacting the culture obtained above or its treated product with phenylpyruvic acid in the presence of (1) an energy source, (2) an inorganic ammolium compound or urea, and (2) oxygen.

Thus, L-phenylalanine is produced in the aqueous liquid. The working conditions include standing or stirring at 10 to 50°C. The energy source and inorganic ammonium compound used are the same as those mentioned above, and the method for recovering mustard phenylalanine from the culture solution or aqueous solution is the ion exchange resin method.

Activated carbon adsorption method etc. are used.

Examples will be described below.

Example 1 The microorganisms shown in Table 1 were used as inoculum, which were statically cultured on a bouillon agar medium at a temperature of 28° C. for 24 hours.

This bacterial cell was inoculated into a test tube containing 5+y+l of the following fermentation medium, and 21Orp was added at a temperature of 28°C.
After shaking culture for 5 hours under shaking conditions of 100 ml, 0.5 ml of sodium phenylpyruvate 50++g/m+ aqueous solution (sterilized by filtration with a Mengren filter) was added to continue the culture for a total of 24 hours. The accumulation of phenylalanine in the supernatant of the culture-finished liquid was determined as shown in Table 1 for each microorganism. The amount of phenylalanine accumulated in the case without the addition of phenylpyruvic acid and potassium is
The concentration of all microorganisms was 0-1 mg/ml or less.

Table 1 Bacillus cereus IFO 31313, 2 Citrobacter freun 3.6 d ATCC 8750 Corynebacterium glenn 2, 5 Micumumicum ATCC 13032 Escherichia coli ATCC 113033, 2 Flavobacterium ribeo 2.8 Reus AATCC 958 Microbacterium ammonium 1 .8 Affilam AT
CC15354 Serratia marcescens 1.2 ATCC19180 Alcaligenes faecalis 3.2 1AM1015 Salmonella tissimurium 3.1 ATCC19585 Enterobaccu cloacula-3,4 1AM1528 Erwinia Herbicola-1,7 ATC(:21434 Kleb Sierra pneumoniae 3.7 ATCC, 87
24 Composition of fermentation medium Nigelcose 5%, ammonium sulfate 1%, urine go,
z%, KH2po, 0.05%, K2'HP O-
0,05%, MgSO4.7H200,025%.

Corn steep liquor 0.05%, Ca CO3
2% (pH 7.2, hydrated with aqueous ammonia).

Example 2 Escherichia coli AT CC11303 as inoculum
Using phenylpyruvic acid sodium salt 50mg/3 times at 4th, 9th and 24th hours of culture, the glucose concentration of the fermentation medium was 10% and the ammonium sulfate concentration was 2%.
Add m+ solution in 0.5 ml portions and continue culturing until a total of 4
The culture was carried out in the same manner as in Example 1 except that shaking culture was carried out for 8 hours, and as a result, 9.8 mg/ml of L-phenylalanine was accumulated in the supernatant of the culture-finished liquid. The amount of L-phenylalanine accumulated when phenylpyruvic acid/sodium salt is not added is O, l m
It was less than 1.

Example 3 Corynebacterium glutamicum ATCC as inoculum
Fermentation medium 20+Tl+ with the following composition using 21673
A 300 ml Erlenmeyer flask (with baffle plate) containing 1. rp
Shaking culture was carried out under shaking conditions of m.

At the 20th and 44th hour of culture, 0.5 ml and 1.5 ml of 50 mg/ml phenylpyruvate/sodium solution were added.
1 was added to each cell and culture was continued for a total of 68 hours. As a result, 8.2 mg/ml of L-phenylalanine was accumulated in the culture supernatant. The amount of phenylalanine accumulated when phenylpyruvic acid/sodium salt is not added is 7.3
mg/m+.

Composition of fermentation medium: Blackstrap molasses (as sugar) 10%, ammonium sulfate 2%.

KH2P0. 0.05%, 2HP0. 0.05%
, Mg5O<・7H,OO,025%, Corn Steep Liquor 0.1%, L-Tyrosine 10mg/ml,
CaC0a 2% (pH 7.2, neutralized with NH40H).

Patent Applicant (102) Kyowa Hakko Kogyo Co., Ltd. Representative Yuki Kinoshita Continuation of page 1 ■Int, CI,' Identification symbol Internal publication procedure amendment (
Method) 1. Display of the case 1982 Patent Application No. 14783 2 Name of the invention Process for producing L-phenylalanine 3 Person making the amendment Relationship to the case Patent applicant Postal code 100 Address 6 Otemachi-chome, Chiyoda-ku, Tokyo 1 name
(102) Kyowa Hakko Kogyo Co., Ltd. (Location: 03-20
1-7211 Extension 2751>Representative: Mikio Kato 4. Date of amendment order: φ day, month q, 1982 (shipment date: 21 + day of the middle month of 1982) 5. Specifications subject to amendment 6. Contents of amendment Engraving of the specification (no change in content) Procedures Amendment September 1980//F3 1. Indication of the case 1982 Patent Application No. 14783 2. Name of the invention Process for producing L-phenylalanine 3. Amendment Relationship with the case of a person who does
-201-7211 Extension 2751) 5. Contents of amendment 1) The microbial salts on page 5, lines 2-13 of the specification are corrected as follows.

"Brevibacterium lactofermentum cere"
us) I FO3131, Citrobacter freundi
6750, Corynebacterium glutamicum (Cor
yne-bacterium+ glutamicum
) A TCC13032, Escherichia coli (Bs
cherichia coli) ATCC11303,
Flavobacterium suaveolens (Flavobacterium suaveolens)
cterium 5uaveolens) ATCC95
8. Microbacterium ammonophilum (Mic
robacteriu+++ ammoniaphil
um) ATCC15354, Seratia marcescens ATCC
19180° Alcaligenes faecalis (8 lcal
I.A.M. 101
5. Salmonella typhimurinum
la typhimurium) AT CC19
585, Enterobacter cloacae (Bntero
bacter cloacae) IAM 152
8. Erwinia herbicola ([irwinia t
+erbicola) ATCC21434, Klebsiella oxytoc
a) Correct to ATCC8724J.

2) Same book, page 6, line 1 Delete "fumaric acid".

3) In the same book, page 9, table 1 [Citrobacter freundii ATCC8750J
[Citrobacter freundii ΔTCC6750
Correct to J.

“Flavobacterium ribeoleus AATCC958
J [Flavobacterium faveolens ΔTCC9
Corrected to 58J.

[Correct Talebsiella pneumoniae ATCC 8724J to "Klebfera oxytophores ATCC 8724J.

Correct "Salmonella chimmurium" to "Salmonella typhimurinium".

Claims (1)

[Scope of Claims] A Brevibacterium, Bacillus, Citrobacter, Corynebacterium, Escherichia, Flavobacterium, Microbacterium, Serratia,
Alkaline earth metals, microorganisms belonging to the genus Salmonella, Enterobacter, Erwinia, or Klebsiella.
By culturing the microorganism as an energy source, ■ an inorganic ammonium compound or urea, and ■ phenylpyruvic acid in the presence of oxygen in the medium or at any time during the cultivation, or B. A culture of the above microorganism or a treated product thereof. Said ■,
By acting on phenylpyruvic acid in an aqueous solution in the presence of components ① and ②, L is added to a culture solution or an aqueous solution.
- A method for producing phenylalanine, which comprises producing phenylalanine and collecting the produced L-phenylalanine.