JPS623793A - Production of l-phenylalanine - Google Patents

Abstract

PURPOSE:To minimize the amount of salt produced in an aqueous medium as a by-product and to increase the yield of L-phenylalanine, by warming a specific mold strain in an aqueous medium containing cinnamic acid, an ammonia donor and a small amount of an acid other than cinnamic acid. CONSTITUTION:A microbial strain Geotrichum capitatum AJ117128 (FERM P-7736), Moniliella suaveolens var suaveolens AJ117129 (FERM P-7737), etc., which is a mold capable of producing L-phenylalanine from cinnamic acid and an ammonia donor, is warmed in an aqueous medium containing cinnamic acid, an ammonia donor and an acid other than cinnamic acid. The amount of the acid other than cinnamic acid is <=0.12mol per 1 mol of the ammonia donor.

Description

[Detailed description of the invention] [Industrial application field] L-phenylalanine (hereinafter abbreviated as phenylalanine).

) is a dipeptide sweetener that has been attracting attention in recent years.
It is an important raw material for 4-rutylphenylalanine methyl ester. The present invention relates to a method for producing phenylalanine,
More specifically, the present invention relates to a method for producing phenylalanine from cinnamic acid and an ammonia donor using microorganisms.

[Conventional technology]

Furthermore, the present applicants have proposed a method using microorganisms belonging to filamentous fungi (Japanese Patent Application No. 59-18869; Japanese Patent Application No. 59-161).
216. Patent application No. 59-215962, Patent application No. 6O-88
58) has been developed.

[Problems to be solved by the present invention]

In the conventionally known reaction of producing phenylalanine from cinnamic acid and an ammonia donor by microorganisms, it is necessary to add expensive acids such as hydrochloric acid or sulfuric acid to the aqueous medium in order to maintain the ... of the aqueous medium between 8 and 11. However, in the phenylalanine purification process, the salts generated by the addition of acid must be removed, which has the drawback of reducing the processing yield of phenylalanine.

The problem to be solved by the present invention is to solve the above-mentioned drawbacks and to establish an efficient and industrially inexpensive method for producing phenylalanine.

[Means for solving problems]

In order to solve the above problems, the present inventors conducted various studies and found that in the conventional method of producing cinnamic acid and ammonia donor 17-enylalanine using filamentous fungi, The ammonium salt produced to adjust - significantly inhibits the production of L-phenylalanine, and lowering the concentration of ammonium salt in the aqueous medium significantly improves the production of L-phenylalanine compared to conventional methods. In addition, due to the low concentration of ammonium salt in the aqueous medium, the process of removing the salt in the purification process of 7-enylalanine can be simplified, and the processing yield of phenylalanine has been found to be improved. The present invention has been made based on this knowledge.゛The microorganisms used in the present invention may be any filamentous fungi, but specifically include the following.

GeoLrichum
capitatum ) AJ 117128 FER
M P-7736 Moniliea Seepeolens Paar Seepeolens (Mon111ella 5uave
olens varsuaveolens) AJ
117129 FERM P-7737 Pellicularia filamentosa
lamenLosa) IFO6254 Gonatobotryum apiculatum
) IFO9098 Syneephalastrum racemosu
m) IFO4814 Endo-4 Endomycesllndneri AJ 6
611 FERM P-7425 Aspergius Siharie II (A@psrgillu♂chsvali
erl) AJ 7221 FERM P-7426
5acchar, dmycopsis fibuli
gera) IFO0105 Eurotium svalieri (Eurotium chsvalieri) I
FO4090 Chromerella cummaensis (Glome)
AJ 63
07 FERM P-7892 Cladosporium colocasia
e) IFo 6698, etc.

A method for allowing these microorganisms to act on cinnamic acid and an ammonia donor is to culture the microorganism and allow the culture, cells, or treated microorganisms of the microorganism to act on cinnamic acid and the ammonia donor in an aqueous medium.

The medium for culturing the above-mentioned microorganisms is a conventional medium containing a conventional carbon source, nitrogen source, and inorganic ions. Furthermore, desirable results are often obtained by adding organic micronutrients such as vitamins and amino acids.

As the carbon source, glucose, carbohydrates such as sucrose, organic acids such as acetic acid, alcohols, and others are used as appropriate. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, and others are used. As the inorganic ions, magnesium ions, phosphate ions, potassium ions, iron ions, and others are used as appropriate.

Cultivation is carried out under aerobic conditions for 1 to 30 days while controlling the temperature within an appropriate range of 15 to 30°C.

In order to cause the culture, cells, or treated product of the present microorganism to act on cinnamic acid and an ammonia donor in an aqueous medium, the culture, cells, or treated product of the microorganism must be added to an aqueous medium containing cinnamic acid and an ammonia donor. Dissolve or suspend the aqueous medium in
The mixture may be left standing for a while or stirred while adjusting the temperature to an appropriate temperature of 0 to 70°C.

Incidentally, in the reaction to produce phenylalanine from cinnamic acid and an ammonia donor, the amount of cinnamic acid used is not particularly limited, but usually from 0.01 to 0.01 when carried out in a batch method.
1. OM, preferably about 0.1 to 0.8M.

Since the ammonia donor, which is the other reaction substrate, produces salt in the reaction solution and does not form stones, it is most desirable to use ammonia to neutralize and dissolve cinnamic acid, but this method does not make it difficult to raise L-phenylalanine. If the amount is above 1, it may be present in the form of an ammonium salt such as ammonium acetate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium carbonate, ammonium nitrate, ammonium formate, or ammonium citrate. In the present invention, acids other than cinnamic acid contained in the aqueous medium are adjusted to have a molar concentration ratio of 0.12 or less with respect to the ammonia donor, so salts produced as by-products in the aqueous medium can be removed by conventional methods. significantly lower than that. For this reason, the amount of L-phenylalanine produced increases. Further, these reaction substrates may be added in portions as the reaction progresses. The reaction is usually carried out in an aqueous medium at a temperature of 20 to 60°C, preferably 30 to 40°C.

Furthermore, as the bacterial cells, a culture solution containing the bacterial cells may be used as is. Alternatively, it may be used once separated from the culture solution and washed or without washing. Examples of bacterial cell treatments include mechanically ground bacterial cells, ultrasonic-treated bacterial cells, freeze-dried bacterial cells, acetone-dried bacterial cells, bacterial cells treated with enzymes such as lysotium, surfactants, toluene, etc. Bacterial cells treated with , protein fractions of bacterial cells, immobilized products thereof, or others can be used as appropriate.

As a method for obtaining such bacterial cells, the above-mentioned culture medium and culture method can be used as they are.

When culturing this microorganism, by adding at least a small amount of cinnamic acid, phenylalanine, or D-phenylalanine to the medium, a bacterial cell with a high ability to produce phenylalanine from cinnamic acid and an ammonia donor can be obtained. There is.

Confirmation and quantification of phenylalanine was performed by a bioassay method using Leuconostoc mecentroides ATCC-8042.

Example 1 Polly J! Goodon 1. OfZ/dl, yeast extract 1.
OE/di.

K2HPO40,31/dl, KH2PO40,
117dl, MgSO4”7H200,051!/
Medium containing dt1 phenylalanine 0.517dt (p
Pour 5QmJ of H6,0) into a 500m flask 115
Sterilize for 15 minutes at °C.

Geotrichum cavitatum AJ 11712 was cultured on potato dextrose agar medium at 25°C for 7 days.
8 FERM P-7736, Moniliea Seveolence Paaseepeolence AJ 117129 F
ERMP-7737, Pelicularia filamentosa IFO6254, Punatogotrium aviculatum IF09098, Synthephalastrum racemosum IF04814, Endolord Ises rindeneri AJ
6611 FERM P-7425, Aspergius cypalieri AJ 7221 FERM P-742
6. Satsukalomycobsis fui 21J r+ IFO
0105, Ni α Chiumushi Harie'J IFO409
01 Glomerella cucmaensis AJ 6307 FE
RM P-7892, Cladosporium colocache I
One platinum loop of FO6698 was inoculated with AJ117.
128 (FERM P-7736), AJ 11
7219 (FERM P-7737), AJ 661
1 (FERM P-7425) at 25℃14
days, IFO6254, IFO4814, AJ722
1 (FERM P-7426), IFO4090, IF
O0105, AJ6307 (FERM P-7892)
In the case of H25℃ for 4 days, IFO9098 and IFo
In the case of 669B, it was cultured at 25°C for 2 days. These culture fluids and bacterial cells were collected by filtration, washed once with the same amount of physiological saline as the culture fluid, and the bacterial cells were collected. Also, for comparison, the conventionally known Rhodotorula gurches IF0
0559 strain was treated with poly(Buto71.OEl/l, yeast extract 1.0 I/dt, 2HPO40, 3fi/dt
, KH2PO40,11/dt, MgSO4-
7H200,051/d1.7:t-=#7,F
Medium (pH 6,0) containing 0.511/dl
001nl flask and sterilized at 115°C for 15 minutes, inoculated with white fungus and cultured at 25°C for 24 hours, collected by centrifugation and washed once with physiological saline in the same volume as the culture solution. Bacterial cells were collected.

These bacterial cells were treated with 3J of cinnamic acid and 55% of 28% ammonia water.
In contrast to the reaction method in which 100 ml of an aqueous medium containing 100 ml of hydrochloric acid (final 8M) and the amount of hydrochloric acid shown in Table 1 were adjusted, the unadjusted reaction method of the present invention resulted in a higher accumulation in the non-adjusted fungi. With this adjustment, L-phenylalanine production decreased.

Example 2 Washed bacterial cells of Cladosporium colocachea IFO6698 prepared in the same manner as in Example 1 were collected.

Cinnamic acid 3011/l, and an aqueous solution containing the ammonia of the spear shown in Table 2 (-10 in pH unadjusted pear and HCt)
Bacterial cells were added to 1 liter of the mixture at a concentration of 509 cells/liter, and the mixture was kept at 30° C. for 3 hours for reaction. As a result, it was found that phenylalanine was produced and accumulated in the amount shown in Table 2, and under the condition of I) 11 without adjustment, a high accumulation was observed even when the amount of ammonia added was low.

Example 3 Cladosporium colocasche IFO as in Example 1
6698 bacterial cells were prepared, and m ammonia water 5 of Example 2 was prepared.
The reaction was carried out under the same reaction conditions as using 50 ml.

After removing the bacterial cells from this reaction-completed liquid, the internal pressure was 180+mHg using a rotary evaporator equipped with a pressure reduction regulator.
The solution was concentrated to a volume at which crystals began to precipitate. Concentrates of 285 m19J and 7 sa ml (B) were obtained, respectively.

These concentrated solutions were then diluted with 35 pl (approximately 1 pl) of HCl solution.
After neutralizing the solution to 100% and removing the precipitated unreacted cinnamic acid by filtration, 28% NH solution was added to solution F, the volume was adjusted to 7, and the mixture was stirred overnight at room temperature to form crystals of L-phenylalanine. It was allowed to precipitate. The crystals tF were separated, washed with a small amount of ice water, and dried to give 19.1' (~ and 10.711
Crystals of I,-phenylalanine (B) were obtained.

Example 4

Claims (1)

[Scope of Claims] 1) Geotrichum, Moniliea, Pericularia, Gonatobothrium, Syncephalastrum, Endomyces, Aspergillus, which have the ability to produce L-phenylalanine from cinnamic acid and an ammonia donor;
Microorganisms belonging to the genus Eurotium, genus Saccharomycopsis, genus Glomerella, or genus Cladosporium are treated with acids other than cinnamic acid at a molar concentration ratio of 0.12 or less with respect to the concentration of cinnamic acid, ammonia donor, and ammonia donor. A method for producing L-phenylalanine, which comprises keeping warm in an aqueous medium containing L-phenylalanine, producing and accumulating L-phenylalanine in the aqueous medium, and collecting the L-phenylalanine. 2) L-phenylalanine according to claim 1, wherein the acid other than cinnamic acid is an acid selected from one or more of the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, nitric acid, formic acid, acetic acid, and citric acid. Production method. 3) The method for producing L-phenylalanine according to claim 1, wherein the concentration of the ammonia donor is at least equimolar to cinnamic acid.