JPH01312996A - Production of l-phenylalanine - Google Patents

Abstract

PURPOSE:To improve the efficiency of the subject process at a low cost by treating a cinnamic acid ester and an ammonia and/or an ammonia donor with an enzyme. CONSTITUTION:A cinnamic acid ester (A) is produced by reacting cinnamic acid with terpene alcohol, etc. A raw material of ammonia is selected to obtain ammonia and/or an ammonia donor (B). A microbial strain belonging to genus Aspergillus, etc., is cultured in a medium containing glucose, peptone, etc., under aerobic condition at pH2-11 and 15-40 deg.C for 1-10 days to obtain a cultured product (C) containing produced phenylalanine ammonia lyase and esterase. The component A is mixed with the component B at a ratio to give a molar ratio of the cinnamic acid of the component A to the ammonia of the component B of 1:(3-100) and the mixture is added to the component C in an amount of 0.01-400wt.% (in terms of A+B) to obtain a mixture (D) of the enzyme and t he substrate. The mixture D is subjected to enzymatic reaction at pH6-11 and 20-40 deg.C and the obtained reaction liquid is purified by ion exchange resin process, etc., to recover L-phenylalanine.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a method for producing L-phenylalanine, specifically, a method for producing L-phenylalanine from cinnamate ester as a raw material by the action of enzymes and/or microorganisms. Relating to a method of manufacturing.

[Conventional technology]

L-phenylalanine is one of the important essential amino acids for humans, and is not only a nutritionally or medically important substance, but is also in increasing demand as a raw material for asparatylphenylalanine methyl ester, a dipeptide sweetener.

Methods for producing L~phenylalanine from cinnamic acid and ammonia or an ammonia donor using the action of enzymes, that is, enzymes and/or microorganisms, are known, for example,
British Patent No. 1489468, JP-A-53-96
Examples include methods described in JP-A No. 388, JP-A-56-26197, JP-A-61-192295, JP-A-61-19496, and JP-A-61-170398.

[Problem to be solved by the invention]

However, these conventional methods use expensive cinnamic acid, which increases production costs, and high concentrations of cinnamic acid inhibit the growth of microorganisms and the action of enzymes. There are problems such as difficulty in efficiently producing L-2 enylalanine, and an economical and highly efficient method for producing L-phenylalanine has been desired.

Therefore, an object of the present invention is to provide an economical and highly efficient method for producing mono-phenylalanine.

[! ! Means for Solving the Problem) The present invention achieves the above object by using a cinnamic acid ester and ammonia and/or an ammonia donor as raw materials when producing L-phenylalanine by the action of an enzyme. This has been achieved by providing a method for producing phenylalanine.

Hereinafter, the method for producing L-phenylalanine of the present invention will be described in detail.

Examples of the cinnamic acid ester used in the present invention include:
Examples include ethyl cinnamate, propyl cinnamate, glycerin ester of cinnamic acid, propylene glycol ester of cinnamic acid, etc. Among these, from the viewpoint of reaction efficiency, workability, economic efficiency, etc., esters of cinnamic acid and terpene alcohol are preferred. is preferred.

Examples of the terpene alcohol preferably constituting the cinnamic acid ester include menthol (Menth).
ol), monoterpene alcohols such as Nerol, sesquiterpene alcohols such as Farnesol and Eudesmol, diterpene alcohols such as netinol and Geranylgeraniol, and cycloartenol (Cycloartenol).
artenol), elaphorbol (Euphorb)
ol), α-Amyrin (cr-Amyrin), β
-Amyrin (β-Amyrin), geraniol (Ce)
rmanico+), /L/Lupeol
, butyrospermol
and the like, preferably triterpene alcohols, more preferably pentacyclic triterpene alcohols, particularly preferably α-amyrin, β-amyrin, lupeol, and butyrospermole.

The above-mentioned cinnamic acid terpene alcohol esters are widely distributed in the natural world, and are particularly present in large amounts in vegetable oils and fats. For example, shea butter can be used.

Cinnamic acid terpene alcohol ester can be preferably used either purified or unrefined. For example, the above shea butter contains about 2fi1% of cinnamic acid terpene alcohol ester, and Since the fractionated soft oil of shea butter contains about 10% by weight of cinnamic acid terpene alcohol ester, these shea butter and the fractionated soft oil of shea butter can be used as they are.

Sono ([h, Any raw material containing cinnamic acid terpene alcohol ester can be preferably used, but if it contains components that inhibit the action of enzymes, remove them before use. Needless to say, it is preferable to do so.

In addition, as the ammonia donor used in the present invention,
For example, various ammonium salts such as ammonium acetate, ammonium chloride, ammonium sulfate, ammonium nitrate, etc. can be mentioned, and when culturing microorganisms and producing L-phenylalanine are carried out simultaneously, peptone, meat extract, yeast extract, etc. can be used. Also included are those commonly used as nitrogen sources for culture.

The action of the enzyme in the present invention is the action due to phenylalanine ammonia lyase activity and esterase activity.

That is, in the present invention, cinnamic acid ester and Ammonia and/or an ammonia donor are added to culture the microorganism, or one or more microorganisms that produce phenylalanine ammonia lyase enzyme and esterase enzyme are cultured and then collected in a medium excluding cinnamic acid ester, Continue culturing the microbial cells in a suitable medium containing cinnamic acid ester and ammonia and/or an ammonia donor, or culture one or more microorganisms that produce phenylalanine ammonia lyase enzyme and hysterase enzyme. Phenylalanine ammonia lyase enzyme and esterase isolated from a culture solution, microorganism cells collected from this culture solution, or a processed product of the microorganism cells, or one or more microorganisms that produce phenylalanine ammonia lyase enzyme and hiesterase enzyme. An enzyme may be allowed to act on a cinnamic acid ester and ammonia and/or an ammonia donor.

In the present invention, a phenylalanine ammonia-lyase enzyme and an esterase enzyme are required, and it is not necessarily necessary to use a microorganism that produces these two enzymes at the same time. Microorganisms that produce esterase enzymes may be used in combination with microorganisms that produce only esterase enzymes or microorganisms that mainly produce esterase enzymes, or a culture solution in which these microorganisms are cultured or microbial cells collected from this culture solution may be used. Alternatively, this microbial cell-treated product may be used. Furthermore, when enzymes are used, it is not necessarily necessary that the enzymes phenylalanine ammonia lyase and hiesterase be produced at the same time, and there is no problem in using enzymes produced separately.

Further, two or more of these microorganisms, a culture solution in which the microorganisms are cultured, microorganism cells collected from the culture solution, a processed product of microorganism cells, and an enzyme may be used in combination.

The above-mentioned microorganisms can be used without any particular restrictions as long as they are one or more types of microorganisms that produce phenylalanine ammonia-lyase enzyme and/or esterase enzyme (However, microorganisms that only or mainly produce phenylalanine ammonia-lyase enzyme, and microorganisms that only produce esterase enzyme) can be used. For example, as a microorganism that produces phenylalanine ammonia lyase enzyme and esterase enzyme, Aspergillus (85
PEIIGILLUS) older brother, Talatosborium (CLA)
DO3PORIUM) fi, ] Fu IJ Nuss (CO
PRINUS) genus, Entomyce, 11. (IENO
OMVCUS) genus, Eurotium (EUI?OTIt)
1M) Genus, Fusarium CFUSAI? lUM), Genus Geotrichum (G OTRIC11t1M), Genus Glomerella (GLOMEI?ELLA), Genus Gonatopotrium (G
ONATO [lOT! ? YU11) genus, Hehe a-7 (
IIEBELOM^) genus, Hyalodendron (HYA)
LOD[! NDRON) genus, Liophyllum (IJOPI)
IYLLIIM), genus Moniliea (formerly LIELLA)
PELL ICULARr
A) Ii.
S) genus, Sporobolomyces (SPOROBOLOMV)
C[! S) genus, Syncephalastrum (SYNCHP)
II^LASTRUM), etc.

In addition, examples of microorganisms that only produce phenylalanine ammonia-lyase enzyme or microorganisms that mainly produce this enzyme include Aspergillus (ASPEL?GrLL[
IS) genus, Cladosporium (CLA00SPO1?I
[IiI) genus, COPRINUS genus, ENDOMYCI!S genus, EUROTIUM genus, Fusarium (PUSARI)
UM) genus, GEOTRICHUM genus,
Genus Glomerella (GLOMERf!LLA), Genus GONATOBOTRYU?I, Genus Heberoma (1111!B[!LOM^), Genus HYALOD[!NDRON], Genus Lyophyllum (
LYOPIIYLLUM) WF2, - [: 2IJ,
11 nia (genus MONILIE [, [, A), genus PRLLICULARIA, genus Foliota (
P11OLIOT8) Genus Rhodotorula (RIIODOT)
ORULA) genus, Rhodosporitium (R110DOS
I+ORIDILIM)i, Satucharomycobsis (S
ACCHAI? OMYCOPSIS) l [, SPOROBOLOMYCES 171
, Synthephalastrum (SYIiCEr'llA[,
ASTI? t1M) II, etc. Microorganisms that only produce esterase enzymes or microorganisms that mainly produce this enzyme include, for example, Pseudomonas (Pseud
omonas genus, Serratia genus, and the like.

The amount of cinnamic acid ester added in the present invention varies depending on the type of microorganism used, but it may be used in a medium in which one or more microorganisms producing phenylalanine ammonia lyase enzyme and esterase enzyme are cultured, or in a medium in which the above microorganisms are being cultured. When adding cinnamic acid ester and ammonia and/or an ammonia donor to the medium before culturing the above-mentioned microorganisms, preferably o, oi% by weight to 400% by weight, more preferably 0.1% by weight to the medium. 50
It is a serious consideration%.

A culture solution in which one or more types of microorganisms producing phenylalanine ammonia-lyase enzyme and esterase enzyme are cultured, microbial cells collected from this culture solution, or a processed product of the microorganism cells, or a phenylalanine ammonia-lyase enzyme isolated from the above-mentioned microorganisms. When the esterase enzyme is made to act on cinnamic acid ester and ammonia and/or an ammonia donor, a culture solution in which these microorganisms are cultured, microbial cells collected from this culture solution, or a processed product of the microorganism cells, or Preferably, cinnamic acid ester is added to the medium used for producing the phenylalanine ammonia lyase enzyme and esterase enzyme isolated from the above-mentioned microorganisms.
00% by weight, more preferably 0.1% to 50% by weight
Good to add.

However, the amount of the medium used as a standard for defining the preferable addition amount of cinnamic acid ester is the amount necessary and sufficient to produce the microorganisms used.

If the amount of cinnamic acid ester added is below the above range, L-
The amount of phenylalanine produced is small, and production efficiency tends to decrease. Moreover, if it exceeds the above range, the efficiency of the enzymatic reaction will deteriorate, and the production efficiency will also tend to decrease.

The amount of ammonia and/or ammonia donor added together with the cinnamic acid ester is such that the number of moles of ammonia and/or the ammonia donor as ammonia is preferably 3 to 3 to the number of moles of the cinnamic acid ester as cinnamic acid.
The amount is 1OO times, more preferably 5 to 80 times.

The medium used for culturing one or more types of microorganisms that produce phenylalanine ammonia-lyase enzyme and esterase enzyme is not particularly limited, and may be a normal nutrient medium containing hydrocarbons, nitrogen, inorganic salts, organic trace elements, etc. Can be used.

Carbon sources for the above medium include carbohydrates such as glucose and sucrose, vegetable oils and fats, organic acids such as acetic acid, etc., and nitrogen sources include ammonia, ammonium acetate,
Examples include various ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium nitrate, peptone, meat extract, and yeast extract.

Furthermore, the above-mentioned medium may be supplemented with inorganic ions such as various phosphates, sulfates, chlorides, etc., and organic trace elements such as vitamins, as required.

Cultivation for increasing the number of the above microorganisms, in other words, cultivation for producing phenylalanine ammonia lyase enzyme and esterase enzyme, may be carried out by a conventional method, for example, under aerobic conditions at a pH of 2 to 11 and a temperature of 15°C.
It may be carried out for 1 to 10 days at an appropriate temperature range of 40°C to 40°C.

However, when the enzyme acts on the cinnamic acid ester and ammonia and/or an ammonia donor, it is preferable to carry out the reaction at a pH of 6 to 11 and at a temperature of 20°C to 40°C.

So, phenylalanine ammonia lyase enzyme and/or
Or, when culturing the microorganism by adding cinnamic acid ester and ammonia and/or an ammonia donor to the medium before culturing one or more microorganisms that produce esterase enzymes,
It is preferable that the pH does not fall below 6 from the middle stage of culture.

In addition, when cinnamic acid ester and ammonia and/or an ammonia donor are added to a medium in which one or more types of microorganisms producing phenylalanine ammonia-lyase enzyme and/or esterase enzyme are cultured, or to a medium in which the above-mentioned microorganisms are being cultured. Alternatively, one or more microorganisms that produce phenylalanine ammonia lyase enzyme and/or esterase enzyme are cultured in a medium excluding cinnamate ester, and then collected, and the microorganism containing cinnamate ester and ammonia and/or an ammonia donor is collected. When culturing is continued in an appropriate medium, or a culture solution in which one or more types of microorganisms producing phenylalanine ammonia lyase enzyme and/or esterase enzyme are cultured, microorganism cells collected from this culture solution, or microorganism cell treatment of this microorganism cell. When a phenylalanine ammonia lyase enzyme and an esterase enzyme isolated from the above-mentioned microorganisms are allowed to act on a cinnamic acid ester and ammonia and/or an ammonia donor,
Preferably, the pH does not fall below 6 after contacting the phenylalanine ammonia-lyase enzyme and the esterase enzyme with the cinnamic acid ester and the ammonia and/or the ammonia donor.

A culture solution in which one or more types of microorganisms producing phenylalanine ammonia-lyase enzyme and esterase enzyme are cultured, microbial cells collected from this culture solution, or a processed product of the microorganism cells, or a phenylalanine ammonia-lyase enzyme isolated from the above-mentioned microorganisms. The reaction solution in the case where the cinnamic acid ester and the ammonia and/or ammonia donor are allowed to act on the cinnamic acid ester and the ammonia and/or ammonia donor is not particularly limited. The reaction conditions are preferably pH 6-11 and temperature 20°C-40°.
It is fine if it is C.

Separation and purification of the 1-phenylalanine accumulated in the culture solution or reaction solution can be carried out using a conventional ion exchange resin method or other known methods in combination with the conventional ion exchange resin method.

(Example) EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples.

In the following examples, phenylalanine was quantified based on the position of ninhydrin coloring by thin layer chromatography and by high performance liquid chromatography (column: Fine).
pak Sil C1B, mobile bed: water 600mff
1/methanol 400d/phosphorus 110.5d/l-sodium pentanesulfonate 50mg).

Example 1 Each of the following microorganisms ■ to ■ was placed in medium 5 (1+j
-
A platinum loop was inoculated and cultured with rotation at 25°C for 5 days.

Medium composition Peptone 1.0% by weight Yeast extract 1.0% by weight, Po
, 0.2% by weight M g S
Oa・7H,OO, 05% by weight Shea soft part oil (contains about 10% by weight of cinnamic acid terpene alcohol ester) 1.0% by weight Remaining water * The number of moles of cinnamic acid and the number of moles of ammonia in the medium The ratio was 1:37.

Cultured microorganisms ■ Cladosporium colocasiae
IFO6698 ■ [urotium chevalieri]
IFO4090■Gonatobotryum apiculaLu
m) rho 9098 ■ Pellicularia filamentosa
) IFO6254■Rhodosporjdiu+m Loruloides
es) IFO0559 ■Rhodotorula texensis
IFO0932■ Saccaromycobsis fivierigera (Sacc) + aromycopsis fi
buligera) IFO0107 ■ Sporobolomyces roseus
IFO1040 The culture solution obtained as described above accumulated phenylalanine in the amounts shown in the table below.

*+Fo: Fermentation Research Institute Preserved Bacterial Strain Example 2 0 Dosboridium toruroides (Rho dos
poridium toruloides.

IFO-0559) was added to the medium 50Ir1 (pI
]6) Into a 500 ml Erlenmeyer flask containing -
A platinum loop was inoculated and cultured with rotation at 25°C for 7 days.

Medium composition Glucose 1.0% by weight Peptone 0.3% by weight NH,C
I 0.3 wt%KH,P0
．． 0.05% by weight, HPo,
0.2 wt% M g S Oa
・7H,OO,05% by weight shea soft part oil (contains about 10% by weight of cinnamic acid terpene alcohol ester) 1o, o% by weight remaining
The ratio of the number of moles of cinnamic acid and the number of moles of ammonia in the water*medium is:
The ratio was 1:6.

The culture solution obtained as described above contains 2.5 tagl-
Noshi - Phenylalanine was accumulated.

Example 3 Talatosborium colocache
porium colocasiaeS TFO-(
i69B) was inoculated into a 500 d Erlenmeyer flask containing 50 d of a medium (pH 6) with the following composition, and incubated at 25°C.
Rotation culture was carried out at C for 7 days.

On days 2.3.4.5 and 6 after roasting, add 5 g of crudely purified cinnamic acid terpene alcohol ester (same as that used in the medium with the composition below) to 3 rails of ammonia water adjusted to pH 10°5. It was added a total of 5 times.

Medium composition Glucose 1. OfI amount% peptone 0.3 weight% NH, CI
0.3 wt%KH□PO'
, 0.05% by weight, IPO,0
, 2% by weight M g S O4.7H,OO, 05% by weight Crude cinnamic acid ester produced from shea soft part oil (approximately 60% by weight as cinnamic acid ester) 10.0% by weight remaining
The ratio of the number of moles of cinnamic acid and the number of moles of ammonia in the water*medium is 1
: It was 6.

In the culture solution obtained as described above, 2(1+g/d) of L-phenylalanine was accumulated.

Example 4 Talatosborium Kolokashe (C1a d.

sporium colocasiae, IFO-6
698) was inoculated into a 500 at Erlenmeyer flask containing 50 d of medium (pH 6) with the following composition, and 2
Rotation culture was carried out at 5°C for 5 days.

Medium composition Glucose 1.0% by weight Peptone 0.3% by weight NH,
CI 0.3 Weight%KH,P
O40.05% by weight, HPo, 0.2% by weight M
g S Oa ・7H,OO,05% by weight L-phenylalanine 0.02% by weight 1% balance
Water The bacterial cells were collected from the obtained culture solution by filtration, 0, OIM-KH, PO, liquid 100.
Clean with 1d.

After adjusting p11 to 10.5 with hydrochloric acid, 4d of 28% ammonia water was filled up to 50ml with water, and 1.0ml of the same crude cinnamic acid ester used in Example 3 was added.
g and the above-mentioned filtered bacterial cells were added, and the mixture was kept at 30°C for 24 hours to react. The reaction solution contained 3 mg/ml of phenylalanine.

〔Effect of the invention〕

According to the method of the present invention, L-
Phenylalanine can be produced.

Claims (1)

[Claims] A method for producing L-phenylalanine, which comprises using a cinnamic acid ester and ammonia and/or an ammonia donor as raw materials when producing L-phenylalanine by the action of an enzyme.