JP2003000294A - Method for producing l-phenylalanine derivative by microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for readily obtaining an L-phenylalanine derivative having high optical purity by using a cinnamic acid derivative having a substituent on a phenyl group as a raw material, and further using a microorganism, and further to provide the L-phenylalanine derivative having the high optical purity by using the method. SOLUTION: This method for producing the phenylalanine derivative represented by formula (1) (symbols in formula (1) are equal to those in the specification) is characterized in that the cinnamic acid derivative is reacted in the presence of ammonia by the action of microorganisms belonging to the genus Cladosporium, the genus Eurotium, the genus Thanatephorus, the genus Gonatobotryum or the genus Sporobolomyces, a component of the microorganism, or a treated substance of the microorganism. The L-phenylalanine derivative having the high optical purity is obtained by this method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an optically active L-phenylalanine derivative obtained by the action of a microorganism and a method for producing the same. In particular, the present invention relates to a method for producing a corresponding optically active L-phenylalanine derivative by adding ammonia to a cinnamic acid derivative. The optically active L-phenylalanine derivative obtained by the present invention is useful as a raw material for the synthesis of medicines, agricultural chemicals and other fine chemicals.

[0002]

2. Description of the Related Art As a method for obtaining an optically active amino acid,
Many organic synthetic reactions utilizing asymmetric catalysts and fermentative production methods utilizing the specificity of microorganisms have been investigated. As one of the production methods for obtaining optically active phenylalanine, there is a method of reacting cinnamic acid with phenylalanine ammonia-lyase, which is an enzyme of a microorganism, to obtain optically active phenylalanine (British Patent No. 1489468,
JP-A-53-96388). These utilize an enzymatic reaction of adding an amino group to the α-carbon of cinnamic acid by the action of an enzyme under a high ammonia concentration, and have an optical purity of L
-Phenylalanine can be produced.

Optically active L having a substituent on the phenyl group
As a method of obtaining a -phenylalanine derivative, a method of introducing a substituent on the phenyl group by using various modification reactions for L-phenylalanine can be considered. However, in such a method, a decrease in yield and purity due to a side reaction of a portion other than the phenyl group, and a decrease in optical purity due to progress of racemization under severe reaction conditions are unavoidable. Further, since it is difficult to obtain the regiospecificity of the substituent to be introduced on the phenyl group, it is not practical because of low yield and difficulty of separation and purification.

Therefore, a method is conceivable in which the above-mentioned phenylalanine ammonia-lyase is used to cause the enzyme to act on a cinnamic acid derivative having a substituted phenyl group in which a desired substituent has been introduced in advance. Since the enzymatic reaction proceeds under mild conditions, it is expected that an optically active L-phenylalanine derivative can be obtained with high purity without causing any adverse effect on the phenyl group and the substituents on the phenyl group due to side reactions. However, the substrate specificity of phenylalanine ammonia-lyase is generally strict, and the reactivity to the substituted phenyl compound is significantly lower than that of the original reaction to obtain L-phenylalanine from cinnamic acid, or may hardly react. Many. A method of obtaining an optically active fluorinated phenylalanine by the action of an enzyme using a cinnamic acid derivative having a fluorinated phenyl group as a raw material (JP-A-63-1).
48992) and a method for obtaining a phenylalanine derivative using a specific microorganism of the genus Rhodotorula (US Pat. No. 59.
81239) and only a few cases have been disclosed.

[0005]

DISCLOSURE OF THE INVENTION The present invention uses a cinnamic acid derivative having a substituent on the phenyl group as a raw material, and a novel method for easily obtaining an L-phenylalanine derivative having a high optical purity by using a microorganism, and a method thereof. An object is to provide an L-phenylalanine derivative having a high optical purity obtained by the method.

[0006]

DISCLOSURE OF THE INVENTION In order to search for a microorganism having an ammonia lyase activity which is highly reactive to a cinnamic acid derivative having a substituent on the phenyl group, the inventors of the present invention screened a novel soil microorganism, and known phenylalanine. The research on the reactivity of the ammonia-lyase-producing microorganisms was conducted intensively. And new genus Cladosporium , Eu
Rotium , Thanatephorus , Gonatobotryum , Sporob
The present inventors have found the ability to convert a cinnamic acid derivative having various substituted phenyl groups into a substituted phenyl type L-phenylalanine derivative in a microorganism belonging to the olomyces genus, and arrived at the present invention.

The genus Cladosporium, the genus Eurotium , Than
It has not been known so far that microorganisms belonging to the genus atephorus, the genus Gonatobotryum, and the genus Sporobolomyces have the ability to convert cinnamic acid derivatives having various substituted phenyl groups into L-substituted phenylalanine derivatives. It is the first thing that the people found out.

That is, the present invention provides the following method for producing an L-phenylalanine derivative and L-phenylalanine derivative obtained by the method.
A phenylalanine derivative is provided. 1) Cladosporium , Eurotium , Thanatephorus , G
The following formula (1), characterized in that a microorganism belonging to any one of the genus onatobotryum and the genus Sporobolomyces , the microbial component or the biological treated product is used.

[Chemical 4] (However, R¹, R², R³, R^Four, R^FiveEach independently
Hydrogen, hydroxyl group, alkyl group (where alkyl is straight chain or branched
It may be omitted, and has 1 to 4 carbon atoms. ), A
Lucoxy group (Alkyl forming alkoxy is linear or
May be branched and has 1 to 4 carbon atoms.
It ), Cyano group, nitro group, halogen, NR ⁶R⁷(R
⁶, R⁷Are each independently hydrogen, straight chain or branched
Is an alkyl group having 1 to 4 carbon atoms, or
Or R⁶And R⁷May form a ring with
The number may be 3 to 5 and may include a hetero atom. ),is there
Or a phenyl group which may have a substituent. However
And R¹, R², R³, R^Four, R^FiveIs all hydrogen at the same time
It never happens. ) L-phenylalanine derivative represented by
Method of manufacturing conductor.

2) The following equation (2)

[Chemical 5] (However, R¹, R², R³, R^Four, R^FiveEach independently
Hydrogen, hydroxyl group, alkyl group (where alkyl is straight chain or branched
It may be omitted, and has 1 to 4 carbon atoms. ), A
Lucoxy group (Alkyl forming alkoxy is linear or
May be branched and has 1 to 4 carbon atoms.
It ), Cyano group, nitro group, halogen, NR ⁶R⁷(R
⁶, R⁷Are each independently hydrogen, straight chain or branched
Is an alkyl group having 1 to 4 carbon atoms, or
Or R⁶And R⁷May form a ring with
The number may be 3 to 5 and may include a hetero atom. ),is there
Or represents a phenyl group which may have a substituent. However
And R¹, R², R³, R^Four, R^FiveIs all hydrogen at the same time
It never happens. ) Cinnamic acid derivative and an
In the presence of Monia,CladosporiumGenus,EurotiumGenus,Thanat
ephorusGenus,GonatobotryumGenus,SporobolomycesGenus
Microorganisms belonging to it, microbial components thereof or microorganisms thereof
It is represented by the formula (1) described in the above item 1 which causes the treated material to act.
A method for producing an L-phenylalanine derivative.

3) R ¹ , R ² , R ³ , R ⁴ and R ^{5 of the} formula (1)
Are each independently hydrogen, a cyano group or a hydroxyl group, but not all are hydrogen at the same time.
Alternatively, the method for producing the L-phenylalanine derivative according to item 2 above. 4) Cladosporium spp. Euro cultivated in a medium containing phenylalanine or a phenylalanine derivative in advance.
genus tium, genus Thanatephorus, genus Gonatobotryum , Sporobol
4. The method for producing an L-phenylalanine derivative according to any one of items 1 to 3 above, which uses a microorganism belonging to any of the omyces genus. 5) The method for producing an L-phenylalanine derivative according to the above item 2, wherein at least a part of ammonia is supplied as a carbonate. 6) The method for producing an L-phenylalanine derivative as described in 2 or 4 above, wherein the pH of the working solution is adjusted to 8.5 to 1.1 using carbon dioxide. 7) The method for producing an L-phenylalanine derivative according to item 2, wherein at least a part of the obtained L-phenylalanine derivative is recovered as an ammonium salt.

8) The method for producing an L-phenylalanine derivative according to the above 5 or 6, wherein at least a part of the obtained L-phenylalanine derivative is recovered as a carbonate. 9) The method for producing an L-phenylalanine derivative represented by the formula (1) according to item 2 above, wherein the microorganism, its microbial component or its microbial treated product is allowed to act in a liquid having a cinnamic acid derivative concentration of 5% by mass or less. 10) The L-phenylalanine derivative is 3-cyano-L
-The method for producing an L-phenylalanine derivative according to any one of items 1 to 3, which is phenylalanine. 11) L-phenylalanine derivative is 4-cyano-L
-The method for producing an L-phenylalanine derivative according to any one of items 1 to 3, which is phenylalanine. 12) The method for producing an L-phenylalanine derivative according to any one of items 1 to 3, wherein the L-phenylalanine derivative is 3-hydroxy-L-phenylalanine. 13) The method for producing an L-phenylalanine derivative according to any one of the above items 1 to 3, wherein the L-phenylalanine derivative is 4-hydroxy-L-phenylalanine.

14) The L-phenylalanine derivative is
4. The method for producing an L-phenylalanine derivative according to any one of items 1 to 3, which is 3,4-dihydroxy-L-phenylalanine. 15) The microorganism used is Cladosporium colocasiae , Eur
otium chevalieri , Thanatephorus cucumeris , Gonatob
5. The method for producing an L-phenylalanine derivative according to the above 1, 2 or 4, which is either otryum apiculatum or Sporobolomyces roseus . 16) The microorganism used is Cladosporium colocasiae IFO
6698, Eurotium chevali eri IFO 4090, Thanatephorus
cucumeris IFO 6254, Gonatobotryum apiculatum IFO 90
98, Sporobolomyces roseus IFO 1040, or the method for producing an L-phenylalanine derivative according to the above 1, 2, or 4, 17) The following formula (1) having an optical purity of 100% (detection limit of 0.1%) obtained by the production method according to any one of items 1 to 16 above.

[Chemical 6] (However, R¹, R², R³, R^Four, R^FiveEach independently
Hydrogen, hydroxyl group, alkyl group (where alkyl is straight chain or branched
It may be omitted, and has 1 to 4 carbon atoms. ), A
Lucoxy group (Alkyl forming alkoxy is linear or
May be branched and has 1 to 4 carbon atoms.
It ), Cyano group, nitro group, halogen, NR ⁶R⁷(R
⁶, R⁷Are each independently hydrogen, straight chain or branched
Is an alkyl group having 1 to 4 carbon atoms, or
Or R⁶And R⁷May form a ring with
The number may be 3 to 5 and may include a hetero atom. ),is there
Or represents a phenyl group which may have a substituent.
However, R¹, R², R³, R^Four, R^FiveIs all hydrogen at the same time
Never be. ) L-phenylalanine represented by
Derivative.

18) R ¹ , R ² , R ³ , R ⁴ , and R ⁵ each independently represent hydrogen, a cyano group, or a hydroxyl group, but they are not all hydrogen at the same time. L-phenylalanine derivative of. 19) The L-phenylalanine derivative is 4-cyano-L
-The L-phenylalanine derivative according to item 18, which is phenylalanine. 20) The L-phenylalanine derivative according to item 18, wherein the L-phenylalanine derivative is 3-hydroxy-L-phenylalanine. 21) The L-phenylalanine derivative according to item 18, wherein the L-phenylalanine derivative is 4-hydroxy-L-phenylalanine. 22) The L-phenylalanine derivative according to item 18, wherein the L-phenylalanine derivative is 3,4-dihydroxy-L-phenylalanine.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in detail below.
It Applied to the present invention,CladosporiumGenus,Eurotium
Genus,ThanatephorusGenus,GonatobotryumGenus is widespread in nature
It is a type of filamentous fungus that is often found. AlsoSporobolomyces
The genus is a type of yeast that is also widely found in nature.
It Phenylalanine ammonia that catalyzes this reaction
Examples of microbial strains showing lyase activity include foundations
Deposited at Fermentation Research InstituteCladosporium colocasiae 
IFO 6698,Eurotium chevalieriIFO 4090,Thanateph
orus cucumerisIFO 6254,GonatobotryumapiculatumI
FO 9098,Sporobolomyces roseusListed each stock of IFO 1040
You can get it. In addition, of the microorganisms used in the present invention
home,ThanatephorusGenus was previouslyPelliculariaGenus and
It was classified asThanatephorus cucume
risWhenPellicularia filamentosaAre the same microorganism
It

Specifically, for example, Eurotium chevalieri
The IFO 4090 strain is grown at a temperature of 15 ° C to 3 ° C by a commonly known method for culturing microorganisms, for example, in a nutrient medium such as 1% peptone.
5 ° C, preferably 18 ° C to 30 ° C for 24 hours to 7
After culturing for about one day, the resulting broth contains 1 ppm to 20%, preferably 10 ppm to 10% of a cinnamic acid derivative having a substituent as a reaction raw material, and 0.5 M to 11 M, preferably 1 M to a final concentration of ammonia. It is added so as to be 9M, and the pH is adjusted to 8.5 to 11, preferably 9 to 10.5, and then continuously for 1 hour to 20 in the same temperature range.
The reaction can be achieved by continuing stirring for about 0 hours.
Acids used to adjust the pH include sulfuric acid, hydrochloric acid,
Inorganic acids such as phosphoric acid, boric acid and carbonic acid, organic acids such as formic acid, acetic acid and propionic acid and salts thereof can be used. At this time, it is useful to use a volatile acid because the reaction solution can be sterilized and distilled off to omit the step of desalting and to easily separate the product. Carbonic acid is suitable as such an acid. Carbonic acid in this case includes carbonic acid produced by dissolving carbon dioxide in an aqueous solution by bubbling or the like. Further, salts of these acids and ammonia can be used as an ammonia source for the reaction liquid, and it is preferable to use ammonium carbonate or ammonium hydrogen carbonate as a part or all of the ammonia source for the reason as described above.

The cinnamic acid derivative having a substituent to be added does not necessarily have to be completely dissolved, but a solvent, a surfactant or the like which improves the solubility or dispersibility in the reaction solution may be used. It can also be added. The cinnamic acid derivative may be added continuously or intermittently depending on the consumption of the cinnamic acid derivative as the reaction proceeds. In this case, the concentration of the cinnamic acid derivative in the reaction solution is not limited to the above. However, it is recognized that the addition of excess cinnamic acid derivative inhibits the enzymatic reaction, and it is desirable that the concentration of the cinnamic acid derivative dissolved in the reaction solution does not exceed 5% at the maximum during most of the reaction period. .

The carbon source of the medium for culturing microorganisms includes sugars such as glucose, sucrose, fructose and molasses, organic substances such as ethanol, acetic acid, citric acid, succinic acid, lactic acid, benzoic acid and fatty acids, or these. Alkali metal salts of n, paraffins and other aliphatic hydrocarbons, and natural organic substances such as peptone, meat extract, fish extract, soybean flour, bran, malt extract and potato extract, alone or in combination. Therefore, it can be used at a concentration of usually 0.01% to 30%, preferably about 0.1% to 10%.

The medium nitrogen source for culturing microorganisms includes, for example, inorganic nitrogen compounds such as ammonium sulfate, ammonium phosphate, sodium nitrate and potassium nitrate, nitrogen-containing organic substances such as urea and uric acid, peptone, meat extract, fish extract, Natural organic substances such as soybean flour, malt extract, and potato extract are used alone or in combination, and usually 0.01% to 30%, preferably 0.1% to 10%.
It can be used at a moderate concentration.

Further, if necessary, a phosphate such as potassium dihydrogen phosphate, a metal salt such as magnesium sulfate, ferrous sulfate, calcium acetate, manganese chloride, copper sulfate, zinc sulfate, cobalt sulfate and nickel sulfate may be added. It is added to improve the growth of bacteria. The concentration of addition depends on the culture conditions,
Generally, 0.01% to 5% for phosphate, 10 ppm to 1% for magnesium salt, 0.1 p for other compounds.
It is about pm to 1000 ppm. In addition, depending on the medium to be selected, for example, yeast extract, casamino acid, yeast nucleic acid as a source of vitamins, amino acids, nucleic acids, etc., from 1 ppm to 1
By adding about 00 ppm, the growth of bacteria can be improved.

Further, in order to improve the reactivity of the fungus to the cinnamic acid derivative, phenylalanine was added to the phenylalanine ammonia-lyase at an amount of 10 ppm to
It is useful to add 1%.

When any of the components is used, the pH of the medium
Is adjusted to 4.5 to 8, preferably 5 to 7.5. In addition, the microbial cells pre-cultured in the medium as described above are collected from the culture solution by a method such as centrifugation or membrane filtration and used for the reaction to reduce contaminants brought in from the culture solution. It is useful for simplifying the fractionation of the product.

The phenylalanine derivative formed in the reaction solution is a commonly used method such as centrifugation, membrane filtration, vacuum drying, distillation, solvent extraction, salting out, ion exchange, various chromatographies depending on the properties of the reaction solution. Is collected in. It can be achieved simply as follows. For example, after removing the bacterial cells or the treated product from the reaction solution by filtration and centrifugation, solvent extraction or adjusting the reaction solution to acidic to precipitate unreacted starting cinnamic acid derivative and remove the precipitate. The pH of the obtained supernatant is again adjusted to near the isoelectric point of the phenylalanine derivative, and the derivative that precipitates is similarly recovered. In this way, the product can be recovered from the reaction solution in good yield and purity. In addition, it is useful to remove excess ammonia from the reaction solution in advance by distillation or to distill off water to increase the concentration in order to improve the product recovery rate at the isoelectric point.

More simply, the pH of the reaction solution is adjusted with a volatile acid. If the conversion rate is sufficiently high, after sterilization, it is as it is.If a large amount of the cinnamic acid derivative as the raw material remains, it is acidified by solvent extraction, or the precipitate is filtered under acidic conditions.
The product can be isolated as a salt of a phenylalanine derivative by distilling off water and acid / base after removal by centrifugation or the like. Carbonic acid and its ammonium salt are preferable as the volatile acid used in such a method.

Depending on the nature of the reaction product, the reaction rate may decrease due to the accumulation of the product in the reaction solution. In such a case, a method of adding water containing ammonia, physiological saline, and a reaction buffer solution to the reaction solution and continuously diluting the reaction solution according to the concentration of the product is preferable. When the reaction rate decreases, the bacteria are collected, the supernatant is recovered as a product solution, and the collected bacteria are returned to the solution or suspension containing the reaction raw materials to recover the reaction speed. You can These methods can be repeated as many times as long as the ammonia lyase activity of the microorganism is maintained.

The present invention also applies to a cell-free extract of a microorganism applicable to the present invention, and a treated product such as a cell-free extract obtained by concentrating and extracting a component which catalyzes the above reaction. It can be carried out. Further, it can also be achieved by immobilizing the microorganism applicable to the present reaction or its extract / extract component on a sparingly soluble carrier and bringing this immobilization product into contact with the raw material solution. Examples of such an immobilization carrier include polyacrylamide, polyvinyl alcohol, poly-N-vinylformamide, polyallylamine, polyethyleneimine, methylcellulose, glucomannan, alginate, carrageenan and the like, and further copolymerization and crosslinked products thereof, microorganisms, etc. Alternatively, compounds capable of forming a poorly water-soluble solid content including the extracted components may be used alone or in combination. Further, it is also possible to use activated carbon, porous ceramics, glass fiber, porous polymer molded body, nitrocellulose membrane, or the like in which a microorganism or its extract liquid / extracted component is held on an object previously formed as a solid. . Performing a continuous reaction using such an immobilized product is useful for avoiding the inhibition of the enzymatic reaction due to the accumulation of products.

The cinnamic acid derivative having a substituent as a raw material of the present invention is represented by the following formula (2)

[Chemical 7] (However, R ¹ , R ² , R ³ , R ⁴ , and R ⁵ are each independently hydrogen, a hydroxyl group, or an alkyl group (the alkyl may be linear or branched, and has 1 to 4 carbon atoms). ), An alkoxy group (the alkyl forming the alkoxy may be linear or branched and has 1 to 4 carbon atoms), a cyano group, a nitro group, a halogen such as chlorine or fluorine, NR ⁶ R. ⁷ (R ⁶ and R ⁷ each independently represent hydrogen, a linear or branched alkyl group having 1 to 4 carbon atoms, or R ⁶ and R ⁷ may form a ring. Of course, this ring represents a phenyl group which has 3 to 5 carbon atoms and may contain a hetero atom), or a phenyl group which may have a substituent, provided that R ¹ , R ² , R ³ and R ⁴ , R
⁵ is not all hydrogen at the same time. ), And preferably R ¹ , R ² , R ³ , R ⁴ , and R ⁵ are each independently hydrogen, a cyano group, or a hydroxyl group (provided that not all are hydrogen). ).

Particularly preferred examples are 3-cyanocinnamic acid, 4-cyanocinnamic acid, 3-hydroxycinnamic acid, 4-
Examples thereof include hydroxycinnamic acid and 3,4-dihydroxycinnamic acid.

The cinnamic acid derivative having a substituent, which serves as a starting material for these reactions, reacts the acetic anhydride with the aldehyde group of the benzaldehyde derivative having the corresponding substituent introduced.
So-called Perkin reaction (Arch. Pharm. (Weinh
eim) 327 (10), p619-625 (1994), etc.) or a method of reacting malonic acid with pyridine solvent in the presence of piperidine (Journal of Chemical Society, p357-360).
(1939) etc.) and various other amendments (Synth. Commun.,
29 (4), p573-581 (1999) etc.) and the like.

L-having a substituent, which is the product of the present invention
The phenylalanine derivative has the following formula (1)

[Chemical 8] (However, R ¹ , R ² , R ³ , R ⁴ , and R ⁵ are each independently hydrogen, a hydroxyl group, or an alkyl group (the alkyl may be linear or branched, and has 1 to 4 carbon atoms). ), An alkoxy group (the alkyl forming the alkoxy may be linear or branched and has 1 to 4 carbon atoms), a cyano group, a nitro group, a halogen such as chlorine or fluorine, NR ⁶ R. ⁷ (R ⁶ and R ⁷ each independently represent hydrogen, a linear or branched alkyl group having 1 to 4 carbon atoms, or R ⁶ and R ⁷ may form a ring. Of course, this ring represents a phenyl group which has 3 to 5 carbon atoms and may contain a hetero atom), or a phenyl group which may have a substituent, provided that R ¹ , R ² , R ³ and R ⁴ , R
⁵ is not all hydrogen at the same time. ), And preferably R ¹ , R ² , R ³ , R ⁴ , and R ⁵ are each independently hydrogen, a cyano group, or a hydroxyl group (however, not all are hydrogen). ).

Particularly preferred examples are 3-cyano-L-phenylalanine, 4-cyano-L-phenylalanine and 3
-Hydroxy-L-phenylalanine (metatyrosine), 4-hydroxy-L-phenylalanine (tyrosine), 3,4-dihydroxy-L-phenylalanine (DOPA).

The L-phenylalanine derivative obtained in the present invention has a high optical purity, and the measuring method shown in the examples shows an optical purity of 100% within the detection limit of 0.1%. Further, in the present invention, the substituents on the phenyl group, such as a cyano group and a hydroxyl group, are not affected under the mild conditions of this reaction and are retained until the end of the reaction, and the desired L
-A phenylalanine derivative can be obtained in good yield with a conversion rate of almost 100%.

Therefore, according to the method of the present invention, an L-phenylalanine derivative having a high optical purity can be obtained by a one-step reaction using a cinnamic acid derivative having a substituent which can be easily obtained by organic synthesis as a substrate. The L-phenylalanine derivative thus obtained is useful as a synthetic intermediate in the field of fine chemicals such as pharmaceuticals and agricultural chemicals requiring high optical purity.

[0033]

The present invention will be described below with reference to examples.
These examples do not limit the scope of the invention.
The L-phenylalanine derivative thus obtained was separated and quantified by reverse phase HPLC under the following analytical conditions. Column: Shodex (registered trademark of Showa Denko KK) RSpak N
N-614 (Showa Denko) Column temperature: 40 ° C Eluent: acetonitrile / water / 50 mM H ₃ PO ₄ -K
H ₂ PO ₄ aqueous solution (pH 3) = 20/70/10 Flow rate: 1.0 ml / min Detection: By UV 210 nm absorption

The optical purity of the obtained L-phenylalanine derivative was analyzed by the optical resolution HP under the following conditions.
Performed by LC. Column: Shodex (registered trademark of Showa Denko KK) ORpak C
RX-853, Column temperature: Room temperature (22 ° C), Eluent: Acetonitrile / water = 15/85, CuSO ₄
2.5 mM, flow rate: 1.0 ml / min, detection method: by UV 256 nm absorption.

Example 1 Cladosporium colocasiae IFO 6698 (sold from the Fermentation Research Institute) was mixed with potato dextrose agar (Di
fco) and cultured for 72 hours in a 25 ° C. constant temperature bath. The formed microbial cells are scraped off, and a liquid medium 100 having the following composition in a 500 mL baffled flask is used.
Suspended in mL.

<Media composition> Glucose 10g Peptone 5g Yeast extract 3g Malt extract 3g L-phenylalanine 0.5g Distilled water 1L pH 6.0

The flask was placed in a constant temperature rotary shaking incubator at 25 ° C. and cultured at 150 rpm for 3 days. The microbial cells obtained were collected by centrifugation at 10,000 g, and the same volume as the culture medium was added to 2M ammonium hydrogencarbonate / 6M ammonia solution (after dissolving 2M ammonium hydrogencarbonate corresponding to a final concentration of 2M in a small amount of water, a final concentration of 6M). Add a considerable amount of concentrated ammonia water and fi
It was suspended in the one which was added up, pH = 10.3). 4-in bacterial suspension
1% (w / v) of cyanocinnamic acid was added, and the reaction mixture was placed in a constant temperature rotary shaking incubator at 30 ° C. and reacted at 120 rpm for 96 hours. When the reaction solution was subjected to reverse phase HPLC, 0.58% accumulation of 4-cyano-L-phenylalanine was detected by comparison with the standard product. According to the gas chromatographic analysis of the derivative, the optical purity is 100% (detection limit 0.1%).
Was calculated.

Example 2 Cladosporium colocasiae IFO 6698 was cultured in the same manner as in Example 1 and suspended in 2M ammonium hydrogen carbonate / 6M ammonia solution (pH = 10.3). 1% (w / v) of 3-cyanocinnamic acid was added to the bacterial suspension, and the suspension was placed in a constant temperature rotary shaking incubator at 30 ° C. and reacted at 120 rpm for 96 hours. When the reaction solution was subjected to reverse-phase HPLC, 0.70% accumulation of 3-cyano-L-phenylalanine was detected by comparison with the standard product. According to gas chromatographic analysis of the derivative, the optical purity was 100% (detection limit 0.1%).

Example 3 Cladosporium colocasiae IFO 6698 was cultured in the same manner as in Example 1 and suspended in 2M ammonium hydrogencarbonate / 6M ammonia solution (pH = 10.3). 4-Hydroxycinnamic acid equivalent to 1% (w / v) was added to the bacterial suspension, and the suspension was placed in a constant temperature rotary shaking incubator at 30 ° C. and reacted at 120 rpm for 96 hours. When the reaction solution was subjected to reverse-phase HPLC, 0.33% accumulation of L-tyrosine was detected by comparison with the standard product. According to gas chromatographic analysis of the derivative, the optical purity was 100% (detection limit 0.1%).

Example 4 Cladosporium colocasiae IFO 6698 was cultured in the same manner as in Example 1 and suspended in 2M ammonium hydrogencarbonate / 6M ammonia solution (pH = 10.3). 3-Hydroxycinnamic acid equivalent to 1% (w / v) was added to the bacterial suspension, and the suspension was placed in a constant temperature rotary shaking incubator at 30 ° C. and reacted at 120 rpm for 96 hours. When the reaction solution was subjected to reverse phase HPLC, 0.80% accumulation of metatyrosine was detected by comparison with the standard product. According to gas chromatographic analysis of the derivative, the optical purity was 100% (detection limit 0.1%).

Example 5 Eurotium chevalieri IFO 4090 (sold from the Fermentation Research Institute) was mixed with potato dextrose agar medium (Difco
(Manufactured by the company) and cultured for 72 hours in a constant temperature bath at 25 ° C.
The formed microbial cells are scraped off, and 100 m of liquid medium having the following composition in a 500 mL baffled flask is used.
Suspended in L.

<Medium composition> Glucose 10g 5 g of peptone Yeast extract 3g Malt extract 3g L-phenylalanine 0.5g Distilled water 1L pH 6.0

The flask was placed in a constant temperature rotary shaking incubator at 25 ° C. and cultured at 150 rpm for 5 days. From the obtained culture solution, microbial cells were collected by centrifugation at 10,000 g,
2M ammonium hydrogen carbonate / 6M ammonia solution in the same volume as the culture solution (after dissolving ammonium hydrogen carbonate at a final concentration of 2M in a small amount of water, then adding concentrated ammonia water at a final concentration of 6M and filling up with water, pH = 10.3). 4-cyanocinnamic acid equivalent to 1% (w / v) was added to the bacterial suspension,
The plate was placed in a constant temperature rotary shaking incubator at 30 ° C. and reacted at 120 rpm for 120 hours. When the reaction solution was subjected to reverse phase HPLC, accumulation of 0.22% of 4-cyano-L-phenylalanine was detected. According to the optical resolution HPLC analysis of the derivative, the optical purity was calculated to be 100% (detection limit: 0.1%).

Example 6 Thanatephorus cucumeris IFO 6254 (sold from the Fermentation Research Institute) was mixed with potato dextrose agar medium (Di
fco) and cultured for 72 hours in a 25 ° C. constant temperature bath. Commercially available potato dextrose broth (Di
(manufactured by fco), and suspended in 100 mL of a baffled flask having a capacity of 500 mL and cultivated at a constant temperature rotary shaking incubator at 25 ° C. for 150 rpm for 5 days. Microbial cells were collected from the obtained culture broth by centrifugation at 10,000 g and suspended in a 2M ammonium hydrogencarbonate / 6M ammonia solution (pH = 10.3). 3-% cyanocinnamic acid 1% (w / v) in bacterial suspension
Corresponding amounts were added, and the mixture was placed in a constant temperature rotary shaking incubator at 30 ° C. and reacted at 120 rpm for 120 hours. Reverse reaction HP
When subjected to LC, accumulation of 0.39% of 3-cyano-L-phenylalanine was detected. Optical resolution of derivative HP
According to LC analysis, the optical purity is 100% (detection limit: 0.1
%)Met.

Example 7 Gonatobotryum apiculatum IFO 9098 (sold from Fermentation Research Institute) was cultured in the same manner as in Example 5, and the obtained cells were added to 2M ammonium hydrogen carbonate / 6M ammonia solution (pH = 10.3). Suspended. 4-Hydroxycinnamic acid (1% (w / v)) was added to the bacterial suspension, and the suspension was placed in a constant temperature rotary shaker at 30 ° C. and reacted at 120 rpm for 120 hours. When the reaction solution was subjected to reverse phase HPLC, it was 0.32%.
Accumulation of L-tyrosine was detected. The optical purity of the derivative is 100% by optical resolution HPLC analysis (detection limit: 0.
1%).

Example 8 Eurotium chevalieri IFO 4090 (sold by the Fermentation Research Institute) was cultured in the same manner as in Example 6 and suspended in 2M ammonium hydrogencarbonate / 6M ammonia solution (pH = 10.3). 3-Hydroxycinnamic acid 1% (w /
v) Corresponding amounts were added, and the mixture was placed in a constant temperature rotary shaking incubator at 30 ° C. and reacted at 120 rpm for 96 hours. When the reaction solution was subjected to reverse phase HPLC, 0.64% of 3-hydroxy-L was obtained.
-Phenylalanine (metathyrosine) accumulation was detected. According to the optical resolution HPLC analysis of the derivative, the optical purity was 100% (detection limit: 0.1%).

Example 9 50 mL of the reaction solution obtained in the same manner as in Example 1 was suction filtered with a filter paper to remove microbial cells. Carbon dioxide was passed through the obtained aqueous solution to adjust the pH to 7, then an equal volume of toluene was added, and stirring and extraction were performed. The aqueous layer obtained by fractionation was distilled off under reduced pressure using an evaporator. The obtained solid substance was analyzed by ¹ H-NMR, elemental analysis, and HPLC. The main component of the solid is ammonium salt of 4-cyano-L-phenylalanine, the content of which is per dry weight.
99.0%, and the recovery rate of 4-cyano-L-phenylalanine from the reaction solution was 93%.

Example 10 50 mL of the reaction solution obtained in the same manner as in Example 2 was suction filtered with a filter paper to remove microbial cells. Carbon dioxide was passed through the obtained aqueous solution to adjust the pH to 7, then an equal volume of toluene was added, and stirring and extraction were performed. The separated aqueous layer was dried under reduced pressure using an evaporator. The obtained solid substance was analyzed by ¹ H-NMR, elemental analysis, and HPLC. The main component of the solid is 4-cyano-L-phenylalanine, the content of which is 98.0% per dry weight, and the recovery ratio of 4-cyano-L-phenylalanine from the reaction solution is 91.3%.
Met.

[0049]

INDUSTRIAL APPLICABILITY According to the present invention, an L-phenylalanine derivative having various substituents on a phenyl group can be obtained simply and efficiently from a cinnamic acid derivative which can be easily synthesized as a raw material.

Claims (22)

[Claims] 1.CladosporiumGenus,EurotiumGenus,Thanatep
horusGenus,GonatobotryumGenus,SporobolomycesAny of the genera
Crab belonging to the above, said microbial component or said biological treatment
The following formula (1) characterized by using a thing [Chemical 1] (However, R¹, R², R³, R^Four, R^FiveEach independently
Hydrogen, hydroxyl group, alkyl group (where alkyl is straight chain or branched
It may be omitted, and has 1 to 4 carbon atoms. ), A
Lucoxy group (Alkyl forming alkoxy is linear or
May be branched and has 1 to 4 carbon atoms.
It ), Cyano group, nitro group, halogen, NR ⁶R⁷(R
⁶, R⁷Are each independently hydrogen, straight chain or branched
Is an alkyl group having 1 to 4 carbon atoms, or
Or R⁶And R⁷May form a ring with
The number may be 3 to 5 and may include a hetero atom. ),is there
Or a phenyl group which may have a substituent. However
And R¹, R², R³, R^Four, R^FiveIs all hydrogen at the same time
It never happens. ) L-phenylalanine derivative represented by
Method of manufacturing conductor. 2. The following formula (2) [Chemical 2] (However, R¹, R², R³, R^Four, R^FiveEach independently
Hydrogen, hydroxyl group, alkyl group (where alkyl is straight chain or branched
It may be omitted, and has 1 to 4 carbon atoms. ), A
Lucoxy group (Alkyl forming alkoxy is linear or
May be branched and has 1 to 4 carbon atoms.
It ), Cyano group, nitro group, halogen, NR ⁶R⁷(R
⁶, R⁷Are each independently hydrogen, straight chain or branched
Is an alkyl group having 1 to 4 carbon atoms, or
Or R⁶And R⁷May form a ring with
The number may be 3 to 5 and may include a hetero atom. ),is there
Or represents a phenyl group which may have a substituent. However
And R¹, R², R³, R^Four, R^FiveIs all hydrogen at the same time
It never happens. ) Cinnamic acid derivative and an
In the presence of Monia,CladosporiumGenus,EurotiumGenus,Thanat
ephorusGenus,GonatobotryumGenus,SporobolomycesGenus
Microorganisms belonging to it, microbial components thereof or microorganisms thereof
It is represented by the formula (1) according to claim 1, which causes a processed material to act.
A method for producing an L-phenylalanine derivative. 3. R ¹ , R ² , R ³ , R ⁴ , and R of formula (1)
^The method for producing an L-phenylalanine derivative according to claim 1 or 2, wherein ⁵ independently represent hydrogen, a cyano group, or a hydroxyl group, but not all are hydrogen at the same time. 4. Preliminarily phenylalanine or phenylalanine
Cultured in medium containing a lanine derivativeCladospori
umGenus,EurotiumGenus,ThanatephorusGenus,Gonatobotryum
Genus,SporobolomycesUsing microorganisms belonging to one of the genera
L-Phenylara according to any one of claims 1 to 3.
A method for producing a nin derivative. 5. The L according to claim 2, wherein at least a part of the ammonia is supplied as a carbonate.
-A method for producing a phenylalanine derivative. 6. The pH of the working solution is set to 8.5 to 8 using carbon dioxide.
The method for producing an L-phenylalanine derivative according to claim 2 or 4, wherein the method is adjusted to 11. 7. The method for producing an L-phenylalanine derivative according to claim 2, wherein at least a part of the obtained L-phenylalanine derivative is recovered as an ammonium salt. 8. The method for producing an L-phenylalanine derivative according to claim 5, wherein at least a part of the obtained L-phenylalanine derivative is recovered as a carbonate. 9. The method for producing an L-phenylalanine derivative according to claim 2, wherein the microorganism, the microbial component thereof or a treated product of the microorganism is allowed to act in a liquid having a cinnamic acid derivative concentration of 5% by mass or less. 10. The L-phenylalanine derivative is 3-
It is cyano-L-phenylalanine, The manufacturing method of the L-phenylalanine derivative in any one of Claims 1 thru | or 3. 11. An L-phenylalanine derivative is 4-
It is cyano-L-phenylalanine, The manufacturing method of the L-phenylalanine derivative in any one of Claims 1 thru | or 3. 12. The L-phenylalanine derivative is 3-
It is hydroxy-L-phenylalanine, The manufacturing method of the L-phenylalanine derivative in any one of Claims 1 thru | or 3. 13. The L-phenylalanine derivative is 4-
It is hydroxy-L-phenylalanine, The manufacturing method of the L-phenylalanine derivative in any one of Claims 1 thru | or 3. 14. The L-phenylalanine derivative is 3,
The method for producing an L-phenylalanine derivative according to any one of claims 1 to 3, which is 4-dihydroxy-L-phenylalanine. 15. The microorganism used is Cladosporium coloc
asiae , Eurotium chevalieri , Thanatephorus cucumeri
s , Gonatobotryum apiculatum , Sporobolomyces roseus
The method for producing an L-phenylalanine derivative according to claim 1, 2 or 4, wherein 16. The microorganism used is Cladosporium coloc
asiae IFO 6698, Eurotium chevalieri IFO 4090, Than
atephorus cucumeris IFO 6254, Gonatobotryum apicul
The method for producing an L-phenylalanine derivative according to claim 1, 2 or 4, wherein the method is atum IFO 9098 or Sporobolomyces roseus IFO 1040. 17. The manufacturing method according to claim 1.
Optical purity obtained by the method 100% (detection limit 0.1%)
The following formula (1) [Chemical 3] (However, R¹, R², R³, R^Four, R^FiveEach independently
Hydrogen, hydroxyl group, alkyl group (where alkyl is straight chain or branched
It may be omitted, and has 1 to 4 carbon atoms. ), A
Lucoxy group (Alkyl forming alkoxy is linear or
May be branched and has 1 to 4 carbon atoms.
It ), Cyano group, nitro group, halogen, NR ⁶R⁷(R
⁶, R⁷Are each independently hydrogen, straight chain or branched
Is an alkyl group having 1 to 4 carbon atoms, or
Or R⁶And R⁷May form a ring with
The number may be 3 to 5 and may include a hetero atom. ),is there
Or represents a phenyl group which may have a substituent.
However, R¹, R², R³, R^Four, R^FiveIs all hydrogen at the same time
Never be. ) L-phenylalanine represented by
Derivative. 18. R ¹ , R ² , R ³ , R ⁴ , and R ⁵ each independently represent hydrogen, a cyano group, or a hydroxyl group,
18. The L-phenylalanine derivative according to claim 17, wherein not all are hydrogen at the same time. 19. The L-phenylalanine derivative is 4-
The L-phenylalanine derivative according to claim 18, which is cyano-L-phenylalanine. 20. The L-phenylalanine derivative is 3-
The L-phenylalanine derivative according to claim 18, which is hydroxy-L-phenylalanine. 21. The L-phenylalanine derivative is 4-
The L-phenylalanine derivative according to claim 18, which is hydroxy-L-phenylalanine. 22. The L-phenylalanine derivative is 3,
The L-phenylalanine derivative according to claim 18, which is 4-dihydroxy-L-phenylalanine.