JP2001224395A - Method for producing d-phenylalanine or d-tyrosine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for the production of D-phenylalanine or D- tyrosine with microorganism or microbial enzyme in high efficiency and yield on an industrial scale at a low cost. SOLUTION: D-phenylalanine or D-tyrosine is produced by treating phenylalanine containing mixed D and L isomers or tyrosine containing mixed D and L isomers with a cultured product of microorganism belonging to the genus Micrococcus, Sarcina or Sporosarcina or its treated product to effect the preferential asymmetric decomposition of L-phenylalanine or L-tyrosine and separating and collecting the remaining D-phenylalanine or D-tyrosine.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to D-
Phenylalanine (D-Phe) or D-Tyrosine (D-Tyr)
A method for producing the same.

[0002]

2. Description of the Related Art D-phenylalanine and D-tyrosine are useful as raw materials for pharmaceuticals and synthetic intermediates.

[0003] The conventional method using microorganisms or enzymes
As a method for producing D-phenylalanine or D-tyrosine, a method of asymmetrically decomposing N-acetyl-DL-phenylalanine or N-acetyl-DL-tyrosine using a microbial enzyme (Applied and Environmental Microbiology, vol.
54, No. 4, p. 984-989), a method of transferring an amino group to α-keto acid by a microbial enzyme (JP-A-62-205790)
Etc. are known. However, these methods have problems in that it is difficult to separate and recover D-phenylalanine or D-tyrosine from the reaction solution, and the raw materials are expensive.

As a method for producing D-phenylalanine by selectively assimilating or asymmetrically degrading L-phenylalanine (L-Phe) of DL-mixed phenylalanine by a microorganism, Rhodotorul (Rhodotorul) is used.
a) Utilizing a microorganism belonging to the genus, Rhodosporidium or Providencia (JP-A-2-65797), Proteus
Utilizing microorganisms belonging to the genus Morganella or Saccharomycopsis (Japanese Patent Laid-Open No. 10-80297), Fusarium
Genus, Gibberella, Aspergillus (Asper
gillus, Pleurotus or Nectria (Japanese Patent Application Laid-Open No. 10-286098), and the enzyme oxidizes only L-phenylalanine in DL-mixed phenylalanine using an enzyme. As a method for producing D-phenylalanine, a method utilizing a snake-derived L-amino acid oxidase (JP-A-11-1038887) is known. However, these methods have a problem that a long time is required for the reaction or a large amount of assimilation / decomposition is observed not only for L-phenylalanine but also for D-phenylalanine and the yield is low. The production method of D-phenylalanine was unsatisfactory.

[0005] In the production of D-tyrosine, D-tyrosine is produced by selectively assimilating or asymmetrically decomposing only L-tyrosine (L-Tyr) of the DL-type mixed tyrosine. -Similar to the method for producing phenylalanine,
JP-A-10-80297, although described in JP-A-10-286098, the accumulation concentration of D-tyrosine is extremely low,
Or there are problems such as low optical purity of D-tyrosine,
It was still unsatisfactory as an industrial method for producing D-tyrosine.

[0006]

SUMMARY OF THE INVENTION The present invention provides a method for industrially and advantageously producing D-phenylalanine or D-tyrosine with high efficiency and high yield using microorganisms and microbial enzymes.

[0007]

Means for Solving the Problems The present inventors preferentially give priority to L-phenylalanine of DL-mixed phenylalanine or L-tyrosine of DL-mixed tyrosine to many microorganisms. Search for microorganisms that asymmetrically and efficiently asymmetrically decompose, and as a result of diligent efforts, newly discovered microorganisms having this ability, D-phenylalanine or
The present inventors have found a method for efficiently producing D-tyrosine and have completed the present invention.

[0008] That is, the present invention relates to a method of reacting a culture of a microorganism having the ability to asymmetrically degrade L-phenylalanine or L-tyrosine or a processed product thereof with DL-mixed phenylalanine or DL-mixed tyrosine. ,
A method for producing D-phenylalanine or D-tyrosine, which comprises separating and collecting the remaining D-phenylalanine or D-tyrosine, specifically, a DL-mixed phenylalanine or a DL-mixed tyrosine, Micrococcus, Sarcina (Sarc)
ina), or by treating a culture of a microorganism belonging to the genus Sporosarcina, or a processed product thereof, to preferentially asymmetrically degrade L-phenylalanine or L-tyrosine, respectively. Remain
A method for producing D-phenylalanine or D-tyrosine, comprising separating and collecting D-phenylalanine or D-tyrosine. Among the above microorganisms, those belonging to the genus Micrococcus are most suitable for the present invention.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The DL-mixed phenylalanine, which is a raw material in the present invention, is not limited as long as it contains D-phenylalanine and L-phenylalanine. Similarly, the DL-form mixed tyrosine may be any as long as it contains D-tyrosine and L-tyrosine, and its abundance ratio does not matter.

The microorganism used in the present invention is DL
Any microorganism may be used as long as it is a microorganism having the ability to efficiently assimilate and degrade L-tyranosine of the mixed body type phenylalanine preferentially and L-tyrosine of the DL mixed body type tyrosine with high efficiency. Such microorganisms, for example, Micrococcus (Micrococcus), Sarsina (Sarcina),
Or a microorganism belonging to the genus Sporosarcina, of which micrococcus (Micrococcu
Microorganisms belonging to the genus s) are preferred.

Specific examples of these microorganisms include the following. Micrococcus luteus ATCC 272 Micrococcus luteus ATCC 11880 Micrococcus luteus ATCC4698 Sarcina sp. FERM P-16591 Sporosarcina ureae

Among these microorganisms, micrococcus
Luteus ATCC 272, Micrococcus Luteus AT
CC 11880 strain and Micrococcus Luteus ATCC 4
698 shares are from the American Type Culture Collection, 12301
Parklawn Drive, Rockville, Maryland 20852, United
States of America).

The Sarsina sp. FERM P-16591 strain is
It has a private number AJ3132 and was deposited on January 16, 1998, with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (ZIP 1-3-1, Higashi Tsukuba, Ibaraki 305-8526, Japan).

[0014] Sporosarcina ureae IFO 12698 strain is an institution for fermentation.
Osaka (Institute for Fermentation, Osaka, address 1)
7-85, Juso-honmachi 2-chome, Yodogawa-ku, Osaka 53
2, Japan).

These microorganisms may be strains newly isolated from nature such as soil and plants, or may be strains artificially bred by treatment with mutagenic drugs or recombinant DNA technology. Is also good.

The microorganism used in the present invention may be cultured by any culture method such as liquid culture or solid culture as long as it has a necessary ability, or the culture solution itself or a culture solution collected from the culture solution. Any of live cells may be used. Furthermore, a processed product of a culture such as a culture solution or a viable cell obtained may be used as long as it has a necessary ability. Examples of the treatment herein include ultrasonic treatment, glass beads, French press, freeze-drying treatment, and treatment with a lytic enzyme, an organic solvent, a surfactant, or the like. Further, if the treated product obtained by these treatments is a crude enzyme or a purified enzyme prepared by a conventional method (liquid chromatography, ammonium sulfate fractionation, etc.) and has the required ability, it is used. You may.

Further, when using the above-mentioned culture or its processed product, it is possible to use them after enclosing them in carrageenan gel or polyacrylamide, or immobilizing them on a membrane of polyethersulfone or regenerated cellulose.

Next, regarding the method for culturing microorganisms according to the present invention, the term “culturing” as used herein refers to culturing to obtain microbial cells, and from L-phenylalanine from DL-mixed phenylalanine or from DL-mixed tyrosine.
It means both cultures for performing asymmetric degradation of L-tyrosine, and these steps may be divided and performed sequentially, continuously, or simultaneously. The method for culturing microorganisms in the present invention can be performed using a medium usually used in this field, that is, a medium containing a carbon source, a nitrogen source, inorganic salts, trace metal salts, vitamins, and the like. Further, depending on the type of microorganism or culture conditions, about 0.1 to 1.0 g / dl amino acid may be added to the medium, or DL-mixed phenylalanine or DL-mixed tyrosine serving as a substrate may be converted into a single carbon source. When used as a single nitrogen source or a single carbon / nitrogen source, the ability to decompose L-phenylalanine and L-tyrosine can be promoted. D in the cells
To increase the permeability of mixed L-form phenylalanine or L-tyrosine into cells, Triton X (Triton
Surfactants such as X) and Tween, and organic solvents such as toluene and xylene can also be used.
As a specific substance used as the medium component, for example,
The carbon source is not limited as long as the microorganism to be used is available. For example, glucose, sucrose, fructose, glycerol, acetic acid, and the like, or a mixture thereof can be used. As the nitrogen source, ammonium sulfate, ammonium chloride, urea, yeast extract, meat extract, corn steep liquor, casein hydrolyzate, or a mixture thereof can be used. As a specific medium composition, for example, glucose 1.0%, ammonium sulfate 0.3%, powdered yeast extract 1.0%, peptone 1.
0%, L-phenylalanine 0.2%, KH2PO4
0.1%, K2HPO4 0.3%, MgSO4.7
H2O 0.05%, FeSO4.7H2O 0.00
1%, MnSO4.4H2O 0.001% (pH 7.
0).

The cultivation temperature is usually within the range where the microorganism to be used grows, that is, at 20 to 45 ° C, but is preferably within the range of 25 to 37 ° C. The pH of the medium is adjusted in the range of 3 to 11, preferably 4 to 8. The aeration conditions are set to conditions suitable for the growth of the microorganism to be used, but aerobic conditions are preferred. Culture time is D-phenylalanine or D-
When the maximum use of tyrosine, that is, when producing D-phenylalanine, the decomposition of L-phenylalanine is maximum and the decomposition of D-phenylalanine is suppressed as much as possible, In the case of production, decomposition of L-tyrosine is the largest and D-
It is desirable to carry out the reaction until the decomposition of tyrosine is suppressed as much as possible, usually for about 12 to 120 hours, preferably about 24 to 96 hours.

DL-mixed phenylalanine or DL-mixed tyrosine serving as a substrate is added all at once, intermittently, or continuously in a concentration range in which the decomposition of L-phenylalanine or L-tyrosine is not suppressed. You.
As for the method of addition, it may be added directly during the cultivation of the strain, or may be used by isolating the cells once after the cultivation and mixing with the cells obtained by the separation or the processed product thereof. At the time of addition, the substrate is added in the form of an aqueous solution or slurry, but may be added as a mixture with an organic solvent or a surfactant for the purpose of increasing solubility or promoting dispersion.
Further, for the purpose of continuing or promoting the metabolism of the microorganism, it may be added by mixing with a medium component such as a carbon source or a nitrogen source.

In addition, D- or
The quantification of L-phenylalanine or D- or L-tyrosine can be promptly measured using a well-known method. That is, thin layer chromatography using Merck, HPTLC CHIR, etc. can be used simply,
To improve the analysis accuracy, Daicel Chemical Industries, "CR
High-performance liquid chromatography (HPLC) using an optical resolution column such as "OWNPAK CR (+)" may be used. Thus, D-phenylalanine or D-tyrosine accumulated in the culture solution or the reaction solution can be collected from the culture solution or the reaction solution and used in a conventional manner. The collection from the culture solution or the reaction solution may be performed by well-known means commonly used in the art in such a case, for example, filtration,
Operations such as centrifugation, vacuum concentration, ion exchange or adsorption chromatography, and crystallization are used in combination as needed.

[0022]

The present invention will be described in more detail with reference to the following Examples, which by no means limit the present invention. In the examples, the optical purity of phenylalanine and tyrosine was measured using an optical resolution column “CROWNPAK CR (+)” (φ4.6 × 15) manufactured by Daicel Chemical Industries.
0 mm). The analysis conditions are as shown below. In the case of phenylalanine Mobile phase: aqueous solution of perchloric acid (pH 2.0) / methanol = 9
/ 1 (V / V) Flow rate: 0.5 ml / min. Column temperature: 30 ° C. Detection: UV 210 nm Under these conditions, D-phenylalanine was obtained for 6.8 minutes.
L-phenylalanine can be fractionated and quantified with a retention time of 8.4 minutes. In the case of tyrosine Mobile phase: aqueous solution of perchloric acid (pH 1.8) Flow rate: 0.5 ml / min. Column temperature: 30 ° C. Detection: UV 210 nm Under these conditions, D-tyrosine can be separated and quantified with a retention time of 8.7 minutes, and L-tyrosine can be separated and quantified with a retention time of 10.7 minutes.

[Example 1] Glucose 1.0%, ammonium sulfate 0.3%, powdered yeast extract 1.0%, peptone 1.
0%, L-phenylalanine 0.2%, KH2PO4
0.1%, K2HPO4 0.3%, MgSO4.7
H2O 0.05%, FeSO4.7H2O 0.00
1%, MnSO4.4H2O 0.001% (pH 7.
0) was inoculated with the microorganisms shown in Table 1 below, cultured at 30 ° C. for 24 hours, and the cells were scraped off. The cells were dried with 2.0% DL-phenylalanine and 0.1% glucose.
1%, ammonium sulfate 0.3%, powder yeast extract 0.1%, KH
2PO4 0.1%, K2HPO4 0.3%, MgS
A sterilized liquid medium containing 0.05% of O4.7H2O (pH 7.0) was inoculated so as to be 5% by weight of wet cells and cultured aerobically at 30 ° C. After 24 hours, D and L-phenylalanine remaining in the culture solution were quantified. The results are as shown in Table 1, and showed that L-phenylalanine was decomposed efficiently.

[0024]

[Table 1]

[Example 2] Glucose 1.0%, ammonium sulfate 0.3%, powdered yeast extract 1.0%, peptone 1.
0%, L-phenylalanine 0.2%, KH2PO4
0.1%, K2HPO4 0.3%, MgSO4.7
H2O 0.05%, FeSO4.7H2O 0.00
1%, MnSO4.4H2O 0.001% (pH 7.
The medium containing 0) was dispensed in 5 ml portions into a large 80 ml test tube and sterilized at 120 ° C. for 10 minutes. After cooling, one platinum loop was inoculated into each of two test tubes with the microorganisms shown in Table 2 below, and cultured at 30 ° C. for 24 hours under aerobic shaking.
Thereafter, 5 ml of a liquid containing 4% of L-phenylalanine or a liquid containing 4% of D-phenylalanine (both are sterilized at 110 ° C. for 10 minutes and cooled) containing 2% of glucose and 0.6% of ammonium sulfate are put into the test tubes. The culture was further continued aerobically at 30 ° C. After 24 hours, add L-
Table 2 shows the results of quantifying the residual L-phenylalanine when phenylalanine was added and the residual D-phenylalanine when D-phenylalanine was added.
Even when the cells were prepared by liquid culture, L-phenylalanine was preferentially degraded in the microorganisms of Examples.

[0026]

[Table 2]

Example 3 A medium was prepared in the same manner as in Example 2, and the microorganisms shown in Table 2 below were placed in two test tubes at a scale of 5 ml using a large test tube of 80 ml. Platinum loops were inoculated, and cultured with aerobic shaking at 30 ° C. for 24 hours. Then, a liquid containing 4% of L-tyrosine containing 2% of glucose and 0.6% of ammonium sulfate, or D-
5 ml of a solution containing 4% of tyrosine (all were sterilized at 110 ° C. for 10 minutes and then cooled) was added, and the culture was further continued aerobically at 30 ° C. Twenty-four hours later, the results of quantification of residual L-tyrosine when L-tyrosine was added to the culture solution and residual D-tyrosine when D-tyrosine was added to the culture solution are shown in Table 2. L-tyrosine was preferentially degraded in tyrosine as in the case of phenylalanine.

[0028]

[Table 3]

Example 4 The same medium as in Example 2 was used for 50 times.
20 ml in a 0 ml Sakaguchi flask, 120 ° C, 10
Sterilized for a minute. After cooling, Micrococcus Luteus
One loopful of ATCC 11880 was inoculated and aerobically shake cultured at 30 ° C. for 24 hours. Then, dry heat sterilization (120
2 g of DL-phenylalanine powder)
Was added and the culture was continued. Table 4 shows the results of quantifying the remaining L-phenylalanine and D-phenylalanine in the culture solution at the lapse of 24, 48, 72, and 144 hours after the addition of DL-phenylalanine, respectively. 72
After elapse of time, L-phenylalanine was completely decomposed, but D-phenylalanine was hardly decomposed even after elapse of 144 hours.

[0030]

[Table 4]

Example 5 In Example 4, 300 ml of a culture solution obtained by adding DL-phenylalanine and continuing culturing for 72 hours was boiled for 10 minutes to completely dissolve D-phenylalanine. Then, centrifugation operation (10,000g
× 10 minutes), and the centrifugal supernatant was concentrated to 80 ml by a vacuum concentration operation to precipitate D-phenylalanine crystals. Precipitated crystals were collected by filtration using filter paper to obtain crude crystals. Crude crystals (4.
7 g) and dissolved by adding 40 ml of water and 4 ml of concentrated sulfuric acid.
300 mg of activated carbon was added thereto, and the solution was decolorized. Next, after removing activated carbon by filtration, 28% was added to the filtrate.
16 ml of aqueous ammonia was added to adjust the pH to 3.5.
Phenylalanine was recrystallized. Thereafter, the precipitated crystals were collected by filtration using a filter paper, dried, and dried.
g of D-phenylalanine was obtained. When subjected to HPLC analysis, the substance purity was 98% and the optical purity was 99% ee or more.

Example 6 The same medium as in Example 2 was used for 50 times.
20 ml in a 0 ml Sakaguchi flask, 120 ° C, 10
Sterilized for a minute. After cooling, Micrococcus Luteus
One loopful of ATCC 11880 was inoculated and aerobically shake cultured at 30 ° C. for 24 hours. Then, dry heat sterilization (120
2 hours), 2 g of DL-tyrosine powder was added,
Further culturing was continued. 24 after addition of DL-tyrosine
Table 6 shows the results of quantification of residual L-tyrosine and D-tyrosine in the culture solution after 48, 72, and 144 hours, respectively. After 72 hours, L-tyrosine was completely decomposed, but D-tyrosine was hardly decomposed even after 144 hours.

[0033]

[Table 5]

Example 7 In Example 6, 20 ml of a culture solution obtained by adding DL-tyrosine and continuing culturing for 72 hours was added.
Then, 10 ml of 6N HCl was added to completely dissolve D-tyrosine. After adding water to 100 ml, the cells were separated by centrifugation (10,000 g × 10 minutes). The centrifuged supernatant was transferred to a beaker, and 6N KOH was added dropwise to the mixture with gentle stirring using a stirrer so that the pH became 6 to 8, to precipitate D-tyrosine crystals. The precipitated crystals were collected by filtration using filter paper. Drying yielded 860 mg of D-tyrosine. When subjected to HPLC analysis, the substance purity was 97% and the optical purity was 99% ee or more.

[0035]

According to the present invention, a culture of a microorganism belonging to the genus Micrococcus, Sarcina, or Sporosarcina to DL-mixed phenylalanine or DL-mixed tyrosine,
Alternatively, by treating the treated product, L-phenylalanine or L-tyrosine can be preferentially asymmetrically decomposed with higher selectivity than ever before, so that D with high optical purity is industrially efficient and inexpensive. -Phenylalanine or D-tyrosine can be produced.

Claims (2)

[Claims] [Claim 1] DL-mixed phenylalanine or DL-mixed tyrosine is added to Micrococcus
genus coccus), genus Sarcina, or sporosarcina
(Sporosarcina) a culture of a microorganism belonging to the genus, or by treating the treated product, preferentially decompose L-phenylalanine preferentially, or L-tyrosine, respectively, and then asymmetrically decompose D-phenylalanine, Alternatively, a method for producing D-phenylalanine or D-tyrosine, comprising separating and collecting D-tyrosine. 2. The method for producing D-phenylalanine or D-tyrosine according to claim 1, wherein the microorganism to be acted is a microorganism belonging to the genus Micrococcus.