JPH0870878A - Method for producing tropolonyl alanine - Google Patents

Abstract

PURPOSE: To efficiently obtain the L-tropolonyl alanine useful as an antimicrobial agent, an insecticide, a metabolism-antagonizing agent, etc., in a good yield by allowing a tyrosinephenol lyase to act on a substrate comprising tropolone, etc., in an aqueous medium. CONSTITUTION: This L-tropolonyl alanine is obtained by allowing a tyrosine phenol lyase such as Erwinia herbicola ATCC 21433 originated from Erwinia herbicola on a substrate comprising (A) tropolone and (B) (i) a fi-chloro-L-alanine such as β-chloro-L-alanine, (ii) pyruvic acid and ammonia, or (iii) a serine such as L-serine in an aqueous medium, and subsequently collecting the L-toropolonyl alanine produced in the aqueous medium.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing L-troponyl alanine, and more specifically, it is produced by the action of tyrosine phenol lyase (also called β-tyrosinase).
-A method for producing troponyl alanine. This L-troponyl alanine is considered to be used as an antibacterial agent or an anti-insect agent, and as an antimetabolite as an amino acid analog.

[0002]

2. Description of the Related Art Although tropolonyl alanine has been reported to be produced by a synthetic method, it has been reported (The Bul-letin of the Che
mical Society of Japan, Vol 33, No8, pp 1084-1086,
(1960)), the manufacture of DL bodies is the main one. Therefore, in order to obtain only the L-form, it was necessary to either optically resolve the produced DL-form or asymmetrically synthesize the L-form. However, none have been successful at this time for technical reasons.

As a method of utilizing tyrosine phenol lyase, a method for producing L-dopa and L-tyrosine is known (Japanese Patent Publication No. 48-16194 and Japanese Patent Publication No. 51-547).
5, Japanese Examined Patent Publication No. 53-43594) and no report on a method for producing L-troponyl alanine.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for efficiently producing only L-troponyl alanine from tropolone, which has not been considered as a reaction substrate until now, by utilizing tyrosine phenol lyase. Especially.

[0005]

Means for Solving the Problems The present inventors have conducted various investigations in order to solve the above problems, and as a result, by the action of tyrosine phenol lyase, (1) tropolone and (2) (a) β-chloro It was found that only L-troponyl alanine was efficiently produced from alanine, (b) pyruvic acid and ammonia or (c) serine, and completed the present invention. That is, the present invention allows tyrosine phenol lyase to act on a substrate composed of (1) tropolone and (2) (a) β-chloroalanine, (b) pyruvic acid and ammonia or (c) serine in an aqueous medium, Producing L-troponyl alanine in an aqueous medium,
A method for producing L-troponylalanine, which comprises collecting L-troponylalanine. The present invention will be described in detail below.

The tyrosine phenol lyase used in the present invention is not particularly limited, but those derived from microorganisms are preferable. Specific examples of microorganisms having the ability to produce tyrosine phenol lyase include the following. Erwinia herbicola ATCC 2
1433, ATCC21434 Citrobacter freundi
i) ATCC 6750 Escherichia coli ATCC 15289 Proteus mirabilis ATCC 152
90 Enterobacter cloacae
ATCC 7256 Flavobacterium
flavescens) ATCC 8315 Achromobacter c
andicans) OUT 8005 Symbiobacterium thermophilum The above-mentioned microorganism is merely an example, and any microorganism can be used as long as it has the ability to produce tyrosine phenol lyase.

(1) Tropolone and (2) (a) β-
A method of allowing these microorganisms to act on a substrate composed of chloroalanine, (b) pyruvic acid and ammonia or (c) serine is (1) when culturing the microorganism in a liquid medium.
Tropolone and (2) (a) β-chloroalanine,
(B) Pyruvate and ammonia or (c) a substrate consisting of serine may be added and reacted while culturing,
Alternatively, after culturing the present microorganism, a culture of the microorganism, a microbial cell, a treated product of the microbial cell, a protein fraction of the microbial cell, an enzyme in various purification steps, or the like may be allowed to act on the substrate.

As a medium for culturing the above-mentioned microorganism, an ordinary medium containing an ordinary carbon source, a nitrogen source and inorganic ions is used. In order to obtain higher enzyme activity, it is desirable to add organic micronutrients such as vitamins and amino acids. In particular, addition of L-tyrosine is effective for inducing tyrosine phenol lyase. The required amount of L-tyrosine varies depending on the microbial strain used, but is usually 0.01% or more, and 0.1% to 0.5%.
The degree is preferred. In addition, it is generally effective to add vitamin B6s to the medium in order to enhance the reaction activity, and the amount of addition is 0.5 mg / dl or more in the medium.

As the carbon source, carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols and the like are appropriately used. As the nitrogen source, ammonia gas, ammonia water, ammonium salt, etc. are used. As the inorganic ion, magnesium ion, phosphate ion, potassium ion, iron ion and the like can be appropriately used if necessary. In addition, culture is usually carried out under aerobic conditions at pH.
Culturing may be carried out for 1 to 3 days while controlling the temperature within an appropriate range of 5.0 to 8.5 and a temperature of 15 to 40 ° C. That is, in order to produce the desired L-troponyl alanine during the culture using the above-mentioned microorganism, (1) tropolone and (2) (a) β-chloroalanine and (b) pyruvic acid, which are substrates, are produced. Then, ammonia or (c) serine may be added, and the culture may be performed at the above pH and temperature. The substrate such as tropolone and β-chloroalanine may be added at the start of the culture, or may be added during the culture.

Further, since a culture obtained by culturing the above-mentioned microorganism in the above-mentioned medium and under the above-mentioned culture conditions contains a high tyrosine phenol lyase activity, the bacterial cell may be used as an enzyme source without being separated, or the bacterial cell may be used once. It may be separated from the culture medium and used without washing or washing. Further, the treated product of bacterial cells can also be used as an enzyme source, and examples of such treated product of bacterial cells include mechanically ground bacterial cells, bacterial cells treated by ultrasonic waves, freeze-dried bacterial cells, and acetone-dried bacterial cells. , Microbial cells treated with an enzyme such as Rhizotium, a surfactant, microbial cells treated with toluene and the like, protein fractions of the microbial cells, and their immobilized products or others are appropriately used. The point is that it can be used as long as the enzyme activity is maintained. Now, the enzyme source acts on a substrate selected from (1) tropolone and (2) (a) β-chloroalanine, (b) pyruvic acid and ammonia or (c) serine in an aqueous medium. To do so, the enzyme source is dissolved or suspended in an aqueous medium containing the substrate,
Each aqueous medium may be left standing or stirred for a while while adjusting the pH to 5 to 10 and the temperature to 0 to 60 ° C.

The amount of tropolone used in the reaction for producing L-troponyl alanine is not particularly limited, but it is usually 0.01 to 0.
It is about 2M, preferably about 0.05 to 0.15M.
As the other reaction substrate, any one of (a) β-chloroalanine, (b) pyruvic acid and ammonia, and (c) serine may be used, and the amount thereof is 1 to 6 times the molar amount of the above tropolone. The degree may be used. All of these may be added at the start of the reaction, and as the reaction progresses,
You may add it sequentially.

The β-chloroalanine used may be any of β-chloro-L-alanine, β-chloro-D-alanine and β-chloro-DL-alanine, and serine may be any of L-serine, D-serine and DL-serine. But it's okay. Further, instead of β-chloroalanine and the like, O-ethylserine, On-propylserine, O-isopropylserine, On-butylserine, O-sec-butylserine, On-amylserine, O-isoamylserine, O
-Cyclopentylserine, O-2-propenylserine,
O-2-propynylserine, O-2-butenylserine,
A serine derivative such as O-3-methyl-2-butenylserine can also be used as a substrate. But preferably,
(A) β-chloroalanine, (b) pyruvic acid and ammonia or (c) serine are preferable. The confirmation and quantification of L-troponyl alanine can be carried out by a usual method using thin layer chromatography and high performance liquid chromatography.

[0013]

EXAMPLES The present invention will be specifically described below with reference to examples. The present invention is not limited to the embodiments.

Example 1 Strain Erwinia herbicola AT cultured overnight at 30 ° C. on broth agar medium (slant)
One slant of CC21434 is added to the same broth medium 500m
A 3 L flask containing 1 was inoculated and shake-cultured at 30 ° C. overnight. 0.5 g / d of mannitol was added to this seed culture solution.
1, glycerol 0.6 g / dl, KH ₂ PO ₄ 0.0
_{5g / dl, MgSO 4 · 7H} 2 O 0.05g / dl,
Soy protein acid hydrolyzate 1.0 g / dl, L-alanine 0.4 g / dl, glycine 0.3 g / dl, DL-methionine 0.1 g / dl, L-glutamic acid 0.4 g / d
1, L-tyrosine 0.1 g / dl, L-phenylalanine 0.2 g / dl, FeSO ₄ .7H ₂ O 0.005 g
/ Dl, ZnSO ₄ .5H ₂ O 0.001 g / dl, pyridoxine hydrochloride 0.025 g / dl (pH)
The jar of 7.5) containing 30 L was inoculated, and the culture was carried out with aeration and stirring at 30 ° C. overnight.

Centrifugation (1
The bacterial cells were collected at 20,000 rpm for 20 minutes at 4 ° C., washed once with physiological saline, and collected under the same centrifugation conditions. 400 ml of 10 mM phosphate buffer (pH 7.0) containing 0.2 mM pyridoxal phosphate (PLP), 5 mM 2-mercaptoethanol and 4 mM EDTA (ethylenediaminetetraacetic acid) was added to 1/4 amount of this microbial cell.
And suspend the cells, sonicate for a total of 20 minutes in ice-cooling to destroy the cells, and at 10,000 rpm at 4 ° C,
Centrifugation was performed for 20 minutes to obtain the supernatant. The supernatant thus obtained was dialyzed against the above-mentioned phosphate buffer overnight, and again centrifuged to obtain the supernatant. This was used as a cell-free extract fraction.

A 30-80% precipitate fraction was prepared from this cell-free extract by treatment with ammonium sulfate, dialyzed, and then subjected to DEA.
E-Sepharose CL-6B (Pharmac
ia) column chromatography, but
yl-Toyopearl 650M (manufactured by Tosoh Corporation) column chromatography was performed to obtain a purified enzyme preparation. 2 mg of this purified preparation of tyrosine phenol lyase
0 mM tropolone, 10 mM β-chloro-L-alanine, 0.05 mM pyridoxal phosphate (PLP), 1
Add to 2 ml of reaction solution containing 00 mM sodium hydroxide and 100 mM phosphate buffer (pH 8.0),
Reacted at 90 ° C for 90 minutes. In the above reaction system, a control group without enzyme and a group without substrate tropolone were also set.

After the reaction, 0.4 ml of concentrated hydrochloric acid was added to stop the reaction, and the enzyme was removed by centrifugation to obtain a supernatant. As a result of performing thin-layer chromatography on these three samples and analyzing the produced amino acids with ninhydrin, new ninhydrin colored spots were observed in the reaction system group, which was not found in the two control groups. Furthermore, when amino acid analysis was performed, β-chloro-
A new amino acid peak different from L-alanine was observed, which revealed the enzymatic synthesis of troponyl alanine. The scheme for producing troponyl alanine from tropolone and β-chloro-L-alanine is shown in FIG.

Example 2 1.6 g of the cell-free extract fraction of tyrosine phenol lyase obtained in Example 1 was added to 100 mM.
Tropolone, 100 mM β-chloro-L-alanine,
0.05 mM pyridoxal phosphate (PLP), 100
Add to 500 ml of reaction solution containing mM sodium hydroxide and 100 mM phosphate buffer (pH 8.0),
The reaction was carried out at 16 ° C for 16 hours. During the reaction, the pH was adjusted to 8.0 with 2N sodium hydroxide. After the reaction, 100 ml of concentrated hydrochloric acid was added to stop the reaction, the enzyme was removed by centrifugation, and the supernatant was concentrated to dryness. After removing unreacted tropolone with ether, troponyl alanine was extracted with methanol and concentrated to dryness. This one
Dissolve in 20 ml of water, Dowex 50W-X
The product in which the target fraction was eluted with (H ⁺ ) was concentrated to dryness and extracted with ethanol. After concentration to dryness, crystallization was performed with water-ethanol. The yield was about 15 mg.
As a result of measuring the amino acid analysis, ultraviolet absorption spectrum, infrared absorption spectrum, and NMR of the crystal sample, it was confirmed that it was troponyl alanine.

The obtained crystal was red, and it was thought that troponyl alanine was chelated by iron ions.
It was dissolved in 0.2 ml of water and iron ions were removed with a 1M NaHS solution. However, the yield was considerably reduced by these series of operations. Further, the enzymatically synthesized purified troponylalanine was treated with L-form-specific and D-form-specific amino acid oxidases, and its degradability was examined. Since it was decomposed by the L-form-specific amino acid oxidase and was not decomposed by the D-form-specific amino acid oxidase, it was confirmed that the present troponyl alanine was L-troponyl alanine.

Example 3 Mannitol 0.5 g / dl,
Glycerol 0.6 g / dl, KH ₂ PO ₄ 0.05 g
_{/ Dl, MgSO 4 · 7H 2} O 0.05g / dl, soybean protein acid hydrolyzate 1.0 g / dl, L-alanine 0.
4 g / dl, glycine 0.3 g / dl, DL-methionine 0.1 g / dl, L-glutamic acid 0.4 g / dl,
L- Tyrosine 0.1 g / dl, L- phenylalanine _{0.2g / dl, FeSO 4 · 7H} 2 O0.005g / d
1, ZnSO ₄ .5H ₂ O 0.001 g / dl, pyridoxine hydrochloride 0.025 g / dl (pH 7.
50 ml of 5) was put into a 500 ml flask and sterilized at 115 ° C for 15 minutes. To this, 2 g of separately sterilized calcium carbonate was added, and 1 platinum loop of Erwinia herbicola ATCC21433, which had been cultured overnight at 30 ° C. in broth agar medium, was inoculated and shake-cultured at 30 ° C. overnight. Cells were collected from 1 L of the culture solution for 20 flasks by centrifugation and washed once with physiological saline to obtain cells.

Approximately 10 g of the cells were treated with 100 mM tropolone, 100 mM β-chloro-L-alanine, 0.05 m.
M pyridoxal phosphate (PLP), 100 mM sodium hydroxide and 100 mM phosphate buffer (pH 8.
0) was added to 500 ml of the reaction solution, and the mixture was reacted at 30 ° C. for 16 hours. During the reaction, the pH was adjusted to 8.0 with 2N sodium hydroxide. After the reaction, the bacterial cells were removed by centrifugation, 100 ml of concentrated hydrochloric acid was further added to stop the reaction, the protein was removed by centrifugation, and the supernatant was concentrated to dryness. After removing unreacted tropolone with ether, troponyl alanine was extracted with methanol and concentrated to dryness. Dissolve it in 50 ml of water and
The target fraction was eluted with wex 50W-X (H ⁺ ) and concentrated to dryness, followed by extraction with ethanol. After concentration to dryness, crystallization was performed with water-ethanol. The yield was about 4 mg. As a result of measuring amino acid analysis, ultraviolet absorption spectrum, infrared absorption spectrum, and NMR using the crystallized troponyl alanine, it was confirmed to be troponyl alanine.

[0022]

[Effect of the present invention]

By allowing tyrosine phenol lyase to act on a substrate composed of (1) tropolone and (2) (a) β-chloroalanine, (b) pyruvic acid and ammonia or (c) serine in an aqueous medium, the desired L -Only troponyl alanine can be obtained.

[Brief description of drawings]

FIG. 1 is a diagram showing a mechanism of producing L-troponyl alanine from tropolone and β-chloro-L-alanine by the action of tyrosine phenol lyase.

Claims (2)

[Claims] 1. (1) Tropolone and (2) (a) β
-Tyrosine phenol lyase is allowed to act on a substrate composed of chloroalanine, (b) pyruvic acid and ammonia or (c) serine in an aqueous medium to produce L-tropolonyl alanine in the aqueous medium, A method for producing L-troponyl alanine, which comprises collecting polonyl alanine. 2. The method for producing L-troponyl alanine according to claim 1, wherein the tyrosine phenol lyase is derived from Erwinia herbicola.