JPS58209989A - Preparation of optically active tryptophan compound - Google Patents

Abstract

PURPOSE:To prepare an optically active tryptophan compound, by the asymmetric hydrolysis of DL-tryptophan amide compound in the presence of a specific metallic salt using a L-tryptophan amide hydrolase produced by specific microorganisms. CONSTITUTION:A DL-tryptophan amide compound of formula I (X is H, OH, halogen, lower alkyl or lower alkoxy) is made to react with a hydrolase of an L-tryptophan amide compound produced by yeast belonging to Rhodotorula genus, Trichosporon genus or Rhodosporidium genus, in the presence of one or more metallic salts selected from manganese, magnesium and cobalt salts. The obtained optically active L-tryptophan of formula II is separated from the system, and the remaining D-tryptophan amide compound is hydrolyzed by conventional process to obtain an optically active D-tryptophan.

Description

[Detailed description of the invention] The present invention is based on D'L-tryptophanamides.

Optically active L~tryptophans and D-) IJ
The present invention relates to a method for biochemically producing gutphans.

It is well known that both (1)-tryptophans and (D-) listophans are optically active substances, and each has different uses. For example, L-) listphanes are heavy compounds as essential amino acids.

It has uses such as amino acid infusions, pressurized animal feeds, sleep-inducing agents, etc. as disclosed in JP-A-58-209989 (2). D-) Listofans are also useful as sweeteners, for example.

Conventionally, many methods 1 for producing tryptophans by organic synthesis have been proposed. However, in the case of organic synthesis, the product is obtained as a mixture of D and [,=, ie, a racemate, so how to efficiently optically resolve this is an extremely important issue.

In the biochemical optical resolution method of DL-)I7putophanes, the present inventors used the amide form of DL-,)IJbutophanes as a substrate, and used it as a substrate for L-)ligtraanamides produced by microorganisms. When ice-melting enzyme (amidase) is applied, only L-)ligtraanamides are hydrolyzed and optically active L''-tryptophans are obtained. If the amide form of butophanes is decomposed by 1 water, the optical activity D
−)! It was previously discovered that j-butophanes can be obtained (see Japanese Patent Application Laid-open No. 13,000/1983).

The present inventors have discovered that in the method for producing optically active tryptophans by asymmetric hydrolysis, the desired product can be produced in an even higher yield.
We have been conducting intensive research with the aim of developing efficient manufacturing methods. As a result, if the asymmetric hydrolysis is carried out using an ice-melting enzyme of L-ligtraanamides produced by yeast belonging to a specific genus and in the presence of specific metal salts, the objective can be achieved. The present invention was completed based on this finding.

That is, the present invention.

General formula (in the formula, X is a hydrogen atom, a hydroxyl group, etc.), a lower alkyl group or a lower alkoxy group.

) DL-)ligtraanamides represented by

Manganese,
By acting in the presence of at least one kind of metal salt selected from magnesium and coal.

The general formula (wherein X has the same meaning as above) is hydrolyzed, and the above L-)! Optically active D-1.j having the same structural formula as butophanes. A novel method for producing liptophans is provided.

According to the present invention, compared to the method described in the above-mentioned Japanese Patent Application Laid-Open No. 57-13000, L-I) butophan amides can be prepared by using inexpensive salts of specific metals. The hydrolase activity is significantly increased, and as a result, the amount of the enzyme used is greatly reduced, and the manufacturing cost of the target product is significantly reduced, which is a great industrial advantage.

DL-tryptophan amides, which are substrates in the present invention, can be prepared by reacting an ester of 1, for example, DL-)IJ butophans with ammonia. Can be easily synthesized.

In addition, the ice-melting enzyme for L-)liptonanamides used in the present invention is an enzyme that asymmetrically hydrolyzes only one L-libutoneamide among the D I--)liptonanamides represented by the above general formula. The name of the enzyme is understood to be amidase. It is essential that the enzyme belongs to the genus Rhodotorula, Trichosporon or Rhodosporidium.

Yeast that can be used for this purpose are useful regardless of their position in the nine classifications. Typical species names of yeast belonging to this genus are listed below, but the yeast used in the present invention is not limited to these exemplified species. In addition, F-note? U
All of the yeasts shown are publicly known and are stored at the Institute for Fermentation (IFO), a preservation organization of the Japan Federation of Microbial Strains Archives (JL/CC).

It can be easily obtained manually through preservation institutions such as the Institute of Applied Microbiology (IAM) of the University of Tokyo.

(1) Rhodotorula genus Rhodotorula minuta burr texensense F○
-1102 (Rhodotorula m1nuta
var, texensis) Rhodotorula rubra
IFO-0471 (Rhodotorul
a rubra) o g) Lula Glucinis
IFO0389 (Rhodotorula glu
tinis) (2),) Trichosporon phthanium IFO-01
73 (Trichosporon cutaneum)
Trichosporon fermentus IFO-11
99 (Tricosporon fermentans
) Trichosporon capitatum IFO-11
97 (Trichosporon capitatum
(3).
F○-0413 (RhodoS'poridiu+n
toruloides) Rhodosboridium diobobatum IFO-1828 (Rhodospor
idium diobovatum) Lotusboritium infilmominium IFO-1057 (R
hodosporidium infirmo-min
iatum) In the present invention, these yeasts are treated with Dr.
- IJ Butofan amides can be made to act on a culture obtained by culturing the yeast in a liquid medium, bacterial cells isolated from the culture solution, or enzymes isolated from bacterial cells or cultures based on various enzyme isolation methods. 9. For example, the enzyme is made to act in the form of a crude enzyme, a purified enzyme, a processed product such as an enzyme-containing extract or a concentrated solution thereof. Furthermore, it can also be used in the form of a bacterial cell or an enzyme immobilized on a carrier.

Carriers that can be used include alginic acid, carrageenan, collagen, cellulose, acetylcellulose, i
Examples include natural products such as cellophane, collodion, and polymeric substances such as polyacrylamide, polystyrene, polyethylene glycol, polypropylene glycol, polyurethane, and polybutadiene. Immobilization is carried out under mild conditions that do not impair amidase activity.
It can be done according to conventional methods.

Examples of the metal salt selected from manganese, magnesium, and cobalt used in the present invention include salts of these metals such as hydrochloride, sulfate, nitrate, and acetate. These metal salts.

Although it is good to use one type, it is also possible to use two or more types in combination. If the amount used is too small, the activity of the ice-melting enzyme of L-to-IJ butophanamides cannot be significantly increased, while even if a large amount is used, the effect of increasing the enzyme activity remains constant, and the amount exceeds the required amount. The use of is uneconomical. Therefore, the amount of these genus salts per reaction solution is usually 0.001 to 1.
It is used at a concentration of 0 mmol, preferably 0.01 to 5 mmol.

In the present invention, the conditions for allowing the yeast to act on DL-tryptophan amides in the presence of these metal salts include a reaction temperature of 20 to 50°C, and a suitable reaction time of 5 to 50 hours. Of course, it is also possible to shorten the time for one reaction by increasing the source of nine reactions or increasing the amount of enzyme. In addition, the reaction is performed at PH5~1
0. Preferably, it is carried out around PH'7 to 9.

In the present invention, the amounts of yeast and bacterial cells used are such that the weight ratio of 1 to DL-tryptophanamide, which is a substrate, is within the range of O,O'01 to 0.02 based on freeze-dried bacterial cells. It is preferable to use However, in order to shorten one reaction time, the amounts of yeast and bacterial cells used may be increased from the above amounts. When using a yeast culture solution, treated product, or immobilized product thereof, the amount to be used may be determined in terms of the amount of freeze-dried bacterial cells. Further, the concentration of the substrate DL-)liptophan and amide used is generally 1 to 40 wt%, preferably 5 to 30 wt%, based on the reaction solution.

In the present invention, when the hydrolysis reaction of IL-tryptophanamides has proceeded to almost 100%, the reaction is stopped and L-tryptophans and D-tryptophanamides are separated from the reaction solution. The separation can be easily carried out by operations such as one-fraction crystallization and solvent extraction.

In addition, in the asymmetric hydrolysis operation, D~tryptophanamides are not affected by microorganisms at all, and almost all of them are recovered. The recovered D-)IJ butophanamides are easily hydrolyzed by conventional method 1, such as heating in the presence of an acid or alkaline aqueous solution, resulting in optical activity D-)! J-gutophanes can be obtained in high yield. Furthermore, the recovered D-)liptonanamides can be reused as a substrate in the present invention by subjecting them to racemization treatment.

Note that 9 L-) IJ butophans and D-) obtained in the present invention! What is the optical purity and yield of 7-pteptanes?

It is extremely high, comparable to or even higher than biochemical optical resolution using known substrates.

Next, examples of the present invention and comparative examples will be given. Note that the yield of L-9 or D-)IJ butophans on the valley side was calculated using a linear equation.

Example 1 In a flask with a shoulder, glycerol 5 wt%, cone steeple force -5 wt%, tlftm ammonium 0.5
wt% + and inorganic salt mixture (MfSO4・7H
202Of', FeSO4H7H205S',
CaCf22t, MnCf2'4H200,2
? , N,MOO4'2H200,1? and N, (4
After adding 100 ml of medium (pH 7,0) containing 0.1 F dissolved in distilled water (1,000 mL) and killing it, Trichosporon cutanium IFO-0173
'f: Two platinum loops were inoculated from the slant medium and cultured with reciprocal shaking at 30°C for 65 hours. The bacterial cells collected from this culture solution by centrifugation were washed twice with distilled water to obtain washed bacterial cells. This was further freeze-dried to obtain a freeze-dried solid.

The freeze-dried cells were then added to distilled water (PH'i', 5) containing 10% DL-tryptophanamide and 1 mmolar manganese chloride.

After adding so that the bacterial cell/substrate (weight ratio) was 0.005, the reaction was carried out at 30° C. for 20 hours. The reaction solution was analyzed by high performance liquid chromatography.

The amount of L-tryptophan produced was measured.

Examples 2 and 3 An experiment was conducted in the same manner as in Example 1, except that magnesium sulfate (Example 2) and cobalt chloride (Example 3) were used instead of manganese chloride as the metal salt.0 Comparison Example 1゜An experiment was conducted in the same manner as in Example 1 for a pond to which manganese chloride was not added.

Examples 4-6 As yeast, Trichosporon phthanium I, FO-0
In place of 173, Rhodotorula minuta var.
An i-test was conducted using the same procedure as in steps 3 to 3.

Comparative Example 2゜For ponds to which no metal salt was added, experiments were conducted in the same manner as in Examples 4 to 6.

Table 1 shows the results of Examples 1 to 6 and Comparative Example 1.2.

/ / Implementation Criteria 7-9゜The concentration of manganese chloride used was 2 mmol (Example 7),
5 mi IJmol (Example 8) and 10 mi! An experiment was conducted in the same manner as in Example 1, except that the amount was changed to J moles (Example 9). As a result, the yield of L-)ributophane was constant at approximately 100% in each Example.

Examples 10-12゜The working concentration of cobalt chloride was +2 mmol (Example 1
0), 5 mmol (Example 11) and 1069 mol (
An experiment was carried out in the same manner as in Example 3, except that Example 12) was changed. As a result, the yield of L-)lyptophan was constant at approximately 40 cm in each Example.

Example 13 Washed bacterial cells of Trichosporon phthanium IFO-0173 prepared in the same manner as in Example 1 were washed with cold acetone to obtain acetone-dried bacterial cells.

- DL-)Liptophanamide is dissolved in distilled 7X containing 1,000,2 mmolar concentration of manganese chloride.

Substrate solutions (pH 7,0) of various concentrations were prepared.

Next, the acetone-dried microbial cells were added to this substrate solution lO− so that the dry microbial cells/substrate (weight ratio) was 0. After adding so that the amount of O1 was obtained, the reaction was carried out at 30° C. for 20 hours. The reaction solution was analyzed by high performance liquid chromatography, and the amount of L-)IJ butophane produced was determined as maJ.

The results are shown in Table 2.

Table 2 Example 14 Lyophilized cells of Trichosporon phthanium IFO-0173 obtained in the same manner as in Example 1 were treated with 10 wt% D
L ~ tryptophanamide and 2 mmolar concentration of manganese sulfate in distilled water (PH 7,5) 10rnI! To,
After adding the cells at various ratios of bacterial cells/substrate (weight ratio), the reaction was carried out at 35° C. for 200 hours. The reaction solution was analyzed by high performance liquid chromatography to measure the amount of L-tryptophan produced.

The results are shown in Table 3.

Table 3: ip-only 15 Freeze-dried bacterial cells of Trichosporon cutaniume FO-01'73 prepared in the same manner as in column 1: 5.0 In
g of 0.2M containing 2 mmolar concentration of manganese chloride
After suspension in Tris-HCl buffer (PH"l', 8) 57.

French press under cooling (20,000psi) I:l
-Therefore, the bacterial cells were crushed. Then, centrifugation (20,0
00f.

5 m7 of supernatant obtained by 30 min)! In, BL
- 250 m9 of tryptophan amide was added, and the reaction was carried out at 40°C for 5 hours.

As a result of analyzing the reaction solution by high performance liquid chromatography,
L-MJ butophane was produced in 90% yield.

Comparative Example 3 A song in which manganese chloride was not used was tested in the same manner as in Example 15. As a result, the yield of L-drift fan was 7%.

Example 6: To 5 ml of cell-free extract of Trichosporon phthanium IFO-0173 prepared in the same manner as in Example 15,
Ammonium sulfate was added to precipitate the precipitate at 25-75% saturation. After the ammonium sulfate precipitate was collected by centrifugation, 250 m
0.2 M with 9 and 2 mmolar concentrations of manganese acetate
) 5 ml of Lis-HCl buffer (pH 8,0) was added, and the reaction was carried out at 45°C for 5 hours.

As a result of analyzing the reaction solution by high performance liquid chromatography,
'L-)'J butophane was obtained with a yield of 59%.

Comparative Example 4 Kazuya conducted an experiment in the same manner as in Example 16 without using manganese acetate. As a result, the yield of L-tryptophan was 3 times higher.

Example 17 10 ml of cell-free extract of Trichosporon phthanium IFO-0173 prepared in the same manner as in Example 15
, Sephadex G-75 column (1.5 m diameter
, length 65 cm) to collect the amidase active fraction, concentrate it to 5-5 using a semi-permeable membrane method, and then add DL to this.
-) In addition to 256 η of liptosanamide and 2 m 9′ of manganese chloride, a reaction was carried out at 50° C. for 5 hours.

As a result of analyzing the reaction solution by high speed, f-seam chromatography, it was found that L-buto7an was obtained in a yield of 47%.

Comparative Example 5 For a pond in which manganese chloride was not used, an experiment was conducted in the same manner as in Example 17. As a result, the generated L −)
IJ butophane was present in trace amounts.

Example 18゜ Washed bacterial cells of Trichosporon phthanium IP''0-01'i'3 prepared in the same manner as in Example 1 (corresponding to lyophilized bacterial cells 85 In9)'iio, IM phosphate buffer (' PH7,0) Suspended in 0.5m, acrylamide monomer = 501n9. 0.
27! After adding and mixing 0.2 ml of a 2.5% aqueous potassium persulfate solution (polymerization initiator), the mixture was allowed to stand at 25° C. for 1 hour to complete gelation.

Next, the obtained gel was crushed, washed with water, and the entire column (diameter 0.
．． 5 m + length 10 cm), distilled water (PH 7.5) containing 5 wt% DL-) liptophanamide and 2 mmol concentration of manganese chloride was added from the top of the column at SV (space velocity) = 2. ．． It was run at a CI rate of 35°C.

In this continuous reaction, up to 1200 hours per reaction time
-) The yield of IJ butophane was maintained at 95% or higher.

Examples 19 to 22 Freeze-dried cells of various yeasts were obtained in the same manner as in Example 1. 5μ of each freeze-dried bacterial cell was added to 10d of distilled water (PH7.5) containing 5wt% of various DL-)liptosanamides and 2mM manganese sulfate, and the mixture was heated at 30℃ for 2 hours.
The reaction was carried out for 0 hours.

The reaction solution was analyzed using high performance liquid chromatography.

The amount of produced L-)') butophans was measured.

The results are shown in Table 4.

(23) Example 23 Freeze-dried cells 20In9 of Trichosporon phthanium IFO-0173 obtained in the same manner as in Example 1 were DL-
Add to 20 g of distilled water (PH 7.0) containing 22 to 22% of tryptophan amide and 8 to 8 manganese chloride, and add 20 g of distilled water at 30°C.
A time reaction was performed.

After the reaction, bacteria were removed from the reaction solution by centrifugation.

As a result of analysis by high performance liquid chromatography, 7983 m? (yield: 98%) and D
-Tryptophanamide 1,010rn9 was included. This reaction solution was extracted with ethyl acetate to recover unreacted D-tryptophanamide.

The aqueous layer after the extraction was adjusted to pH 5.9 with 2N concentrated sulfuric acid, concentrated under reduced pressure until the total amount was about 5-5, and cooled on ice to precipitate L-)ligtophan (630 m9 (mp280).
~282°C (decomposition), [α) 20-335° (C=0.
5 Wednesday)] was completed.

On the other hand, 500 JLl of recovered D-tryptophanamide
Add 20mek of 2N-NctOH to y and incubate at 90℃ for 3
The mixture was heated for a period of time to carry out a hydrolysis reaction.

After the reaction, the pH was adjusted to 6.0 with ftL acid, and then cooled with water to precipitate D=)Lyptophan 4601n9 [isolated yield 9
2chi, [α) 20 + 33.3° (C = 0.5 water]]
I achieved 1 result.

Patent applicant: Ube Industries Co., Ltd.

Claims (2)

[Claims] (1) General formula (wherein X is a hydrogen atom, a hydroxyl group, etc.)・Rhodan atom. Indicates a lower alkyl group or a lower alkoxyl group. ) to DL-) ribtonoanamides represented by The hydrolysis of L-ligtonamides represented by the above general formula produced by yeasts belonging to the genus Rhodotorula, Trichosporon or Rhodosboridium is performed using a metal selected from pancreatic acid, manganese, magnesium, and cobalt. It is characterized by acting on the presence of at least one type of salt. A method for producing optically active L-) IJ butophans represented by the general formula (wherein X has the same meaning as above). (2) DL-) liptonoanamides represented by the general formula (wherein X represents a hydrogen atom, a hydroxyl group, a rhodane atom, a lower alkyl group or a lower alkoxyl group). At least a metal salt selected from manganese, magnesium, and cobalt; 'j'r obtained by reacting in the presence of one or more
--Characterized by hydrolyzing guttophans V. A method for producing photoactive D-) IJ butophans represented by the general formula (where χ has the same meaning as above).