JPS63123398A - Production of l-alpha-methylphenylalanines - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as a raw material for producing hypotensor, etc., industrially advantageously, by treating a DL-alpha- methylphenylalaninamide with a cell of Mycobacterium methanolica or a treated material thereof and asymmetrically hydrolyzing. CONSTITUTION:A DL-alpha-methylphenylalaninamide shown by formula I (R<1> and R<2> are H, lower alkyl or R<1> and R<2> are mutually bonded to form 5-6-membered ring) is treated with a cell of Mycobacterium metanolica (FERM-P8823, etc.) of a treated material thereof and an L-alpha-methylphenylalaninamide is asymmetrically hydrolyzed to give an L-alpha-methylphenylalanine shown by formula II.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention uses Mycobacterium meta/rica, a new M species, to obtain the corresponding L-α- The present invention relates to a method for producing methylphenylalanines.

L-units of α-methylphenylalanines have pharmacological effects, and L-3,4-dihydroxy-α-methylphenylalanine is known as a typical example thereof. This substance, commonly referred to as L-α-methyldopa, is an excellent antihypertensive agent, and L-α-methylphenylalanines are its raw materials.

[Prior art, problems to be solved by the invention] Conventionally, L
-As a method for producing α-methylphenylalanine, α
- Physical methods using diastereomer methods or direct crystallization methods and biochemical methods using microorganisms have been investigated as methods for optically resolving racemic forms of methylphenylalanines. However, all of these methods are unsatisfactory as industrial processes due to high production costs due to low yields.On the other hand, in recent years, DL-α-methylphenylalanine amides have been used as substrates. A method is known in which L-α-methylphenylalanines are obtained by allowing amidase produced by microorganisms to act on L-α-methylphenylalanines (JP-A-57-11116495). However, in this method, the activity of the microorganism used is not high enough to be used for industrial production.

[Means and actions for solving problems]

The present inventors screened the natural world for microorganisms that efficiently convert DL-α-methylphenylalanine amides into L-α-methylphenylalanines as a substrate, and were able to obtain a strain suitable for that purpose.
The present invention using this strain was completed. Although this strain belongs to the genus Mycobacterium, it was determined to be a new bacterial species based on its mycological properties, and was named Mycobacterium methanolica.

That is, the present invention is based on the general formula (1) (wherein R1 and R2 are the same or different and are a hydrogen atom, a lower alkyl group, or
are alkylene groups, and can also be bonded to each other to form a 5- to 8-membered ring. ) The L-α-methylphenylalanine amides represented by the general formula 1 ( However, in the formula, R and R2 are the same as in the general formula +.

D represented by the general formula (2), which is a substrate in the present invention
Representative examples of L-α-methylphenylalanine amides are listed below, but the substrates of the present invention are not limited thereto.

DL-3,4-dihydroxy-α-methylphenylalaninamide H2 DL-4-hydroxy-3-methoxy-α-methylphenylalaninamide H2 DL-5,4-dimethoxy-α-methylphenylalaninamide NH2 DL-3,4- Methylenedioxy-α-methylphenylalanine amide These substrates are asymmetrically hydrolyzed by microorganisms to obtain L-α-methylphenylalanines corresponding to each substrate.

In addition, L-α-methyldopa is based on these substrates, D
L-! 1,4-dihydroxy-α-methylphenylalaninamide and DT, -5゜4-methylenedioxy-
It can be obtained from α-methylphenylalanine amide.

That is, DL-5,4-dihydroxy-α and DL
When -5,4-methylenedioxy-α-methylphenylalanine is used as a substrate, the methylenedioxy bond of L-5,4-methylenedioxy-α-methylphenylalanine obtained according to the present invention is removed by a conventional method. =L-3,4-')hydroxy-α-methylphenylalanine (L-α-methyldopa) is easily obtained by hydrolysis, for example by treatment with a mineral acid such as hydrochloric acid in the presence of phenol. I can do it.

The bacteria used in the present invention may be any strain as long as it belongs to Mycobacterium methanolica.

Mycobacterium methanolica was discovered by the present inventors 1.
Mycobacterium methanolica BT8 is a representative strain of bacteria belonging to this new bacterial species.
4 (Il Koken Bibori No. 8823), BT-143 (Il Koken Bikyo No. 8824), P-23 (Il Koken Bikki No. 8
825), the same P-26 (P1 Research Institute No. 8826), and the same P-85 (P1 Research Institute No. 8827).

Mycological Properties (1) Microscopic Morphology Cultured at 67°C for 3 days in broth liquid medium and broth agar medium.

■ Cell shape and size Usually short rods. Width 0.5~0.8u, length 1~3u0V
type of dividing cells are observed.

■ No motility.

■ Presence or absence of spores: Not produced.

■ Gram staining Gram positive ■ Anti-acidity positive (2) Growth status in various media ■ Broth agar plate culture Cultivate at 37℃ for 3 days.

Shows medium growth.

Colony shape: circular in shape, size 2-3? [, The ridges are hemispherical, the structure is homogeneous, the surface is rough, the edges are wavy, the color is yellowish-white and lack luster, the transparency is opaque, and the hardness is buttery.

■Methanol-containing agar plate culture Cultured at 37°C for 3 days.

Same as broth agar plate culture.

■ Meat juice agar slant culture Cultured at 37°C for 3 days.

It shows medium growth uniformly along the inoculation line.

Medium ridges, rough surface, wavy margins, yellowish-white color with no gloss, opaque transparency, and buttery hardness.

■ Methanol-containing agar slant culture Cultured at 37°C for 3 days.

Same as broth agar slant culture.

■ Meat juice liquid culture Cultivated at 37℃ for 6 days.

Forms a white cream painting platform. It also forms a film.

■ Peptone water liquid culture Cultivate at 37℃ for 3 days.

Same as broth liquid culture.

■ Methanol-containing liquid culture Cultured at 37°C for 3 days. Grows vigorously.

Forms a white cream painting platform. It also forms a film.

■ Meat juice gelatin puncture culture Cultured at 20°C for 4 weeks.

Grow. However, it does not have gelatin liquefaction properties.

■ Lidomus milk culture Cultivate at 37°C for 4 weeks.

It grows, and the culture solution changes to alkaline (pH 688 to pH 8).
, 3) but without peptonization.

('i) 1% Ogawa medium culture, cultured at 37°C for 3 days.

Grows vigorously. The characteristics of the village are smooth.

OHA medium (hydroxylamine hydrochloride 5 o ug
7Tei supplemented with Ogawa medium) Culture at 37°C for 5 days.

Grows vigorously.

6 PA8 medium (sodium para-aminosalicylate
Culture (Ogawa medium supplemented with 1 ml of 2W) at 37°C for 7 days.

It grows vigorously and the medium turns black.

Only strain P-23 shows no blackening of the medium.

0 picric acid medium (modified method 8a with addition of 0.24 picric acid)
(UTON agar medium) Culture at 37°C for 2 weeks.

It grows vigorously and the medium turns reddish brown.

Only the P-25 strain did not turn reddish-brown. Cultured in OPNB medium (Ogawa medium supplemented with 500"I/me of balanitrobenzoic acid) at 37°C for 7 days.

Grows vigorously.

(i3 EB medium (1 t Ogawa medium supplemented with ethambutol 5t19/-) Cultured at 37°C for 7 days.

Grows vigorously.

(3) Physiological properties ■ Nitrate reduction Reduces nitrate to nitrite.

■ MR test negative ■ vp test negative ■ Indole formation negative ■ Hydrogen sulfide formation positive ■ Starch hydrolysis negative ■ Use of nitrogen sources Ammonium salts, nitrates, urea, and peptone are used as nitrogen sources.

■ Pigment generation　　　　　　　　　　No generation.

■ Urease Positive 0 Catalase Positive 0 Production of ammonia Produce.

◎ Denitrification reaction Negative 0 Oxidase reaction Negative 00-F test Negative 0 Growth range It grows in the pH range of 5 to 9. pH 6-8 is preferred.

It does not grow at 5°C or 43°C. 25-40°C is preferred.

0 Attitude towards oxygen Aerobic 0 Salt tolerance Grows vigorously in triplex +% NaCl-containing medium.

6 in volume% NaC6-containing medium, B'r-84, B'l
``-143 strain grows weakly, but P-23, P-26,
P-85 strain does not grow.

0 Vitamin requirement None O Photochromic test negative ■ Dark color test negative Tween 80 hydrolysis test Negative 0 Contains mycolic acid Positive ■ GC (guanine + cytosine) tt BT-8466, 2mo1% BT-14366, 2mo14 P 25 67. 2mo1%P-2666,
8mol1% P-8568, 7molcl) ■ Main bacterial fatty acid composition Straight chain fatty acid 016:0 Heptanounsaturated fatty acid C14:1. , 8:.

10-methyl fatty acid 10-methyl C19:.

■ Quinone type Menaki/N MK 9 (Hz)
Structure of O cell wall me%0- Contains diaminopimelic acid.

0 Separation Source Soil Verges Manual of Determinative
Bacteriology (Bergev's Manu-al
Determinative Bacteriolo
gy) 8th edition [Editor: Buchanan
n), Gibbons, Cow
an), Hort, and Liston.
5 tons), Murray, Niven, Ravin and Stanier (8tainierC)
-kins), (197a)) c, these strains are bacilli, non-motile, Durham positive, acid-fast, contain mycolic acid, and are aerobic. Mycobacterium spp.
).

This is also supported by the GC content, cell fatty acid composition, quinone type, and cell wall structure.

However, when compared with known bacterial species belonging to the genus Mycobacterium, they are similar in terms of ability to assimilate carbon sources including methanol, production of acids from carbon compounds, salt tolerance, growth temperature, etc. is not found. Furthermore, no bacteria belonging to the genus Mycobacterium that has the ability to assimilate methanol have been found, and this new bacterial species is the first.

The experimental method was in accordance with the above-mentioned Burgeis manual and ``Bacteriology Practice Summary'' (195B) edited by the Institute of Medical Science Alumni Association.

In addition, the following compositions were used as a methanol-containing agar plate medium and a methanol-containing agar slant medium. That is, (NT(4)2804 3.fil, KH2PO41
,4g, NazHPO42,19, Mg804-7'H
2O0,29,CaC1z・2Hz030111F,F
eC6H5O?・xH2O30 da, MnCl2 ・4
H205#, Zn804/7H205W9, CuSO4
・5H200, 5nG + Oyohiteifuko (Dirco)
Bacto-agar 15
Dissolve g in pure water 11, adjust this to pH 7.1 and IK
After sterilizing with 47cmtQ for 20 minutes, methanol 1
0 g was added aseptically to prepare a plate culture medium or a slant culture medium.The above-mentioned medium without agar was used as the liquid medium containing methanol.

Bacteria belonging to this new bacterial species were isolated from the soil, which was the source of isolation, in a conventional manner using the agar plate medium containing metal/alcohol as described above.

The medium used for culturing this bacterium (the bacterium used in the present invention, hereinafter the same) must contain at least a carbon source that can be assimilated by this bacterium, and an appropriate amount of nitrogen source and inorganic salts. Either a synthetic medium or a natural medium may be used as long as the medium contains the following.

The carbon source is not particularly limited as long as it can be assimilated by this bacterium, such as alcohols such as methanol and ethanol, and glucose.

sugars such as fructose, sorbitol, and glycerin;
Organic brews such as succinic acid, sugar-containing substances such as molasses, peptone, and extract of sucrose can be used. As nitrogen sources, inorganic nitrogen compounds are used, such as, for example, ammonium salts, nitrates, and/or organic nitrogen compounds, such as, for example, amides, urea, corn steep liquor, casein, peptone, yeast extract and meat extract. . In addition, as the amide, the amide used as a substrate in the present invention is preferable.

Amides may also act as inducers of enzyme activity.

In addition, examples of inorganic components include calcium salts, magnesium salts, potassium salts, sodium salts, phosphates,
Manganese salts, zinc salts, iron salts, copper salts, molybdenum salts, cobalt salts, boron compounds and iodine compounds are used.

The culture conditions are a temperature of 20 to 42°C, preferably 25 to 40°C.
℃, pHs ~9, preferably 6-8. Culture is carried out aerobically under these conditions.

If cultured outside of these conditions, the growth of this bacterium will be impaired, but this does not preclude cultivation under these conditions.

In the present invention, Mycobacterium methanolica is DL
- In order to act on α-methylphenylalanine amides, use a culture solution obtained by culturing microorganisms in a liquid medium, bacterial cells isolated from the culture solution, or a crude enzyme (amidase) or purified enzyme isolated from the culture solution or bacterial cells. , enzyme-containing extract, or its concentrate (sometimes things other than bacterial cells are collectively referred to as treated products) are used to act. Furthermore, bacterial cells, enzymes, and the like can be immobilized on a carrier for use.

The carriers used for this immobilization include natural products such as alginic acid, carrageenan, collagen, cellulose, acetyl cellulose, agar, cellophane, and collodion, or polyacrylamide, polystyrene, polyethylene glycol, polypropylene glycol, polyurethane, and polybutadiene. Examples include polymeric substances.

Immobilization is carried out by conventional methods, but it must be carried out under mild conditions that do not impair amidase activity.

In the present invention, the reaction temperature is 20 to 50°C, preferably 30°C, as reaction conditions for asymmetric hydrolysis of DL-α-methylphenylalaninamide by the action of Mycobacterium meta/rica cells or a treated product thereof. ~45°C, and the reaction pH is 4-10, preferably 7-9.

The reaction time is usually sufficient from 30 minutes to 1.5 days, and preferably about 1 hour to 1 day.

It is also possible to further shorten the reaction time by raising the reaction temperature, increasing the amount of bacterial cells or their processed material, etc.

In the present invention, the amount of microorganisms used is the substrate DL-
It is preferable to use it so that the weight ratio of dried bacterial cells to α-methylphenylalaninamide is within the range of 01 to 1.

In addition, it is not prohibited to set it outside this range. When using a culture solution containing microbial cells, a treated product, or an immobilized product thereof, the amount to be used may be determined by converting it to the amount of M of dry microbial cells.

The concentration of DL-α-methylphenylalanine amide used as a substrate is generally not limited as long as it is below the saturation concentration of 7957761 DT, -α-methylphenyl used as a raw material, but it is preferably added to the reaction solution. 1wt against
'A' or above, the upper limit is set to be about 1 o wt% at most.

In the present invention, when the hydrolysis reaction of L-α-methylphenylalanine amides is completed, the reaction is stopped as soon as possible, and the target substance L-α is extracted from the reaction product liquid.
- Separate and recover methylphenylalanines and unreacted D-α-methylphenylalanine amides.

The separation can be easily carried out by operations such as fractional crystallization and solvent extraction.

In addition, in the above-mentioned asymmetric hydrolysis operation, D in the substrate
-α-Methylphenylalanine amides are not affected by bacterial cell-treated products or immobilized products, but if the reaction time is significantly prolonged, they may be affected by bacterial cells and produce D-α-methylphenylalanine. Because there is %T
When the hydrolysis of j-α-methylphenylalanine amides is completed, the reaction is stopped as soon as possible, and D-α
- A large amount of methylphenylalanine is removed by a conventional method such as changing p) of the reaction product solution and/or changing the temperature of the reaction product solution to remove α-methylphenylalanine.

Further, unreacted D-α-methylphenylalanine amide can be racemized, for example, by heating, and used again as a substrate.

〔Example〕

The present invention will be explained in more detail with reference to Examples.

Example 1 Glycerol 50g, corn stew.

Liquor 50 g, (NH4)2804
5 g, MIZSO4-7H200, 2 g, Fe
SO41H200, o s ji.

CaCl2-2H200,02ji,Mn(j!2
・4H202m9, NaMoO4・2) T2O1 immediately, N
Dissolve aCρ 1 shoulder in 1f of pure water, and set p)T to 7.0V.
Ko7i! Transfer 100 liters of the prepared medium into an 11-volume Erlenmeyer flask (100 liters) and add 1 ml of the culture solution of each strain of Mycobacterium meta/rica precultured in the same medium to the medium that has been killed at 1 Kq/r for 20 minutes at 2 G. The cells were grown in a tank, cultured with shaking at 50°C for 65 hours, and the culture solution was centrifuged at 1800 rpm for 10 minutes to obtain bacterial cells.

1.25 g of the above-mentioned bacterial cells (calculated as dry bacterial weight) was added to 100+n/ of pure water containing 2.5 g of DL-5,4-methylenedioxy-α-methylphenylalaninamide (% pH).
After adjusting the temperature to 9, shake at 40°C for 2 hours! The reaction was carried out. After the reaction is completed, the reaction product liquid is heated to 18,000 r.
The mixture was centrifuged at pm for 10 minutes to obtain a supernatant. This supernatant liquid was analyzed by high performance liquid chromatography, and the produced L-3,4
The yield of -methylenedioxy-α-methylphenylalanine was determined, and the amount of D-3,4-methylenedioxy-α-methylphenylalanine in the supernatant was also determined.

Note that the obtained T, -3,4-methylenedioxy-α-
When the specific optical rotation of methylphenylalanine was measured,
[α] Maro was +21.5 to 22.5°.

The results are shown in Table 1.

Table 1 Yield for L3.4-methylenedioxy-α-methylphenylalanine amide contained in the substrate (
This also applies to the following examples).

Kurihata Molar ratio of the D-1*-D form of 3,4-methylenedioxy-α-methylphenylalanine produced. However,
The denominator indicates 9 bodies (this also applies to the following examples).

Example 2 T) T, -3,4-methylenedioxy-α-methylene 1 The results are shown in Table 2.

Table 2 Example 3 In pure water containing 2.59 DL-5,4-methylenedioxy-α-methylphenylalaninamide, 0.251 in terms of dry bacterial weight! In addition, ivy.

The results are shown in Table 3.

Table 3 Example 4 Glucose 10g, polypeptone 5g, yeast extract 5g, KT (2PO419, ML2SO4・7H20
0.4fl, FeSO47H200.03g, MnCl
2・4T(200,03,j9.T)T,-ri,
4-Methylenedioxy-α-methylphenylalaninamide 5I was dissolved in pure water at 1/r and 1 oome of a medium with the pH adjusted to 7.0 was placed in a 1a volume □ Erlenmeyer flask, and I
Mycobacterium methanolica P-2 precultured in a medium sterilized with K9/Crn2 G for 20 minutes.
J ml of the culture solution from No. 3 was inoculated and cultured at 30°C for 65 hours. This culture solution was centrifuged at 18,000 rpm for 10 minutes to obtain bacterial cells.

To 100 m/100 m of supplementary water containing 2.5 g of DL-3,4-methylenedioxy-α-methylphenylalanine amide, 1.25 J (calculated as dry bacterial weight) of the above bacteria was added, and the pH was adjusted to 9.
After adjusting the temperature, the mixture was shaken and reacted at 40° C. for 48 hours. .

After the reaction is completed, the reaction product solution is heated at 1800 rpm for 10
Centrifugation was performed for a minute to obtain a supernatant. When this supernatant was analyzed by high performance liquid chromatography, it was found that 98 units of L-1,4-methylenedioxy-α-methylphenylalanine were produced relative to the 5-amide amide in the substrate. Also, D-
The amount of 3,4-methylenedioxy-α-methylphenylalanine was 0.4 molar relative to L-3,4-methylenedioxy-α-methylphenylalanine.

Example 5 Methanol 10.9%KH2PO41,4,li+
.

(NH4'rz80439.NazHPO42, 1g,
Mg804-7H200,2,9, FeC6H50-
xT (2030j19, CaC/2・2H2050'l
T? , ZnFi047H205119, MnC/14H
Medium 200 in which 20511G', Cu804・5H20Q, and 5b7 were dissolved in 11 parts of pure water and the pH was adjusted to 7.1.
+q/ to 1/! Pour into Erlenmeyer flask, IKf/m2Q
A culture of Mycobacterium methanolica P-23 pre-cultured in the same medium was inoculated into a medium that had been sterilized for 20 minutes at 30°C, and cultured at 30°C for 4 days.
The cells were centrifuged at 0 rpm for 10 minutes to obtain bacterial cells.

To 100 ml of pure water containing 2.5 g of DL-3,4-methylenedioxy-α-methylphenylalaninamide was added 0.259 in terms of the dry weight of the bacterial cells, and the pT was adjusted to 9. The reaction was carried out by shaking at 40°C for 48 hours. After the reaction was completed, the reaction product solution was heated at 1800 Orp.
The supernatant was obtained by centrifugation at m for 10 minutes. When this supernatant was analyzed by high performance liquid chromatography, it was found that 63 units of L-3,4-methylenedioxy-α-methylphenylalanine were produced with respect to the 5-amide amide in the substrate. Moreover, D-3,4-dimethoxy-α-methylphenylalanine was 0.3 molti with respect to L-3,4-methylenedioxy-α-methylphenylalanine.

Example 6 In the same manner as in Example 1, Mycobacterium methanolica
BT-F3a was cultured to obtain bacterial cells.

Using this bacterial cell, DI, -3゜4-dihydroxy-α-methylphenylalaninamide, DL-
The reaction was carried out in the same manner as in Example 1, except that 4-hydroxy-3-methoxy-α-methylphenylalaninamide and DL-5,4-dimethoxy-α-methylphenylalaninamide were used, and the reaction time was 24 hours. . The results are shown in Table 4.

〔Effect of the invention〕

According to the present invention, L-α-methylphenylalanines such as L-α-methyldopa, which are used as pharmaceuticals, can be easily, stably, and efficiently obtained.

Patent applicant: Mitsubishi Gas Chemical Co., Ltd. Representative Kazuyoshi Nagano

Claims (1)

[Claims] General formula I ▲ Numerical formulas, chemical formulas, tables, etc.▼ (However, in the formula, R^1 and R^2 are the same or different and are a hydrogen atom, a lower alkyl group, or R^1
and R^2 are both alkylene groups and bond to each other,
A 5- to 8-membered ring can also be formed. ) DL-α-methylphenylalanine amides represented by the formula II are asymmetrically hydrolyzed by the action of Mycobacterium methanolica cells or their treated products, resulting in general formula II ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (However, R^1 and R^2 in the formula are the same as in the above general formula I.) It is characterized by obtaining L-α-methylphenylalanine represented by Method for producing L-α-methylphenylalanines.