JP2721536B2 - Method for obtaining D-β-hydroxy amino acid - Google Patents

Description

The present invention relates to a method for obtaining D-β-hydroxy amino acid. D-β-hydroxy amino acids are antibiotics,
It is useful as a raw material for synthesizing various medicines such as enzyme inhibitors, agricultural chemicals, and other various physiologically active substances.

[Problems to be solved by the prior art and the invention] D-β-hydroxyamino acids are produced by optical resolution of DL-amino acids produced by an organic synthesis method or racemization of L-amino acids. In that case, since the D-amino acid is produced through a long process, the labor is troublesome and the yield is low.

Glycine and aldehyde compounds, which are produced inexpensively and in large quantities, are reacted in the presence of D-threonine aldolase (an enzyme that decomposes D-threonine into glycine and acetaldehyde and also has an activity of catalyzing the reverse reaction). A method for producing the corresponding D-β-hydroxyamino acid is known (JP-A-58-116690).
However, in this method, the aldehyde compound is glycine or D
A non-enzymatic side reaction with -β-hydroxy amino acid (hereinafter abbreviated as amino acid) causes a low reaction yield and a large amount of unreacted glycine remains.

[Means for solving the problem]

The first invention of the present invention provides a method for preparing a glycine metal complex and an aldehyde compound in the presence of D-threonine aldolase.
This is a method of obtaining a D-β-hydroxyamino acid after reacting and converting to a D-β-hydroxyamino acid metal complex.

The second invention uses nickel and / or cobalt as the metal of the glycine metal complex.

From the metal complex of D-β-hydroxy amino acid, D-β-hydroxy amino acid can be easily obtained by demetalation by a known method.

The method of the present invention using a glycine metal complex as a substrate has the following advantages. First, since nickel and / or cobalt forms a stable complex with the amino acid, a side reaction between the amino acid and the aldehyde compound is prevented. Suppressed and D-β-hydroxyamino acid can be obtained in high yield.

Second, when the reaction is performed using the glycine metal complex as a substrate, a higher substrate conversion can be obtained as compared with the case where free glycine is used.

Third, a high concentration of a glycine nickel complex or glycine cobalt complex decomposes an enzyme that acts as a catalyst for an undesirable reaction contained in the cells, for example, L-allothreonine aldolase (decomposes L-allothreonine into glycine and acetaldehyde). Strongly inhibits and inactivates L-threonine aldolase (an enzyme that also degrades L-threonine to glycine and acetaldehyde, which also has the activity of catalyzing the reverse reaction). Therefore, it is possible to use cells or a crudely purified enzyme.

As in the present invention, an enzymatic reaction using a high-concentration metal complex as a substrate is not known, and therefore, the method of the present invention is a novel enzymatic reaction method.

As the glycine metal complex used in the present invention, a glycine nickel complex or a glycine cobalt complex is preferable in terms of the reactivity with D-threonine aldolase and the yield of D-β-hydroxy amino acid. As other metal complexes, metal complexes such as cadmium, iron, manganese and magnesium can be used. Further, these complexes can be used arbitrarily, and among them, a nickel complex and a cobalt complex are preferably used in combination. The general formula of the glycine nickel complex or glycine cobalt complex preferably used in the present invention is [M (NH ₂ CH ₂ CO ₂ ) m] · nH ₂ O (where M is a metal and M is nickel) m = 2,
n = 0 to 2, when M is cobalt, m = 3 and n = 0 to 2). Further, a glycine metal complex containing two or more metals, for example, a glycine metal complex containing both nickel and cobalt can be used.

The glycine metal complex used in the present invention can be synthesized by an ordinary method such as, for example, adding glycine and an alkali to an aqueous solution of a metal salt solution and heating.

The aldehyde compound used in the present invention is an aliphatic aldehyde, an alicyclic aldehyde, an aromatic aldehyde, a heterocyclic aldehyde compound, and the like. For example, acetaldehyde, 1-propanal,
-As cycloaliphatic aldehyde compounds such as butanal, 2-methylpropanal, 1-pentanal, 1-hexanal, 1-heptanal, chloroacetaldehyde, fluoroacetaldehyde, nitroacetaldehyde, and ethoxyacetaldehyde; cyclopentylaldehyde, cyclopentenylaldehyde, cyclohexylaldehyde , Cyclohexenyl aldehyde, cyclohexyl acetaldehyde, cyclohexenyl acetaldehyde, etc., aromatic aldehyde compounds such as benzaldehyde,
2-thiothiophene as heterocyclic aldehyde compound For example, phenaldehyde, bromo-2-thiophenealdehyde, 4-formylimidazole, 4-methyl-5-formylimidazole can be used.

The D-threonine aldolase used in the present invention is an enzyme which acts on D-threonine to decompose into glycine and acetaldehyde. No. 6200), Arisrobacter DK-19 (6201)
No.) and Xanthomonas oryzae
yzae) (IAM1657) and the like have the ability to produce this D-threonine aldolase.

The microorganism can be cultured according to a conventional method. The medium used for the culture is a carbon source necessary for the growth of microorganisms,
This is a normal medium containing a nitrogen source, an inorganic substance, and the like. Furthermore, the addition of organic micronutrients such as vitamins and amino acids often produces desirable results.

Cultivation is performed under aerobic conditions for 1 to 10 days while controlling the pH to an appropriate range of 4 to 10 and a temperature of 20 to 60 ° C.

In the reaction, a culture solution of microorganisms, culture cells separated from the culture solution, dried cells, cell lysate, etc., as well as crude purified enzymes fractionated with ammonium sulfate, organic solvent, etc., were isolated. Enzymes, furthermore, microbial cells, processed microbial cells, immobilized products of the enzymes themselves, and the like can be used.

A glycine metal complex and an aldehyde compound corresponding to D-
The method of converting the β-hydroxy amino acid metal complex into the above-mentioned glycine metal complex and an aldehyde compound in an aqueous medium is performed by a conventional method using a culture solution of the microorganism, a bacterial cell, a treated bacterial cell, an enzyme, or a conventional method. What is necessary is just to contact with the thing fixed. As the aqueous medium at the time of such a reaction, for example, water, a buffer, a water-containing organic solvent and the like can be used.

The concentration of the aldehyde compound in the reaction solution may be such that the enzyme is not significantly inhibited or deactivated.
Is preferred.

The glycine metal complex may be about equimolar to the aldehyde compound, but in order to increase the reaction yield of glycine,
It is better to use less than the aldehyde compound. The addition of the glycine metal complex and the aldehyde compound can be performed at any stage of the reaction, and can be performed by any of batch, continuous, and divided means.

The reaction concentration is preferably from 10 to 70 ° C, more preferably from 10 to 40 ° C. The pH during the reaction is preferably maintained at 5 to 10, preferably 5.5 to 7.0. Pyridoxal-5 'as a coenzyme
-When phosphoric acid is added to the reaction system, the enzyme activity can be increased to promote the reaction.

In addition, by adding magnesium ions or manganese ions to the reaction system, the enzyme activity and the stability of the activity can be enhanced to promote the reaction. The reaction is preferably performed in a batch system or may be performed in a continuous system. Thus, the reaction is 0.
It takes about 5 to 50 hours.

In order to collect the D-β-hydroxyamino acid thus obtained from the reaction solution, for example, after dissolving the amino acid metal complex, the protein is removed by ultrafiltration or the like, and separated by an ion exchange resin or the like. Conventional methods can be used, such as a recovery method, and a method of concentrating the reaction solution to precipitate the amino acid metal complex, and then dissolving and removing the metal.

For example, if the reaction mixture is adsorbed on a strong cation exchange resin and then eluted with an aqueous solution of an alkali metal salt, the three components can be simultaneously separated and recovered efficiently and with high yield.

The D-β-hydroxyamino acid separated from the reaction solution by the above operation can be purified by a method such as ion exchange resin treatment, activated carbon treatment, crystallization, and the like, and can be isolated and purified by concentrating the reaction solution.

〔Example〕

Next, the method of the present invention will be described in more detail with reference to examples. In the examples,% indicates% by weight. The symbols Thr and Allo represent threonine and arosthreonine, respectively.

Enzyme production example Polypeptone 0.5%, Yeast extract 0.5%, KH ₂ PO ₄ 0.1%
Was prepared in a 5 volume culture tank, and sterilized by heating at 120 ° C. for 15 minutes. This medium was inoculated with Arthrobacter DK-19 (No. 6201, manufactured by B.I.K.) and cultured at 30.degree. C. for 20 hours with aeration and stirring while maintaining the pH at 7.5.

After completion of the culture, the cells were centrifuged from the culture solution, washed with physiological saline, and the wet cells were washed with 0.1 mM pyridoxal-5 '.
- phosphoric acid, and 1.0 mM MnCl pH 7.5 in 0.1M Tris containing ₂ - was suspended in hydrochloric acid buffer solution 100 ml. 20K of this cell suspension
The cells were sonicated four times at 3 Hz for 3 minutes to disrupt the cells, and the suspended substances were removed by centrifugation to prepare a crude enzyme solution.

Next, while stirring the crude enzyme solution, cold acetone was added thereto, and the precipitate in the range of acetone concentration of 45 to 65% was fractionated and dissolved in 10 ml of the above buffer solution to prepare an acetone fractionated enzyme solution.

Example of heat-treated bacterial cell production The cells were collected by centrifugation from the culture solution 3.0 of Arisrobacter (Arthrobacter) DK-19 obtained by culturing in the same manner as in Enzyme Production Example 1. Wash with water, and wash the wet cells with 0.01 mM pyridoxal-5'-phosphate and 1.
It was suspended in 100 ml of an aqueous solution containing 0 mM MnCl ₂ and adjusted to pH 6.0. Next, this bacterial cell suspension was subjected to a heat treatment at 55 ° C. for 120 minutes, and the cells were collected by centrifugation, washed with water, and suspended in 10 ml of the above aqueous solution to prepare a heat-treated bacterial cell suspension.

Example 1 Glycine nickel complex [Ni (NH ₂ CH ₂ CO ₂ ) ₂ ] · 2H ₂ O, glycine cobalt complex [Co (NH ₂ CH ₂ CO ₂ ) ₃ ] · was added to 10 ml of the crude enzyme solution obtained in the enzyme production example. Add cobalt hydroxide and nickel hydroxide to 2H ₂ O or glycine aqueous solution and heat
Glycine-cobalt nickel complex [Co.
Ni · (NH ₂ CH ₂ CO ₂ ) ₅ ] · 4H ₂ O and 0.15 g of 0.5 M Tris-HCl buffer (pH 7.5) containing 0.2 g of acetaldehyde in 1
0 ml was mixed and reacted at 30 ° C. for 10 hours in a closed container.

After the reaction was completed, 20 ml of 0.3N hydrochloric acid was added, insoluble materials were removed by centrifugation, and the reaction solution diluted to about 10 times was added to Dowex A-1.
(Na-type, 100-200 mesh, Dow Chemical), demetallized, and glycine by HPLC (Hitachi amino acid analysis system),
And the mixture of threonine isomers was quantified. Each threonine isomer is converted to a diastereomer by reacting with N-carboxy L-phenylalanine anhydride (NCA) and then converted to H
PLC (column: ODS6.0 × 250mm, mobile phase: 0.1M KH ₂ PO ₄ −K ₂ H
PO ₄ (pH 5.0) / CH ₃ CN = 96/4, flow rate: 1.0 ml / min, detection: 210 n
m). For comparison, a reaction was carried out in the same manner as described above using 0.1 g of glycine as a substrate.

 Table 1 shows the results of these analyses.

Example 2 Arthrobacter obtained in an enzyme production example
3.0 g of glycine nickel, 0.1 mmol of MnCl _{2 and} 0.1 ml of acetaldehyde were added to 100 ml of the enzyme solution of DK-19 (Microtechnical Laboratories No. 6201).
Add 5 ml 10 times every 30 minutes (total 5.0 ml), and add 30 ml with stirring.
The reaction was carried out at ° C. Keep closed during the reaction, 0.2N
The reaction pH was kept at 6.5 with NaOH.

20 hours after the start of the reaction, the pH of the reaction solution was lowered to 2.0 with concentrated hydrochloric acid, and insolubles were removed by centrifugation. Then, suspended substances and high molecular weight substances were removed with an ultrafiltration membrane having a molecular weight cutoff of 50,000. . Next, the reaction solution was evaporated under reduced pressure to remove unreacted acetaldehyde, and a 10 ml activated carbon column (Turmicol GL-3) was used.
After passing through 0), the solution was passed through a 100 ml column filled with H ⁺ type Dowex50Wx8 (50-100 mesh, manufactured by Dow Chemical Company), washed with deionized water, and adsorbed with 0.2M ammonia water. Was eluted, and a fraction containing β-hydroxy-α-aminobutyric acid was collected and concentrated, and then methanol was added to precipitate crystals to obtain 0.8 g of crystals. NMR, IR,
Elemental analysis confirmed that the product was β-hydroxy-α-aminobutyric acid. Furthermore, when threonine isomer analysis was performed in the same manner as in Example 1, all were in D-form,
The ratio of D-threonine to D-arothreonine was 2.5.

Example 3 Using glycine nickel as a glycine metal complex,
Various aldehyde compounds were reacted in the same manner as in Example 1.

After completion of the reaction, the generated β-hydroxyamino acid was separated and quantified by thin-layer chromatography. Thin-layer chromatography shows that the ratio of 1-propanol and 25% ammonia water is 2:
Chromatography was performed on a silica gel thin layer using the mixed solution of No. 1 as a developing solvent, the color was developed with ninhydrin, and quantified using a densitometer (CS-910, Shimadzu Corporation). Table 3 shows the results of these analyses.

Identification of the generated β-hydroxyamino acid was performed by ultrafiltration of the reaction solution, purification of the ion-exchange resin column, development by thin-layer chromatography under the above conditions, extraction of the product, vacuum drying, and C ¹³ -NMR. , And H ¹ -NMR.

The optical purity of the produced β-hydroxyamino acid is N-
After reacting with carboxy L-phenylalanine anhydride (NCA) to convert to diastereomer, analysis by liquid chromatography using an ODS column, liquid chromatography using an optical resolution column (Chiralpack, manufactured by Daicel) And NMR using shift reagent
It was determined by analysis. When the β-hydroxyamino acids produced were analyzed by the above method, they were all D-forms.

Example 4 Arthrobacter obtained from an enzyme production example
3.0 g of glycine nickel, 0.1 mmol of MnCl ₂ and 0.5 ml of o-fluorobenzaldehyde were added 10 times every 30 minutes (total 5.0 ml) to 100 ml of the enzyme solution of DK-19 (Microtechnical Laboratories No. 6201).
The reaction was carried out at 30 ° C. under stirring. During the reaction, the sealed state was maintained, and the reaction pH was maintained at 6.5 with 0.2N-NaOH.

20 hours after the start of the reaction, the pH of the reaction solution was lowered to 2.0 with concentrated hydrochloric acid, and insolubles were removed by centrifugation. Then, suspended substances and high molecular weight substances were removed with an ultrafiltration membrane having a molecular weight cutoff of 50,000. . Next, this reaction solution was applied to a 50 ml activated carbon column (Tsurumi Coal GL).
-30) to adsorb the product, wash thoroughly with deionized water, elute the adsorbed material with a mixed aqueous solution of 20% acetic acid and 5.0% phenol, and elute the o-fluorophenylserine Was collected.

Next, the eluted portion of the o-fluorophenylserine was diluted to 1.0 with deionized water to make H ⁺ form Dowex50Wx8 (50
From 100 Mesh, manufactured by Dow Chemical Company)
After washing with deionized water, the adsorbed substance was eluted with 0.5 M aqueous ammonia, and the eluted portion of o-fluorophenylserine was collected.

Next, the ratio of 1-propanol and 25% ammonia water is 2:
Chromatography was performed on a thin layer of silica gel using the mixed solution of 1 as a developing solvent, and the product was extracted and crystallized with ethanol to obtain 0.1 g of a crystal. The crystals were confirmed to be o-fluorophenylserine by IR, NMR and elemental analysis. When the optical purity was measured by the same method as in Example 3, all the compounds were in D-form.

Example 5 Using Pseudomonas DK-2 (Microtechnical Laboratory No. 6200), culturing was performed in the same manner as in the enzyme production example, and using the acetone fractionated enzyme solution prepared from the culture solution, the same procedure as in Example 1 was performed. React in the same way to separate amino acids contained in the reaction solution.
Quantified.

 Table 4 shows the results of these analyses.

Example 6 Alcaligenes faecalis
IFO 12669), and cultured in the same manner as in the enzyme production example.
The reaction was carried out in the same manner as described above, and the amino acids contained in the reaction solution were separated and quantified.

 Table 5 shows the results of these analyses.

Example 7 Pseudomonas DK-2
No. 200), Alcaligenes f.
aecalis) (IFO12669) and Xanthomonas oryzae (IAM1657), cultivated in the same manner as in the enzyme production example, using an acetone fractionated enzyme solution prepared from the culture solution, using glycine nickel and benzaldehyde as substrates, An enzymatic reaction was carried out in the same manner as in Example 1, and the product was separated and quantified. Table 6 shows the results of these analyses. The resulting phenylserine was all in the -form.

Example 8 (Example of microbial cell reaction) The cells were centrifuged from the culture solution 3.0 of Arisrobacter (Arthrobacter) DK-19 (Microtechnical Laboratories No. 6201) obtained by culturing in the same manner as in the enzyme production example. Collect and wash the body with water, 0.01 mM
Pyridoxal-5'-phosphate, and suspended in an aqueous solution 100ml containing 1.0 mM MnCl _2, pH was adjusted to 6.0. Next, this cell suspension was subjected to a heat treatment at 55 ° C. for 120 minutes, washed with water and collected by centrifugation, and suspended in 10 ml of the above aqueous solution. Table 7 shows the results of the reaction and analysis performed using this cell suspension in the same manner as in Example 1.

Example 9 (Example of heat-treated cells, glycine nickel) 3.0 g of glycine nickel complex [Ni (NH ₂ CH ₂ CO ₂ ) ₂ ] · 2H ₂ O was added to 100 ml of the cell suspension obtained in the heat-treated cell production example. And 0.5 ml of acetaldehyde was added 10 times every 30 minutes (total 5 ml), and the reaction was carried out at 30 ° C. for 20 hours with stirring. Keep the sealed condition during the reaction, and adjust the pH of the reaction solution with 0.2N-NaOH.
Was maintained at 6.0. When the amino acids contained in the reaction solution were quantified, the amount was 1.24 g of D-β-hydroxyaminobutyric acid and 0.97 g of gucillin.

After completion of the reaction, the pH of the reaction solution was reduced to 2.0 with concentrated hydrochloric acid,
After removing insolubles by centrifugation, suspended substances and high molecular weight substances were removed with an ultrafiltration membrane having a molecular weight cutoff of 50,000. Next, the reaction solution was evaporated under reduced pressure to remove unreacted acetaldehyde, and the solution was passed through a 10 ml activated carbon column (Turmicol GL-30) to prepare a purified reaction solution. Next, this reaction solution was
The solution was passed through a 65 ml column packed with Dowex50Wx8 (50-100 mesh, Dow Chemical Co.) diluted in H ⁺ form and washed with 300 ml of deionized water, and adsorbed with 0.2 M aqueous sodium chloride. Was eluted. Eluate volume from 50ml to 25
D-β-hydroxyaminobutyric acid eluted in 0 ml fraction, 2
The mixture of D-β-hydroxyaminobutyric acid and glycine eluted in the 50 to 300 ml fraction, and glycine eluted in the next 300 to 600 ml eluted fraction. After glycine was eluted, when a 1.7 M aqueous sodium chloride solution was passed through the column, nickel was eluted in a fraction of 600 to 840 ml.

The eluted fraction of D-β-hydroxyaminobutyric acid was evaporated to dryness and recrystallized from ethanol to obtain 0.95 g of D-β-hydroxyaminobutyric acid crystals.

The fraction eluted with the mixture of D-β-hydroxyaminobutyric acid and glycine was similarly crystallized to obtain a mixture of 0.23 g of D-β-hydroxyaminobutyric acid and 0.18 g of glycine. The glycine eluted fraction was crystallized in the same manner to obtain 0.71 g of glycine crystals.

The eluted nickel was converted into nickel carbonate by reacting with sodium carbonate, and almost all of the amount used for the enzyme reaction was recovered.

Example 10 (Example of heat-treated cells, glycine cobalt) Glycine cobalt complex [Co (NH ₂ CH ₂ CO ₂ ) ₃ ] · 2H ₂ O
Using 3.0 g as a substrate, an enzyme reaction was carried out in the same manner as in Example 1,
An enzyme reaction solution containing 1.36 g of D-β-hydroxyaminobutyric acid and 0.88 g of glycine was obtained.

D-β-hydroxyaminobutyric acid, glycine, and cobalt were eluted and separated from this reaction solution in the same manner as in Example 9.

Next, the eluted fraction of D-β-hydroxyaminobutyric acid was evaporated to dryness and recrystallized from ethanol to obtain 0.94 g of D-β-hydroxyaminobutyric acid crystals.

The fraction eluted with the mixture of D-β-hydroxyaminobutyric acid and glycine was similarly crystallized to obtain a mixture of 0.24 g of D-β-hydroxyaminobutyric acid and 0.17 g of glycine. The glycine eluted fraction was crystallized in the same manner to obtain 0.66 g of glycine crystals.

The eluted cobalt was converted to cobalt carbonate by reacting with sodium carbonate, and almost all of the amount used for the enzyme reaction was recovered.

〔The invention's effect〕

According to the method of the present invention, the corresponding D-β-hydroxyamino acid can be produced from glycine and an aldehyde compound which are produced in large quantities, so that the method is extremely excellent as an industrial method for producing D-β-hydroxyamino acid. is there.

Claims (2)

(57) [Claims] 1. A method for reacting a glycine metal complex with an aldehyde compound in the presence of D-threonine aldolase to convert it to a D-β-hydroxyamino acid metal complex and then obtaining D-β-hydroxyamino acid . 2. The method for obtaining D-β-hydroxyamino acid according to claim 1, wherein the metal of the glycine metal complex is nickel and / or cobalt.