JP3006615B2 - Method for producing D-β-hydroxy amino acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to the reaction of glycine with an aldehyde compound in the presence of D-threonine aldolase to form a corresponding D-β
A method for producing hydroxyamino acids. D-β
-Hydroxyamino acids are useful as raw materials for synthesizing various drugs such as antibiotics and enzyme inhibitors, agricultural chemicals, and other various physiologically active substances.

[Problems to be solved by conventional technology and invention]

Conventionally, as a method for producing D-β-hydroxyamino acid, a method of preferentially crystallization of a racemic mixture (Japanese Patent Publication No.
No. 25006) is known, but the operation is complicated,
Also, the yield is extremely low. On the other hand, as a method using an enzyme or a microorganism, a method in which a 5-substituted hydantoin compound is reacted with a microorganism to convert the compound into D-β-hydroxyaminobutyric acid (JP-A-61-212292), D-β-hydroxyaminobutyric acid A method of causing asymmetric hydrolysis by reacting a microorganism with an amide (Japanese Patent Application Laid-Open No. 61-274690) is known, but these methods require expensive starting materials.

[Means for solving the problem]

The present invention provides a D-β, comprising reacting glycine with an alicyclic aldehyde, an aromatic aldehyde or a heterocyclic aldehyde in the presence of D-threonine aldolase.
-A process for producing hydroxyamino acids.

The alicyclic aldehyde, aromatic aldehyde or heterocyclic aldehyde used in the present invention is, for example, an alicyclic aldehyde such as cyclopentyl aldehyde, cyclopentenyl aldehyde, cyclohexyl aldehyde, cyclohexenyl aldehyde, cyclohexyl acetaldehyde, cyclohexenyl acetaldehyde, etc. Heterocyclic aldehydes such as benzaldehyde, fluorobenzaldehyde, chlorobenzaldehyde, bromobenzaldehyde, nitrobenzaldehyde, chloronitrobenzaldehyde, hydroxynitrobenzaldehyde, methoxybenzaldehyde, fluoromethoxybenzaldehyde, tolualdehyde, trifluorotolualdehyde, and phenylacetaldehyde As 2-thiophenealdehyde, Romo 2-Chi off ene aldehyde, 4-off Ol mill imidazole, 4-methyl-5 full ol mill imidazole can be used.

Glycine used in the present invention may be produced by a usual method, and the production method is not limited.

The D-threonine aldolase used in the present invention is an enzyme that acts on D-threonine to decompose into glycine and acetaldehyde. For example, Alcaligenes faecalis (IFO12669), Pseudomonas DK-2 (fine-crafted) No. 6200), Ahrismobacter (DK-19)
No. 01) and Xanthomonaso oryzae
ryzae) (IAM1657) have the ability to produce this D-threonine aldolase.

The microorganism used in the present invention can be cultured according to a conventional method. The medium used for the culture is a usual medium containing a carbon source, a nitrogen source, an inorganic substance, and the like necessary for the growth of the microorganism.
Further, the addition of organic micronutrients such as vitamins and amino acids often provides desirable results. The cultivation may be carried out under aerobic conditions at pH 4 to 10 and at a temperature in the range of 20 to 60 ° C. for 1 to 10 days.

For the enzyme reaction, cultured cells, dried cells, cell lysate, etc., separated from the culture solution of microorganisms, crudely purified enzymes fractionated with inorganic salts, organic solvents, etc., isolated enzymes Further, cells, cells treated with the cells, immobilized products of the enzymes themselves, and the like can also be used.

The method of performing the enzyme reaction is a culture solution of the above-described microorganism, glycine and aldehyde compound in an aqueous medium, cells,
What is necessary is just to make it contact with the processed cells, the enzyme, or those obtained by immobilizing them by a usual method. As the aqueous medium at the time of such a reaction, for example, water, a buffer, a water-containing organic solvent, or 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, and is preferably 0.05 to 2.0 M / L. Glycine, one of the substrates, may be about equimolar to the aldehyde compound. Such glycine and aldehyde compounds can be added at any stage of the reaction,
It can be implemented by any of batch, continuous, and divisional means.

The reaction temperature is preferably from 10 to 70 ° C, more preferably from 10 to 40 ° C. The pH during the reaction is 5 to 10, preferably 5.5
7.0. When pyridoxal 5'-phosphate is added to the reaction system as a coenzyme, the enzyme activity can be increased to promote the reaction.

In some cases, the reaction yield can be increased by adding 2-mercaptoethanol or sodium bisulfite to the reaction system.

The reaction system may be a batch system or a continuous system.
Thus, the reaction is completed in about 0.5 to 50 hours.

The D-β-hydroxyamino acid thus obtained can be collected from the enzyme solution by a conventional method such as centrifugation or ultrafiltration to remove bacteria, remove proteins, and separate with activated carbon or ion exchange resin. -It can be performed by a method such as purification.

〔Example〕

 In the examples,% indicates% by weight.

Enzyme production example 1 Polypeptone 0.5%, yeast extract 0.5%, KH ₂ PO ₄ 0.1%
PH 7.5 medium was prepared, and 3
Was added and sterilized by heating at 120 ° C. for 15 minutes. In the culture medium, Arisrobacter (Arthrobacter) (DK-19
No. 6201), and cultured at 30 ° C. for 20 hours with aeration and stirring while maintaining the pH at 7.5.

After completion of the culture, the cells were collected from the culture by centrifugation, washed with water, and washed with 0.01 mM pyridoxal-5'-phosphate and 1.0 mM.
0.01 M Tris-HCl buffer solution (pH 7.5) containing manganese chloride 10
The suspension was subjected to ultrasonic crushing at 20 KHz for 3 minutes four times, and the suspended substance in the crushing frame was removed by centrifugation to prepare a crude enzyme solution.

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

Enzyme Production Example 2 Pseudomonas DK-2
No. 200), Alcaligenes f.
aecalis) (IFO12699) and Xanthomonas oryzae (IAM1657)
A bacterial cell and an acetone fractionated enzyme solution were prepared in the same manner as described above.

Example 1 Athrobacter (Arthrobacte) obtained in Enzyme Production Example 1
r) 2.0 mmol of glycine, 2.0 mmol of aldehyde compound, 2.0 mmol of 2-mercaptoethanol, 2.0 μmol of pyridoxal-5'-phosphoric acid in 10 ml of enzyme solution of DK-19 (Microtechnical Laboratories No. 6201), Manganese chloride (divalent) 20 μmol, and pH
10 ml of 6.8 0.5 M-HEPES buffer (Dctite Good's buffer)
A substrate solution was added and reacted at 30 ° C. for 20 hours.

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 1 shows the results of these analyses.

The product β-hydroxyamino acid was identified by ultrafiltration of the reaction solution, purification by an ion-exchange resin column, development by thin-layer chromatography under the above conditions, extraction of the product, vacuum drying, and C ¹³ − Performed by NMR and H ¹ -NMR analysis.

The optical purity of the produced β-hydroxyamino acid is N-
After reaction with carboxyalanine anhydride (NCA) to convert to diastereomer, analysis by liquid chromatography using a CDS column, analysis by liquid chromatography using an optical resolution column (Chiralpack, manufactured by Daicel), and shift It was measured by NMR analysis using a reagent. When the β-hydroxyamino acids produced were analyzed by the above method, they were all D-forms.

Example 2 Pseudomonas D obtained in Enzyme Production Example 2
K-2 (Microtechnical Laboratories No. 6200), Alcaligenes faecalis (IFO12669), and Xanthomonas oryzae (IAM165)
Using the enzyme solution of 7), glycine and benzaldehyde were used as substrates, and an enzymatic reaction was carried out in the same manner as in Example 1, and the product was separated and quantified. Table 2 shows the results of these analyses. The phenylserine formed was all D-form.

Example 3 Athrobacter (Arthrobacte) obtained in Enzyme Production Example 1
r) 30 g of glycine, 3.0 ml of 2-mercaptoethanol, 0.1 mmol of manganese chloride and 0.5 ml of o-fluorobenzaldehyde were added to 30 ml of the enzyme solution of DK-19 (Microtechnical Laboratories No. 6201) in 30 ml.
Add 10 times per minute (total of 5.0 ml) and stir at 30 ° C
And reacted for 20 hours. Keep closed during the reaction,
The reaction pH was kept at 6.5 with 0.2N-NaOH.

After completion of the reaction, the pH of the reaction solution was reduced to 2.0 with concentrated hydrochloric acid,
After removing insoluble matter by centrifugation, suspended substances and high molecular weight substances were removed with a 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).
The product is adsorbed by passing it through the column and washed thoroughly with deionized water. The adsorbed substance is eluted with a mixed aqueous solution of 20% by weight of acetic acid and 5.0% of phenol, and the elution portion of o-fluorophenylserine is collected. did.

Next, the eluted portion of the o-fluorophenylserine was diluted with deionized water 1.0 and passed through a 100 ml column packed with Dowex 50Wx8 (50 to 100 mesh, manufactured by Dow Chemical Co.) in H ⁺ form, After washing with deionized water, the adsorbed substance was eluted with 0.5 M ammonia water, and an elution 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. Further, the optical purity was measured by the same method as in Example 1, and all of them were D-forms.

〔The invention's effect〕

According to the method of the present invention, D-β-hydroxyamino acids corresponding to glycine and aldehyde compounds which are produced in large quantities can be produced at once, which is extremely excellent as an industrial method for producing D-β-hydroxyamino acids. ing.

────────────────────────────────────────────────── (5) Continuation of the front page (51) Int.Cl. ⁷ Identification code FIC12R 1:64) (56) References JP-A-50-63192 (JP, A) JP-A-49-117684 (JP, A) JP-A-58-116690 (JP, A)

Claims (1)

(57) [Claims] 1. D-β characterized by reacting glycine with an alicyclic aldehyde, aromatic aldehyde or heterocyclic aldehyde in the presence of D-threonine aldolase.
-A process for producing hydroxyamino acids.