JPH0559717B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for isolating L-threonine by reacting a mixture of threonine isomers with D-threonine aldolase alone or with D-threonine aldolase and L-allothreonine aldolase.

L-threonine is one of the essential amino acids for humans and animals, and is in great demand as a food and feed additive because its content in various animal and plant proteins is relatively low.

Conventionally, the organic synthesis method for L-threonine had the disadvantage that isomers were produced as by-products and it was difficult to separate them, but the present inventors have developed a method for selectively decomposing D-threonine and D-allothreonine. A novel enzyme that generates glycine and acetaldehyde, D
-We discovered a method for isolating L-threonine by letting threonine aldolase act on DL-threonine, and filed it as Japanese Patent Application No. 1984-209984.
L-allothreonine aldolase, which specifically decomposes L-allothreonine, and the above-mentioned D-threonine aldolase are allowed to act on a mixture of threonine and DL-allothreonine.
He discovered a method to obtain only L-threonine in pure form by decomposing isomers other than threonine, and filed the application as Japanese Patent Application No. 1985-209985. These methods decompose isomers other than L-threonine to generate glycine and acetaldehyde,
If a mixture of threonine isomers containing L-threonine is used as a raw material, L-threonine can be easily isolated, and the by-products glycine and acetaldehyde are also highly marketable. This is an excellent method in which these by-products can be reused as synthetic raw materials if combined with the method for synthesizing threonine.

However, when this enzymatic reaction is carried out using a conventional method such as a column method, the acetaldehyde produced during the enzymatic reaction condenses with glycine, or acetaldehyde self-condenses, resulting in an extremely low recovery rate of acetaldehyde and glycine, resulting in coloration of the reaction solution. There was also a significant problem. Furthermore, if aldehyde is present during the enzymatic reaction, a reverse reaction occurs with threonine aldolase, and acetaldehyde and glycine recombine to produce isomers other than L-threonine. There was a problem that the activity decreased.

As a result of various studies to solve the above problems, the present inventors found that L-threonine was contained in a reactor containing D-threonine aldolase alone or D-threonine aldolase and L-allothreonine aldolase. It has been found that side reactions caused by acetaldehyde and adverse effects on enzymes can be suppressed by adding a mixture of threonine isomers and decomposing isomers other than L-threonine while removing the acetaldehyde produced.

Therefore, the present inventors conducted an enzyme reaction while directly removing enzymes and bacterial cells from the reaction solution in which they coexisted by a method such as distillation. The efficiency of removing the acetaldehyde produced from the reaction solution, such as the speed and energy efficiency of removing the acetaldehyde produced, the extent of side reactions caused by acetaldehyde, and the inefficiency of the reactor due to the need for a large reactor. A fully satisfactory result could not be obtained. As a result of further research on these points, it was discovered that the above-mentioned problems were caused by the enzymatic reaction and the removal of the generated acetaldehyde being carried out in the same reaction system. Therefore, with this method, it is difficult to satisfy all of the enzyme usage efficiency, the removal efficiency of the acetaldehyde produced, and the efficiency of the reactor at the same time, so the enzyme reaction step and the step of removing the acetaldehyde produced are separated. It has been found that it is necessary to remove the acetaldehyde produced by the enzymatic reaction in the shortest possible time to keep the acetaldehyde concentration in the reaction solution as low as possible.

That is, unlike the method of carrying out an enzyme reaction while directly removing the produced acetaldehyde from the reaction solution in which the enzyme coexists by a method such as distillation, this method uses D-threonine aldolase alone or D-threonine aldolase and L-threonine aldolase internally. A mixture of threonine isomers containing L-threonine is supplied to a reactor which is connected in series with a containing reactor and a separator that continuously removes acetaldehyde produced by the reaction, and the acetaldehyde produced is removed by the separator. If isomers other than L-threonine are decomposed while keeping the concentration of acetaldehyde contained in the reaction solution low by immediately removing it, various problems caused by the acetaldehyde produced can be resolved and only L-threonine can be purified with extremely high efficiency. The present inventors have discovered that it can be obtained in a suitable form, and have completed the present invention.

To carry out the method of the present invention, for example, a reactor containing D-threonine aldolase alone or D-threonine aldolase and L-allothreonine aldolase (hereinafter abbreviated as enzyme) and an enzymatic reaction are used. A mixture of threonine isomers containing L-threonine (hereinafter abbreviated as raw material) is prepared either all at once or continuously using an enzyme reaction device in which separators are connected in series to continuously remove generated acetaldehyde from the reaction solution. At the same time, the reaction liquid inside the reactor is continuously fed to an acetaldehyde separator to continuously remove the generated acetaldehyde from the reaction liquid. The reaction solution is continuously circulated back to the original reactor. By repeating the above-described circulation operation, all isomers other than L-threonine can be finally decomposed.
Further, by carrying out the above operation by withdrawing a part of the reaction liquid while continuously supplying the raw material to the reactor, L-threonine can be continuously obtained from a mixture of isomers containing L-threonine. Furthermore, when multiple reaction devices are connected to perform a continuous reaction, the enzyme reaction devices are connected in series, the raw material is continuously supplied to the first reactor, and the L from the final stage reactor is
- Continuously discharge the reaction solution in which all isomers other than threonine have been decomposed. The reaction liquid supplied to each reactor is supplied from the separator in the previous stage, and at the same time isomers other than L-threonine are decomposed, the reaction liquid inside the reactor is continuously supplied to the separator to continuously generate acetaldehyde. Either the entire amount of the reaction liquid is continuously supplied to the next reactor, or a part of it is continuously supplied to the next reactor, and the remaining reaction liquid is recycled to the original reactor. circulate. By the above reaction operation, L-threonine can be continuously obtained from a mixture of isomers containing L-threonine.

When the raw material used in the present invention contains L-allothreonine, both D-threonine aldolase and L-allothreonine aldolase are used. Further, when L-allothreonine is not contained, D-threonine aldolase alone may be used. Here, D-threonine aldolase is D-
It is an enzyme that acts on threonine and D-allothreonine to decompose it into glycine and acetaldehyde, and L-allothreonine aldolase is an enzyme that decomposes threonine and D-allothreonine into glycine and acetaldehyde.
It is an enzyme that acts on allothreonine and breaks it down into glycine and acetaldehyde. In addition, these enzymes may be in any form that can exhibit enzymatic activity, such as isolated purified products, semi-purified products, crude extracts, cultured products,
Live microbial cells, freeze-dried microbial cells, acetone-dried microbial cells, heat-treated microbial cells, or these microbial cells may be used as they are after being immobilized by known means. The immobilization method is carrier binding method,
Known methods such as a crosslinking method, an entrapping method, and an encapsulation method can be widely used.

Examples of microorganisms containing D-threonine aldolase include Alcaligenes Faecalis IFO-12669 and Pseudomonas DK-2.
6200 and Arthrobacter
Examples include DK-19 Microtechnical Research Institute No. 6201.
In addition, examples of microorganisms containing L-allothreonine aldolase include Bacillus DK-315, FEK No. 6202, and Pseudomonas DK-2, FEK No. 6202.
No. 6200, Arylobacter DK-19 Microtechnical Research Institute
No. 6201, Alcaligenes faecalis IFO for the genus Alcaligenes
No. 12669 can be mentioned.

The reactor in the present invention is a reactor containing D-threonine aldolase alone or D-threonine aldolase and L-allothreonine aldolase, and when D-threonine aldolase and L-allothreonine aldolase are used, for example, D - It may be a mixture of two enzymes, such as an immobilized enzyme column reactor in which threonine aldolase and L-allothreonine aldolase are immobilized at the same time, or an immobilized D-threonine aldolase column reactor and immobilization. Chemical L-
Allothreonine aldolase column reactors may be separated, such as reactors connected in series or in parallel.

The structure of the reactor is to house the enzyme inside,
It has an outlet that allows the reaction solution to flow out of the reactor without losing the enzyme to the outside of the reactor, and an inlet that allows raw materials to flow into the reactor,
Examples of methods for retaining the enzyme inside the reactor include continuous ultrafiltration, continuous centrifugation, and immobilization of enzymes or bacterial cells. The type of apparatus can be appropriately selected from tank type, packed type, fluidized bed type, moving bed type, etc.

The removal of acetaldehyde according to the present invention is performed when the concentration of acetaldehyde in the reaction solution in the reactor is 50×10 ^-3
It is preferable to remove acetaldehyde from the reaction solution so that the amount is less than mol/mol, preferably less than 20×10 ^-3 mol/mol. When the concentration of acetaldehyde is high, the recovery rate of glycine and acetaldehyde decreases due to self-condensation of acetaldehyde and condensation reaction of acetaldehyde and glycine, and the coloring of the reaction solution is also significant. Furthermore, a reverse reaction by enzymes occurs, and acetaldehyde and glycine recombine, resulting in L
- Isomers other than threonine are produced, and the enzyme is also significantly inactivated by acetaldehyde.

The acetaldehyde separation device in the present invention may be any device that continuously separates and removes acetaldehyde from the reaction solution, such as a distillation device, a solvent extraction device, a separation device using a separation membrane, an adsorption device using an adsorbent, and a chemical conversion device.・Generally known methods such as a removal device and an enzymatic conversion removal device can be mentioned.

A distillation device is a device for removing acetaldehyde from a reaction solution by distillation, and can be widely used, such as an equilibrium flash distillation device, a bubble column distillation device, a packed column distillation device, and a wet wall column distillation device. As an effective use of distillation energy, it is advantageous in terms of energy economy to use distillation methods such as a vapor recovery method, a vapor recompression method, a back pressure utilization method, a multiple effect method, and a combination method of the above-mentioned methods.

The pressure of the distillation operation can be appropriately selected depending on the purpose, but if the distillation is carried out under reduced pressure, side reactions such as self-condensation of acetaldehyde and condensation of acetaldehyde and glycine can be prevented from occurring during the distillation. Distillation is generally carried out at a pressure of 10 to 150 mmHg and a temperature of 10 to 60°C.

The extraction device of the acetaldehyde remover is a device that removes acetaldehyde from a reaction solution by an extraction operation, and can be widely used, such as a cocurrent extraction device, a countercurrent extraction device, and a mixed flow extraction device. The extractant to be used may be one that is poorly soluble in water, does not dissolve threonine or glycine, and dissolves only acetaldehyde, such as alcohols, ethers, etc.
Examples include ketones, amines, cyclic alkanes, benzene and benzene derivatives, phenol derivatives and bicyclic compounds. These extractants can be used alone or in combination of two or more. The ratio of reaction solution and extractant is acetaldehyde reaction solution,
It can be appropriately selected depending on the partition coefficient of the extractant into both phases.
Further, the extraction phase can be reused as an extractant by removing acetaldehyde by a method such as distillation.

A separation device using a separation membrane is a device that removes acetaldehyde from a reaction solution using a separation membrane, such as a separation device using a reverse osmosis membrane, a separation device using a liquid membrane, a separation device using an ion exchange membrane, and a pervaporation device. ) method, etc.

An adsorption device using an adsorbent is a device that removes acetaldehyde from a reaction solution through an adsorption operation. For example, an adsorption device that uses an inorganic adsorbent such as activated carbon, or an adsorption device that uses an inorganic adsorbent such as activated carbon, or an adsorption device that uses an inorganic adsorbent such as an ion exchange resin or an adsorption resin that has an amino group. Examples include adsorption devices that use adsorbents.

A chemical conversion/removal device is a device that converts acetaldehyde in a reaction solution into other substances using organic synthesis chemistry, such as a catalytic reduction device, a reaction device that adds sodium bisulfite to acetaldehyde and converts it into sulfite chloride. Examples include equipment.

An enzymatic conversion/removal device is an enzymatic reaction device that converts acetaldehyde into other substances through an enzymatic reaction. Examples of enzymatic reactions include the reaction of oxidizing acetaldehyde to acetic acid using acetaldehyde dehydrogenase or aldehyde oxidase, and the reaction of converting acetaldehyde into acetic acid with alcohol dehydrogenase. Examples include reactions that reduce acetaldehyde and convert it into alcohol.

The acetaldehyde removal method and device described above may be used alone or in combination of two or more, and can also be used in combination with the threonine and glycine separation method and device.

The mixture of threonine isomers containing L-threonine used in the method of the present invention may be obtained by any known method, but in the method of the present invention, isomers other than L-threonine are decomposed and L-threonine is -Threonine is obtained, and the byproducts are glycine and acetaldehyde. Therefore, a threonine synthesis method using glycine or glycine and acetaldehyde as starting materials is suitable because these byproducts can be reused as raw materials for synthesis.

In order to cause D-threonine aldolase alone or D-threonine aldolase and L-allothreonine aldolase to act on a mixture of threonine isomers, the mixture of threonine isomers and the enzyme may be brought into contact in an aqueous medium. The concentration of the threonine isomer may be at a level that does not significantly inhibit the enzyme activity, but is preferably about 0.1 to 2 mol/mole.
The solvent for the reaction is, in principle, water, but an organic solvent may be included to the extent that it does not inhibit the enzymatic reaction. The pH during the reaction varies depending on the enzyme used, but in the case of the enzyme produced by the microorganisms shown, it is 6.
A pH of around 10 is preferable.

The reaction temperature of the enzymatic reaction depends on the enzyme used, but in the case of enzymes produced by microorganisms, it is 10 to 55.
A range of 0.degree. C. is preferred. Further, the operating pressure for the enzyme reaction can be appropriately selected from reduced pressure to increased pressure.

In the case of the microbial enzymes shown, as coenzymes
The coexistence of about 10 ^-3 to 10 ^-6 M of pyridoxal 5'-phosphorus enzyme can promote the enzyme reaction and also stabilize the enzyme activity. Furthermore, when mercaptoethanol, sulfite ions, dithiothreotol, Mn ²⁺ , CO ²⁺ , Fe ²⁺ , Mg ^{2+ ,} etc. coexist, enzyme activity can be activated and stabilized.

After the reaction is complete, the reaction solution is treated with an ion exchange resin,
L-threonine crystals can be obtained in a pure form by crystallization, purification and decolorization using activated carbon, etc., and concentrating the purification and decolorization liquid. Glycine can be easily separated and recovered from the reaction solution by, for example, chromatography using an ion exchange resin or fractional crystallization using metal complexation.

Next, the present invention will be explained with reference to examples, but the present invention is not limited thereto.

In the examples, threonine, allothreonine, and glycine were prepared in a ratio of tertiary-butyl alcohol: methyl ethyl ketone: water: concentrated ammonia: 4:3:
A method of performing paper chromatography using a 2:1 mixture as a developing solvent, developing color with ninhydrin, cutting out spots of glycine, threonine, and allothreonine, and extracting with methanol containing 0.005% copper nitrate for colorimetric determination and amino acids. L-threonine was quantified using an analysis method using an automatic analyzer (JEOL: JLC-6AH).
The microbial assay was performed using ATCC8043 (A. faecalis) as the quantitative bacterial strain.

In addition, acetaldehyde was quantified by gas chromatography using a PEG6000 column (6m).

For enzyme activity, add 10 mg of bacterial cells or 0.1 g of immobilized enzyme preparation to 50 mM of threonine isomer corresponding to each enzyme activity.
Pyridoxal 5'-phosphoric acid was dispersed in 5 ml of phosphate buffer of pH 8.5 containing 0.1 mM, and after reacting at 35°C with shaking for 60 minutes, the amount of glycine produced in the reaction solution was determined by quantitative determination. . The enzyme activity that produced 1 μmol of glycine per minute was defined as 1 U (unit).

Furthermore, the residual activity rate refers to the ratio of enzyme activity remaining after the reaction to the enzyme activity used in the reaction.

Example 1 (Obtaining L-threonine from D,L-threonine) (Preparation of enzyme) Polypeptone 0.5%, yeast extract 0.2%,
_{KH2PO4 0.1} %, _MgSO4 0.05%, glutamic acid 0.1
% and 0.2% D-threonine was prepared, and the medium was put into a 5-volume culture tank and sterilized by heating at 120° C. for 10 minutes. D to this medium
- Arilobacter DK-19 FIKEN Bacteria No. 6201 was inoculated as a microorganism containing threonine aldolase, and cultured at 30° C. for 20 hours with aeration and stirring while maintaining the pH at 8.

Next, the above medium 20 was put into a 50 volume culture tank and heat sterilized at 120°C for 10 minutes. Inoculate the above culture solution 2 into this medium and hold at 30°C while keeping the pH 8.
The cells were cultured for 20 hours under aeration and stirring.

After culturing, centrifuge the bacterial cells from the culture solution,
After washing with 0.9% saline, 45 g of wet bacterial cells were obtained.

Next, 40 g of this wet bacterial body was suspended in 0.5 M acetate buffer 4 of pH 5.0 containing 40 × 10 ^-3 mol of cobalt chloride.
After heating at 55℃ for 3 hours, centrifugation and 0.01×
Contains 10 ^-3 moles/pyridoxal 5'-phosphate
The L-threonine deaminase was completely inactivated by washing with 0.01M phosphate buffer at pH 6.0.

Next, the wet bacterial cells containing 0.02×10 ^-3 mol of pyridoxal 5'-phosphate and 6 g of etugalbumin were added.
Prepared by suspending in 2 0.01M phosphate buffer solution with pH 6.0, dispersing 6 g of chitosan in 3 parts of water and stirring at room temperature for 20 minutes, then adding 3 ml of acetic acid, stirring, and dissolving. After adding the chitosan aqueous solution and stirring at 10℃ for 20 minutes, 0.1N sodium hydroxide was gradually added to adjust the pH of the solution to 9.0, gelling the chitosan, and forming the bacterial cell/chitosan complex. body was prepared.

This complex is then recovered from the solution by centrifugation and the pH
After washing with 7.0 1M phosphate buffer and dehydration,
It was extruded through a hole with a diameter of 0.8 mm and sized into a spherical shape.

Yield: 44.6g This molded product was immersed in 500ml of a 0.5M phosphate buffer solution with a pH of 7.0 containing 0.1 mol/mol glutaraldehyde and 0.01×10 ^-3 mol/pyridoxal 5'-phosphate at 5°C for 30 minutes. , a curing reaction is carried out with shaking, and after the reaction is completed, it is washed with water, and a pH7.0 solution containing 0.01×10 ^-3 mol/pyridoxal 5'-phosphoric acid is added.
43.5 g of immobilized D-threonine aldolase agent (wet weight, specific activity)
0.85U/g) was obtained.

(Reaction and Removal of Acetaldehyde by Distillation) The reaction was carried out based on the flow sheet shown in FIG. Reactor 1 has a diameter of 1.9 cm with outer shell;
A 45 cm long column was packed with 40 g of the immobilized D-threonine aldolase agent prepared as described above (wet weight total activity 34 U).

Separately, dissolve 4.76 g of DL-threonine crystals in 100 ml of water containing 0.1 x 10 ^-4 mol of pyridoxal 5'-phosphoric acid, adjust the pH to 8.5 with 1N sodium hydroxide, and add the substrate solution to 100 ml vacuum distillation flask 2. I put it in.

The distillation flask was equipped with a liquid level regulator, and the amount of liquid in the flask was maintained at 100 ml by externally supplying water that was reduced by distillation.

The substrate solution in the distillation flask was passed through the upper part of the reaction column at a flow rate of 1 hour per hour using the pump 5, and the reaction liquid flowing out from the lower part of the reaction column was supplied to the reboiler 3.

Next, the reaction liquid was heated and evaporated in the reboiler 3, and the reaction liquid and vapor were supplied to the fractionation column 4.

After separating the vapor and the reaction liquid in a fractionation column, the water vapor containing acetaldehyde is condensed in a condenser 8,
The reaction solution from which acetaldehyde had been removed was trapped in receiver 9, and was allowed to flow down into flask 2, where it was again supplied to the reaction column. The distillation pressure is controlled by the vacuum regulator 10.
While maintaining the acetaldehyde concentration in the reaction solution at the outlet of the reaction column at 60 mgHg or less at 10 mM or less, hot water at 35°C was flowed through the outer shell of the reaction column, and the above circulation reaction was carried out for 48 hours. After the reaction was completed, the reaction liquid was extracted from the reaction apparatus, and the reaction apparatus was further washed with 0.5 ml of water, and the reaction liquid and washing liquid were combined to obtain a reaction-completed liquid.

(Obtaining L-threonine) Next, this reaction solution was neutralized with diluted hydrochloric acid, and then passed through a column filled with 100 ml of activated carbon and washed with water.

This decolorized reaction solution was converted into H ⁺ type Dowe×50W×
8 (The Dow Chemical Company registered trademark)
Pass the liquid through a column filled with 100ml, and after washing with water,
The adsorbed threonine-containing fraction was eluted with 0.1N ammonia water, and the eluate was dried under reduced pressure.
Add 5 ml of water to this dry substance to dissolve it, and then dissolve it in ethanol.
20 ml was gradually added to separate the precipitated crystals.

The dry weight of the obtained crystals was 2.2 g, and it was confirmed by paper chromatography that they were pure L-threonine. The specific optical rotation was also [α] ²⁷ _D = -28.7 (H ₂ O), which was consistent with that of pure L-threonine.

(Obtaining glycine) 0.1N ammonia was further passed through the above column,
The glycine-containing fraction was eluted, and the eluate was dried under reduced pressure. 1 ml of water was added to the dried product to dissolve it, and 10 ml of methanol was gradually added to separate the precipitated crystals.

The dry weight of the obtained crystals was 1.3 g, and analysis by paper chromatography revealed that they contained only glycine.

On the other hand, 0.6 g of acetaldehyde was recovered from the reaction solution by vacuum distillation.

(Comparative example) The reaction and isolation procedure was carried out in the same manner as above except that acetaldehyde was not removed from the reaction solution, and L-threonine (containing 5% allothreonine and 5% D-threonine) was obtained. crystal of
1.4 g and 0.6 g of glycine crystals were obtained. On the other hand, after the reaction, the reaction solution was distilled under reduced pressure, but acetaldehyde could not be recovered.

Example 2 In the same manner as in Example 1, the immobilized D-threonine aldolase agent was used repeatedly and the reaction was carried out 20 times to measure the total activity of the immobilized D-threonine aldolase agent. Total activity is 29U, residual activity rate 85.3%
It was hot.

As a comparative example, the reaction was repeated in the same manner as above except that acetaldehyde was not removed from the reaction solution, and the total activity of D-threonine aldolase after repeated use 20 times was 1.7 U, with a residual activity rate of 5%. It was hot.

Example 3 (Obtaining L-threonine from D,L-threonine) [Example of solvent extraction and removal of acetaldehyde (Figure 2)] An immobilized D-threonine aldolase agent was prepared in the same manner as in Example 1, and 40 g of this (wet weight, specific activity 0.85U/g) in a reactor with outer shell (1.9cm diameter)
column) 1.

Separately, add 4.76 g of DL-threonine crystals to 0.1×10 ^-4
200 ml of water containing moles of pyridoxal 5'-phosphate
After dissolving in the solution, adjust the pH to 8.5 with 1N sodium hydroxide.
The prepared substrate solution was placed in a 200 ml jacketed flask 2.

The substrate solution in flask 2 is passed through the top of the reactor at a flow rate of 100 ml per hour using pump 5, and the effluent is poured into a container with a diameter of 1 cm and height filled with a 2 mm metal ring.
Supplied to the upper part of a 100 cm packed column countercurrent extractor 12,
On the other hand, 1-pentanol was supplied from the bottom at a flow rate of 100 ml/hour to extract acetaldehyde in countercurrent, and the reaction liquid exiting the extractor was circulated to flask 2.

35 on the outside of the reaction column and on the outside of flask 2
The above circulation reaction was carried out for 48 hours by flowing warm water at .

After the reaction was completed, the reaction liquid was extracted from the reaction apparatus, and the reaction apparatus was further washed with 1 part of water, and the reaction liquid and washing liquid were combined to form a reaction-completed liquid.

Next, this reaction-completed liquid was separated in the same manner as in Example 1, and crystals of L-threonine were obtained.
2.1 g of glycine crystals, and 0.6 g of acetaldehyde were obtained from the extract.

Also, the specific rotation of threonine crystal is [α] ²⁷ _D = -
The value was 28.7 (H ₂ O), which was consistent with pure L-threonine.

Example 4 (Obtaining L-threonine from D,L-threonine and D,L-allothreonine) As a microorganism containing L-allothreonine aldolase, Bacillus DK-315 and Bacillus microorganisms 6202
No. 1, cultured in the same medium as in Example 1,
42 g of wet bacterial cells were obtained.

Next, 40 g of this wet bacterial body was heat-treated at PH8, 35°C for 6 hours, and then fixed in the same manner as in Example 1 to immobilize L.
- 42.7 g of allothreonine aldolase agent (wet weight, specific activity 0.71 U/g) was obtained. 40 g of this was packed into reaction column A similar to Example 1.

In addition, an immobilized D-threonine aldolase agent was prepared in the same manner as in Example 1, and 40 g (wet weight, specific activity: 0.85 U/g) of this agent was filled into reaction column B, which was the same as column A above, and column A. connected in series.

Separately, 4.76 g of DL-threonine crystals and 2.38 g of DL-allothreonine were dissolved in 100 ml of water containing 0.1×10 ^-4 mol of pyridoxal 5'-phosphate, and the substrate solution was adjusted to pH 8.5 with 1N sodium hydroxide. When reaction and isolation were carried out in the same manner as in Example 1, 2.1 g of L-threonine crystals, 3.9 g of glycine crystals, and 0.18 g of acetaldehyde were obtained.

Also, the specific optical rotation of threonine crystal is [α] ²⁷ _D = -
28.7 (H ₂ O), consistent with pure L-threonine. As a comparative example, the reaction and isolation procedure was carried out in the same manner as described above except that acetaldehyde was not removed from the reaction solution. As a result, crystals of L-threonine containing 9% allothreonine and 5% D-threonine were obtained. 1.8 g and 1.7 g of glycine crystals were obtained. On the other hand, after the reaction, the reaction solution was distilled under reduced pressure, but acetaldehyde could not be recovered.

Example 5 In the same manner as in Example 4, the immobilized D-threonine aldolase agent and the immobilized L-allothreonine aldolase agent were repeatedly used to conduct the reaction 20 times to measure the total activity of the immobilized enzyme agent. Immobilization D-
The total activity of the threonine aldolase agent was 29 U, with a residual activity rate of 85.3%, and the total activity of the immobilized L-allothreonine aldolase agent was 24 U, with a residual activity rate of 84.5%.

As a comparative example, the reaction was repeated in the same manner as above except that acetaldehyde was not removed from the reaction solution, and the total activity of D-threonine aldolase after repeated use 20 times was 1.7 U, residual activity rate was 5%, and The total activity of L-allothreonine aldolase was 1.1 U, and the residual activity rate was 3.9%.

[Brief explanation of the drawing]

1 and 2 are schematic explanatory diagrams showing simplified representative embodiments of the present invention. 1... Reactor, 2... Reaction liquid flask, 3...
Reboiler, 4... Fractionation column, 5... Reaction liquid pump, 6... Liquid level regulator, 7... Feed water reservoir, 8... Condenser, 9... Distillate receiver, 1
0...Automatic vacuum regulator, 11...Vacuum pump,
12... Packed column countercurrent extractor, 13... Extractant supply pump, 14... Extractant reservoir, 15... Extractant receiver after extraction.

Claims (1)

[Claims] 1 Course leonine aldolase or this and L-
A threonine isomer mixture containing L-threonine is supplied to an enzyme reaction device that is connected to a reactor containing allothreonine aldolase and a separator for the acetaldehyde produced, and while continuously removing the acetaldehyde produced, A method for obtaining L-threonine, which comprises decomposing the isomer.