JP2000253893A - Production of d-serine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain D-serine useful as an intermediate for medicaments or the like at a high optical purity and in a high yield by reacting microorganism cells (or their treated product) having tryptophan synthase activity with a reactant liquid containing DL-serine or the like and by treating a reaction product under a specific condition. SOLUTION: D-serine is obtained by reacting microorganism cells such as Saccharomyces cerevisiae ones (or their treated product) having tryptophan synthase activity with a reactant liquid containing (preferably 1-15 wt.%) DL- serine and (preferably <=10 wt.% and >=1.1 mol times the quantity of L-serine in the DL-serine) thiophenol at a temp. of 20-60 deg.C to synthesize s-phenyl-L- cysteine from L-serine and by collecting D-serine remaining in the solution of a reaction product. Reaction is preferably carried out in an aqueous solution (pH 9.0-10.5).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an enzymatic DL-
The present invention relates to a method for producing D-serine from serine.
According to the present invention, D-serine can be produced with high efficiency, and separation from S-phenyl-L-cysteine, which is a reaction by-product, is easy, so that high yield and high optical purity can be obtained. It is possible to obtain D-serine.

[0002] D-serine is an amino acid that is expected as a highly useful compound in industry as an intermediate material for pharmaceuticals, foods, agricultural chemicals and the like. On the other hand, S-phenyl-L-cysteine obtained as a by-product is also one of important amino acids as a raw material of a pharmaceutical intermediate and is an amino acid of high industrial value.

[0003]

2. Description of the Related Art As a method for producing D-serine, a method for obtaining D-serine from DL-serine by an optical resolution method is known, but there is a problem with the yield of optical resolution and the like. However, it is difficult to say that this method is industrially advantageous. Also, regarding the D-serine production method by the enzymatic method,
Production method using microbial cells having tyrosinase activity (JP-A-5-91895) and method for fractionating D-serine from DL-serine in the presence of tryptophan synthase (JP-A-62-19098) It has been known.

However, in the case of Japanese Patent Application Laid-Open No. Hei 5-91895, in order to obtain D-serine in high yield, it is necessary to add phenol as a raw material to L-serine in DL-serine in excess. And production efficiency is poor. In the case of JP-A-62-19098, after the reaction is completed, the produced L-cysteine is oxidized and converted to L-cystine to separate it from D-serine, or L-cysteine is added by adding a carbonyl compound such as acetone. And a method of forming a complex with cysteine and separating the complex from D-serine. In this method, the step of separating D-serine from by-products is complicated, and there is a problem as an industrial production method.

The present invention relates to a method for producing D-serine, which is important as an intermediate material for pharmaceuticals, foods, agricultural chemicals and the like, from DL-serine by an enzymatic method, and to a method for efficiently producing high yield and high optical purity. It is intended to produce D-serine.

[0006]

The inventors of the present invention have conducted various studies to solve the above-mentioned problems. As a result, the present inventors have found that a microbial cell having tryptophan synthase activity or a treated product thereof can be obtained from a reaction solution containing DL-serine and thiophenol. And L-serine is converted to S-phenyl-L-
After synthesizing cysteine, D-serine remaining in the reaction solution and S-phenyl-L-cysteine produced were separated by filtration, and it was found that D-serine with high yield and high optical purity could be obtained. Was completed.

That is, the present invention is as follows. 1) Microbial cells having tryptophan synthase activity or a processed product thereof are allowed to act on a reaction solution containing DL-serine and thiophenol to synthesize S-phenyl-L-cysteine from L-serine and remain in the reaction solution. A method for producing D-serine, which comprises obtaining D-serine. 2) The method according to 1), wherein the reaction is carried out in an aqueous solution having a pH of 9.0 to 10.5. 3) The method according to 1) or 2), wherein the reaction is carried out at 20 to 60 ° C. 4) The concentration of thiophenol is set to 10% by weight or less, and DL
-The production method according to 1), wherein it is added in an amount of 1.1 times or more the amount of L-serine in serine. 5) The method according to 1), wherein the reaction is carried out at a DL-serine concentration of 1 to 15% by weight.

In the present invention, by utilizing the difference between the solubility of S-phenyl-L-cysteine and the solubility of D-serine, D-serine can be easily and efficiently separated by filtration.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The tryptophan synthase producing bacteria used in the present invention include microorganisms of prokaryotic cells such as Escherichia coli or D having enhanced productivity of the enzyme.
It may be a transformed microorganism created by applying the NA recombination technique. Alternatively, the tryptophan synthase may be obtained from eukaryotic microorganisms such as Saccharomyces cerevisiae or mold, and is created by applying DNA recombination technology to enhance the productivity of the eukaryotic microorganisms. Or a transformed microorganism. Preferably, Escherichia coli MT-
10242 (FERMBP-20), Neurospora crassa ATCC 14692, and the like. More preferably, it is a microorganism having a tryptophan synthase activity capable of synthesizing 1 g or more of tryptophan per 1 g of dried microorganism cells per hour.

As a medium for culturing a tryptophan synthase-producing bacterium, any of a synthetic medium and a natural medium can be used as long as it contains a carbon source, a nitrogen source, an inorganic substance and, if necessary, a small amount of a micronutrient. is there. However, it is generally preferred to add trace amounts of tryptophan, anthranilic acid or indole to the medium.

[0011] As the carbon source and the nitrogen source used in the medium, any type can be used as long as the bacteria used can be used.

As the carbon source, various carbohydrate compounds such as glucose, fructose, glycerol, sucrose, maltose, mannose, starch, starch hydrolyzate and molasses can be used.

Examples of the nitrogen source include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate, and ammonium acetate, or meat extract, yeast extract, corn steep liquor, casein hydrolyzate, fish meal. Alternatively, natural organic nitrogen sources such as digests thereof, defatted soybean meal or digests thereof can be used. In many cases, natural organic nitrogen sources can be both nitrogen and carbon sources.

The inorganic substances include potassium monohydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, magnesium sulfate,
Sodium chloride, ferrous sulfate and the like can be used as needed.

The cultivation is performed under aerobic conditions such as shaking culturing or aeration and stirring deep culturing. The culturing temperature is preferably in the range of 20 to 50C, particularly preferably 30 to 37C. It is desirable to maintain the pH of the medium during culturing near neutrality. The culture time is usually 1 to 3 days.

The method for extracting tryptophan synthase from cultured cells of Escherichia coli is described in The Journal of Biological Chemistry (The Jou).
rnal of Biological Chem
Istry) (Vol. 252, No. 19, p. 65)
94-6599 (1977)), and a method for extracting Neurospora crassa from cultured cells is described in Vol. 250, N
o. 8, 2941-2946 (1975), the tryptophan synthase used in the present invention need not necessarily be pure.

That is, a culture of a tryptophan synthase-producing bacterium, a viable cell collected from the culture by centrifugation or the like, a dried cell thereof, or a cell obtained by crushing, autolyzing, sonicating, or the like. Processed cells,
Furthermore, extracts from these cells and crudely purified enzymes obtained from the extracts can also be used.
Further, these immobilized enzymes or immobilized cells may be used.

The concentration of DL-serine in the reaction solution is not particularly limited, but is preferably 1 to 15% by weight. The concentration of thiophenol in the reaction solution must be within a range that does not inhibit the enzymatic reaction, and is preferably used at a concentration of 10% by weight or less.

Further, in order to obtain D-serine having high optical purity, it is preferable that the added thiophenol is 1.1 times or more mol of L-serine in DL-serine. Note that thiophenol may be added sequentially during the reaction. At the time of the reaction, it is desirable to add pyridoxal phosphate, which is a coenzyme, in addition to the substrate in a range of 0.1 to 100 ppm.

The amount of tryptophan synthase to be added to the reaction solution varies depending on the method for separating, purifying or treating the enzyme as described above, but is not particularly limited.
It can be changed as appropriate depending on the enzyme activity, reaction time, and other conditions.

The D-serine synthase reaction of the present invention is carried out at pH
The reaction is performed under an alkaline condition of 9.0 to 10.5. The pH is preferably in the range of 9.0 to 10.0. As the alkali to be used, sodium hydroxide, potassium hydroxide, aqueous ammonia and the like can be used. If the pH is below 9.0,
Since the reaction rate of the enzyme for synthesizing S-phenyl-L-cysteine is extremely low, unreacted L-serine remains in the reaction solution and D-serine with high optical purity cannot be obtained. When the pH exceeds 10.5, S-phenyl-L
-Since the reaction rate of the enzyme for synthesizing cysteine decreases again, unreacted L-serine remains, and D-
-Serine cannot be obtained.

[0022] The temperature of the enzymatic reaction is preferably 20 to 60 ° C.
The reaction is particularly preferably performed at 30 to 40 ° C. in view of enzyme stability and reaction rate. Note that the reaction is preferably performed in a nitrogen atmosphere to prevent oxidation of thiophenol.

The reaction is carried out by standing or stirring by a batch method or a continuous method using an immobilized enzyme. When reacting at a thiophenol concentration of 5% or more,
It is better to stir gently. When the reaction is carried out at a thiophenol concentration of 5% by weight to 10% by weight, it is desirable to perform the stirring at a power of 20 KW / m ³ or less. The reaction time is usually 5 to 40 hours.

S-phenyl-L-cysteine has low solubility in water, and usually, as the enzymatic reaction proceeds,
-Phenyl-L-cysteine crystallizes and precipitates in the reaction solution. The pH of the reaction-terminated liquid is adjusted to be acidic with a suitable acid such as hydrochloric acid or sulfuric acid, and after the precipitated crystals are dissolved, thiophenol unreacted with microbial cells or enzymes is removed by activated carbon adsorption treatment or the like. Then, if neutralized with an appropriate alkali, hardly soluble S-phenyl-L-cysteine crystals precipitate, so that D-serine and by-product S-phenyl-L-cysteine can be easily separated by filtration. can do.

When dissolving the S-phenyl-L-cysteine crystals in an acid, the pH is preferably adjusted to 1.5 or less. This is because S-phenyl-L-cysteine cannot be dissolved at a concentration of 1% by weight or more unless the pH of the aqueous solution is 1.5 or less, resulting in poor volumetric efficiency.

In the activated carbon adsorption treatment, in order to adsorb and remove microbial cells or enzymes or components derived from microbial cells, and to sufficiently remove some unreacted thiophenol not adsorbed by activated carbon, air or oxygen is simultaneously added to the solution. Is preferably ventilated. This is because the thiophenol is oxidized to an insoluble solid by aeration, and can be removed by filtration. The ventilation rate is preferably 0.01 to 6.0 times the volume ratio / Hr. The activated carbon adsorption treatment temperature is 40 to 90 ° C, particularly preferably 40 to 60 ° C.
Perform at ° C.

The activated carbon used is not particularly limited, and may be PM-SX, PM-PA, PM-KI, PM-K
S, PM-AA (Mitsui Pharmaceutical) WPH, PCB-
G, ADP (Toyo Calgon), Egret A, Egret M, Egret C, Carboraffin (Takeda Pharmaceutical), Taiko S
Type, Taiko K type (above, Nimura Chemical) and the like can be used. The amount of activated carbon to be added is usually 0.5 to 6% based on the weight of the solution, but depends on the amount of the enzyme or microbial cells used as the source of tryptophan synthase in the enzyme reaction and the amount of unreacted thiophenol. The amount of the activated carbon can be changed. Further, there is no particular limitation on the use of radiolite or the like as a filter aid during the activated carbon adsorption treatment.

[0028]

The method of the present invention will be described below with reference to examples.

Experimental Example 1 Tryptophan synthase-producing Escherichia
Coli MT-10242 (FERM BP-20)
With 50 ml of medium having the composition shown in Table 1.
A 0 ml Sakaguchi flask was inoculated and cultured with shaking at 30 ° C. for 24 hours. 600 ml of this culture solution (for four flasks) was charged with 20 L of 10 L of medium having the composition shown in Table 2.
Jar fermenter at 30 ° C, pH 6.8
(Control with concentrated aqueous ammonia), and aeration culture was performed for 40 hours while glucose was sequentially added. After completion of the culture, the cells were collected by centrifugation, and the obtained wet cells were used as a tryptophan synthase source.

[0030]

[Table 1] Adjusted to pH 7.0

[0031]

[Table 2]

Example 1 Escherichia coli MT-10 was prepared in the same manner as in Experimental Example 1.
242 (FERM BP-20) was obtained. DL
385 g of an aqueous solution containing 54.4 g of serine and 8 mg of pyridoxal phosphoric acid was heated to 35 ° C, and adjusted to pH 9.5 with 45% NaOH. To the aqueous solution, the above centrifuged cells were added to a concentration of 0.96% in terms of dry cells, and distilled water was added to a total weight of 468.7 g.
The reaction was carried out by stirring under a nitrogen atmosphere for 27 hours. At the time of preparation, 15.7 g (about 3.2% concentration of the reaction solution) of thiophenol was added, and after 4 hours and 7 hours, 7.8 g (about 1.5% concentration of the reaction solution) was added.
The reaction scale was 500 g. After completion of the reaction, concentrated hydrochloric acid was added to the reaction solution to adjust the pH to 0.5, and then distilled water 52
7 g was added to completely dissolve the precipitated crystals of S-phenyl-L-cysteine. Temperature was raised to 50 ° C and activated carbon was 27.4
g and Radiolight 22.4 g were added, and the mixture was stirred at 50 ° C. for 3 hours while passing air through the liquid. Thereafter, the mixture was filtered, and the filtrate was adjusted to pH 3.0 with 45% NaOH, and the temperature was adjusted to 30 ° C.
For 1 hour. The crystallization liquid was filtered to separate S-phenyl-L-cysteine crystals and D-serine. The amount of D-serine in the filtrate was 22.0 mg / g and the optical purity was 9
It was 8.7% ee and the yield was 95%. D-
The optical purity analysis of serine was performed by liquid chromatography, and the analysis conditions were as shown below.

Optical purity analysis conditions for D-serine Column; Daicel CROWNPAK ^™ CR (+) Mobile phase; Perchloric acid aqueous solution (pH 1.3) Flow rate; 0.4 ml / min Temperature; 0 ° C. Detection reagent; OPA reagent Ex = 365 nm Em = 4
55 nm

[0034]

According to the present invention, L-serine in DL-serine is efficiently converted to S-phenyl-L-cysteine, and unreacted D-serine is easily converted to S-phenyl-L-cysteine by filtration. By separating it from cysteine, D-serine with high optical purity can be obtained in high yield. The establishment of a method for producing D-serine on an industrial scale according to the present invention has revealed a method for obtaining the compound which is expected as various intermediates, and the present invention is significant.

Claims (5)

[Claims] A microbial cell having tryptophan synthase activity or a processed product thereof is allowed to act on a reaction solution containing DL-serine and thiophenol to synthesize S-phenyl-L-cysteine from L-serine, and the reaction solution A method for producing D-serine, which comprises obtaining D-serine remaining therein. 2. The process according to claim 1, wherein the reaction is carried out in an aqueous solution having a pH of 9.0 to 10.5. 3. The reaction according to claim 1, wherein the reaction is carried out at 20 to 60 ° C.
Production method as described. 4. The process according to claim 1, wherein the concentration of the thiophenol is 10% by weight or less, and the thiophenol is added in an amount of 1.1 times or more of L-serine in DL-serine. 5. The method according to claim 1, wherein the reaction is carried out at a DL-serine concentration of 1 to 15% by weight.