JP3155448B2 - Method for producing L-amino acid or salt thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing an L-amino acid or a salt thereof.

[0002]

2. Description of the Related Art L-amino acids are compounds useful as various pharmaceutical intermediates and food additives. Among these, as an advantageous method for selectively obtaining the L-amino acid from a racemic form of a chemically synthesized amino acid, an amino group of each amino acid constituting the racemic form is once modified with a protecting group, and then the enzyme A method may be considered in which only the protective group in the L-form of the racemic amino acid in which the amino group is protected is eliminated by the reaction, and the above-mentioned target compound is collected. At this time, as the protecting group for the amino group, the following general formula (I)

[0003]

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[0004] (where R is an alkyl group or a benzyl group) may be used.

[0005] Here, as a method for obtaining an amino acid by using an amino acid whose amino group is protected by a group represented by the above general formula (I) as a substrate and hydrolyzing the protective group by enzymatic action to remove it, the amino acid is removed. Cultural and biological chemistry (Agric. Biol. Che
m. ), 49, 3643 (1985). That is, this publication includes N-benzyloxycarbonyl-D, L-amino acids or Nt-
A method of enzymatically decomposing only the L-form from butoxycarbonyl-D, L-amino acid is disclosed.

[0006]

According to this method, the reaction is carried out at a reaction temperature of 5 under acidic conditions of pH 5.8.
The reaction is performed 5 times with a reaction time of 10 minutes. However, when an amino acid whose amino group is protected by the group represented by the general formula (I) is exposed under such acidic and high temperature conditions, the protecting group is chemically hydrolyzed and eliminated. Become like Therefore, when the above-mentioned racemic substrate is subjected to a reaction under such acidic and high-temperature conditions, if the reaction is continued for a long time, the D-form is also chemically hydrolyzed.
-There was a problem that amino acids were also produced as by-products. When the D-amino acid is by-produced in this way, it is necessary to perform a complicated purification operation to isolate the L-amino acid from the reaction product.

Against this background, the present invention provides a method for producing an L-amino acid by enzymatically hydrolyzing only an L-amino acid having a protected amino group, thereby suppressing the by-product of a D-amino acid. Aim.

[0008]

Means for Solving the Problems The present inventors have intensively studied to solve such a drawback. As a result, it has been found that the above-mentioned problems can be solved by allowing a culture solution of specific microorganisms, cells or treated cells to act on the substrate mixture in a specific pH and temperature range, and completed the present invention. I came to.

That is, the following general formula (I)

[0010]

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(Wherein R is an alkyl group or a benzyl group). When the compound is acted on a mixture of a D-form and an L-form of an amino acid or a salt thereof in which the amino group is protected, the L-form A microorganism belonging to the genus Corynebacterium, the genus Microbacterium, the genus Bacillus, the genus Schizosaccharomyces having the ability to selectively hydrolyze L-amino acids or salts thereof, or a culture solution or cell treatment thereof The product is pH 6-10, temperature 10-50 ° C
Under the conditions described above, reacting a mixture of the D-form and the L-form of an amino acid or a salt thereof in which the amino group is protected with the group represented by the general formula (1), and collecting the produced L-amino acid or a salt thereof A method for producing an L-amino acid or a salt thereof, characterized in that:

In the present invention, R in the group represented by the general formula (I) is an alkyl group or a benzyl group.
Here, the alkyl group is not particularly limited, but preferably includes an alkyl group having 1 to 4 carbon atoms such as a methyl group, an ethyl group, an isopropyl group, and a t-butyl group.

In the present invention, as the amino acid protected by the protecting group represented by the general formula (I), known amino acids can be used without any limitation. To illustrate these specifically,
Glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, cystine, methionine, asparagine, glutamine, aspartic acid,
Examples include glutamic acid, lysine, arginine, phenylalanine, tyrosine, proline, hydroxyproline, tryptophan, histidine and the like. Examples of salts of these amino acids include sodium salts, potassium salts, ammonium salts and the like.

In the present invention, the D-form of an amino acid or a salt thereof whose amino group is protected by a group represented by the general formula (I)
When a mixture of the bodies (hereinafter, also simply referred to as a substrate mixture) belongs to the genus Corynebacterium, Microbacterium, Bacillus, or Schizosaccharomyces, and the L-form is allowed to act on the substrate mixture, the L-form is selectively treated. A microorganism having an ability to produce an L-amino acid or a salt thereof by hydrolysis, or a culture solution or a treated product of the microorganism is allowed to act. Thereby, in the substrate mixture, only its L-form is hydrolyzed,
It is possible to selectively obtain an L-amino acid or a salt thereof.

At this time, in the present invention, the pH of the medium for carrying out the enzymatic reaction is 6 to 10, preferably 6 to 9, and the temperature is 1 to 10.
0 to 50 ° C, preferably 15 to 45 ° C. In other words, the enzyme reaction of hydrolysis derived from the specific cells has an optimum pH of 6 to 10, most often between 6 and 9. Therefore, by setting the pH of the reaction medium within this range as described above, the L-amino acid or a salt thereof can be obtained at a favorable reaction rate. Then, by performing the reaction in such a pH range and at the above mild reaction temperature, the protecting group in the D-form amino acid in the substrate mixture is eliminated by chemical hydrolysis, or
Racemization of the L-amino acid once generated is suppressed. As a result, in the present invention, an L-amino acid or a salt thereof can be favorably obtained without by-produced D-form.

Here, when the pH of the reaction medium is lower than 6, the reaction rate is lowered and the chemical hydrolysis reaction is accelerated. When the pH is higher than 10, the reaction rate is also lowered. And the risk of racemization of the produced L-amino acid or a salt thereof occurs. On the other hand, if the temperature of the reaction medium is lower than 10 ° C., a sufficient reaction rate cannot be obtained. If the temperature is higher than 50 ° C., the chemical hydrolysis reaction is accelerated and the enzyme is lost. There is also a danger of being active.

In the present invention, the mixing ratio of the D-form and the L-form in the substrate mixture is not particularly limited.
Usually, a so-called racemic body in which the D-form and the L-form are mixed in an equal amount is used.

On the other hand, in the present invention, the microorganism can be used without any limitation as long as it has the above-mentioned properties. Specifically, Corynebacterium aquaticum (Corynebacterium aquaticum IFO12154), Microbacterium arboresens
rescens IFO3750), Bacillus sphaericus (Bacill
us sphaericus IFO3525) and Schizosaccharomyces pombe IFO0358 are preferably used.

As the medium used for culturing the above microorganisms, known media can be used. For example, glucose, sucrose, fructose, glycerol, sorbitol, molasses, carbon sources such as soluble starch, meat extract, yeast extract, polypeptone, nitrates,
There is no problem as long as it contains a nitrogen source such as ammonium salts and inorganic salts such as potassium phosphate monobasic, potassium phosphate dibasic, sodium chloride, magnesium sulfate, iron sulfate and cobalt sulfate.

The form of the medium may be either liquid or solid. Further, the culture method may be any of static culture, shaking culture, and aeration-agitation culture, but liquid culture by aeration-agitation culture is suitable for mass culture. The culture temperature is between 10 and
Culture at 50 ° C., preferably 15-45 ° C., usually for 10-48 hours.

In the present invention, the method of reacting the above-mentioned microorganism, or a culture solution or a treated product of the microorganism with the above-mentioned substrate mixture is not limited to a method of acting on a substrate which is usually carried out in an enzyme reaction utilizing a microorganism. Adopted.
For example, there is a method in which the above-mentioned substrate mixture is added to a culture medium of the microorganism to make it act. in this case,
The substrate mixture may be added to the medium from the beginning or may be added during the culture.

Further, the microorganism, or a culture solution or treated product thereof may be allowed to act on the substrate mixture in an inorganic or organic solvent that does not inhibit the reaction, preferably in an aqueous solvent. In the present invention, the treated cells are, for example, purified cells such as washed cells, dried cells, ground cells, autolysed cells, ultrasonically treated cells, or cell extracts. The obtained enzyme and the like are used without any particular limitation. Here, as the cells, cell extracts, enzymes obtained by purifying the cell extracts, and the like, those immobilized by a known method for immobilizing cells and enzymes can also be used.

In the present invention, the microorganism,
Alternatively, the concentration of the substrate mixture when the culture solution or the treated product of the cell is allowed to act is not particularly limited. Usually, in the range of 0.01 to 20% by weight, preferably 0.05
It may be appropriately selected from 10 to 10% by weight.

In the present invention, the concentration of the microorganism or the treated microorganism when the microorganism, or the culture solution or the treated bacterial cell is allowed to act on the substrate mixture may vary depending on the degree of purification of the treated bacterial cell. Can not be determined, but is usually appropriately selected from the range of 0.05 to 10% by weight in terms of protein amount. At this time, it is also possible to add a surfactant such as Triton (manufactured by Hanoi Chemical Co., Ltd.), Nonion (manufactured by NOF Corporation), or Span (manufactured by Kanto Kagaku) in order to improve the contact between the substrate mixture and the enzyme. The duration of action is determined by the substrate concentration and the type of strain acting, and therefore cannot be unconditionally determined. However, it is usually sufficient to act for 3 to 100 hours.

As described above, after the hydrolysis reaction of the substrate mixture is performed, the amount of the produced L-amino acid may be determined. The quantification of the reaction product is not particularly limited. For example, after completion of the reaction, the cells are separated by centrifugation,
The reaction solution is subjected to high performance liquid chromatography (hereinafter, HPL).
Called C. ) Can be easily quantified by analysis.

Most commonly, L-amino acids are collected from a large amount of the reaction product by concentrating the reaction solution obtained by centrifugation to a supersaturated solution of amino acids and then by crystallization.

The reaction solution obtained by this reaction contains D-protected with the group represented by the general formula (I).
-Contains amino acids without undergoing hydrolysis.
This protected D-amino acid can be extracted from the reaction solution with an organic solvent such as methylene chloride, ethyl acetate, and diethyl ether, and the obtained extraction solution is concentrated and then isolated by crystallization. Can be.

That is, the present invention is also effective as a method for producing a D-amino acid protected by the group represented by the above general formula (I).

[0029]

According to the present invention, a culture solution, cells or treated cells of microorganisms belonging to the genus Corynebacterium, the genus Microbacterium, the genus Bacillus, the genus Schizosaccharomyces can be prepared by the above-mentioned general formula (I) ), A L-amino acid or a salt thereof can be selectively obtained by acting on a mixture of the D-form and the L-form of an amino acid or a salt thereof in which the amino group is protected by the group shown in (1). This reaction can be carried out at a favorable reaction rate and in a state in which by-products of D-amino acids are well suppressed, and the present invention is extremely useful.

[0030]

Hereinafter, the present invention will be described with reference to examples.
The present invention is not limited to these embodiments.

Example 1 The cultivation of the fungus was carried out by using 1.0% peptone, 0.7% meat extract, 0.1% peptone, and 0.1% peptone.
5% yeast extract, 0.3% sodium chloride, 0.03%
Two to three platinum loops of Corynebacterium aquaticum IFO 12154 were inoculated into 5 ml of a medium of cobalt sulfate, pH 7, and cultured with shaking at 30 ° C for 24 hours. 5 ml of this culture solution was added to 1.0% peptone, 0.7% meat extract, 0.5% yeast extract, 0.3% sodium chloride,
The mixture was added to a 2 L shoulder flask containing 500 ml of a 0.03% cobalt sulfate, pH 7 medium, and cultured with shaking at 30 ° C. for 24 hours. After the culture, the supernatant was removed by centrifugation, and the cells were washed once with 0.85% saline under cooling, and then the cell suspension was freeze-dried to obtain freeze-dried cells.

25 mg of the freeze-dried cells obtained was
mM Nt-butoxycarbonyl-D, L-methionine aqueous solution (pH adjusted to 8 with potassium hydrogen phosphate)
100 μl of 50 mM aqueous cobalt sulfate solution
l, 40 μl of 10% Triton X-100, water 1760
Add a total of 2 ml of the reaction solution to a test tube, and add
After shaking for a time, the reaction was performed. After the reaction, the reaction solution 500μ
1 was collected, the cells were precipitated by centrifugation, and the supernatant was analyzed by HPLC to determine the amount of the obtained amino acids. The amino acids in the reaction solution were all L-methionine, and the amino acid concentration was 10 mM. (100% yield).

The amount of Nt-butoxycarbonyl-D-methionine in this reaction solution was determined to be 10 mM, indicating that it had not been hydrolyzed at all.

Comparative Example 1 A freeze-dried cell obtained by the same operation as in Example 1 was 25 m
g, 400 mM Nt-butoxycarbonyloxy-
100 μl of D, L-methionine aqueous solution (pH adjusted to 5.8 with acetic acid and sodium acetate), 100 μl of 50 mM aqueous cobalt sulfate solution, 10% Triton X-100
Was added to a test tube, and the reaction was carried out by shaking at 55 ° C. for 24 hours. After the reaction, 500 μl of the reaction solution was collected, and centrifuged,
The cells were precipitated, the supernatant was analyzed by HPLC, and the obtained amino acids were quantified. As a result, the methionine concentration was only 2 mM (yield 20%).
3 mM was D-methionine.

Example 2 Nt-butoxycarbonyl-D, L shown in Table 1 in place of Nt-butoxycarbonyl-D, L-methionine
-The same operation as in Example 1 was performed using an amino acid and a salt thereof. After reacting for 24 hours, all amino acids produced by hydrolysis were L-amino acids, and Nt-butoxycarbonyl-D-amino acids were present in the reaction solution without any hydrolysis. Table 1 shows the yields of L-amino acids.

[0036]

[Table 1]

Example 3 N-benzyloxycarbonyl-D, shown in Table 2 in place of Nt-butoxycarbonyl-D, L-methionine
The same operation as in Example 1 was performed using an L-amino acid and a salt thereof. After reacting for 24 hours, the amino acids produced by the hydrolysis were all L-amino acids, and the N-benzyloxycarbonyl-D-amino acids were present in the reaction solution without any hydrolysis. Table 2 shows the yields of L-amino acids.

[0038]

[Table 2]

Example 4 Nt-butoxycarbonyl-D, L used in Example 1
-The same operation as in Example 1 was performed except that the concentration of methionine was changed to the concentration shown in Table 3. L formed after 24 hours
Table 3 shows the concentration and yield of -methionine in the reaction solution.
In addition, D-methionine was not confirmed, and all were present in the reaction solution as Nt-butoxycarbonyl-D-amino acids.

[0040]

[Table 3]

Example 5 Corynebacterium aquaticum used in Example 1
The same operation as in Example 1 was performed except that the bacteria shown in Table 4 were used instead of the IFO12154 bacterium. Table 4 shows the yield of L-methionine formed after 24 hours. In addition, D-methionine was not confirmed, and all were present in the reaction solution as Nt-butoxycarbonyl-D-amino acids.

[0042]

[Table 4]

Example 6 In place of the medium used in Example 1, 0.5% peptone, 2%
% Meat extract, 0.03% cobalt sulfate, pH 6 medium 5
ml, Corynebacterium aquaticum IFO12
The same operation as in Example 1 was performed except that the yeasts shown in Table 5 were used instead of the 154 bacteria. L formed after 24 hours
Table 5 shows the yield of -methionine. In addition, D-methionine was not confirmed, and all were present in the reaction solution as Nt-butoxycarbonyl-D-amino acids.

[0044]

[Table 5]

Example 7 Corynebacterium aquaticum IFO12154 was cultured in the same manner as in Example 1, suspended in 50 mM potassium phosphate buffer (pH 7), disrupted by ultrasonication, centrifuged, and the supernatant was removed. The solution was dialyzed to obtain a cell-free extract.

100 μl of the obtained cell-free extract (protein equivalent: 1.56 mg), 50 μl of a 400 mM aqueous solution of Nt-butoxycarbonyl-D, L-methionine (pH adjusted to 8 with potassium hydrogen phosphate), 50 mM Was added to a test tube, and the reaction was carried out by shaking at 30 ° C. for 24 hours. After the reaction, the reaction solution was analyzed by HPLC, and the obtained amino acids were quantified. As a result, all the amino acids in the reaction solution were L-methionine, and the amino acid concentration was 5.7 mM (57% yield). Also, the Nt-butoxycarbonyl-D-amino acid was not hydrolyzed at all, and was all present in the reaction solution.

Example 8 Nt-butoxycarbonyl-D, L used in Example 7
The same operation as in Example 7 was performed except that Nt-butoxycarbonyl-D, L-amino acid shown in Table 6 was used instead of -methionine. The amino acids formed after 24 hours were all L-amino acids, and the Nt-butoxycarbonyl-D-amino acids were not hydrolyzed at all and were present in the reaction solution. The yields of L-amino acids are shown in Table 6.

[0048]

[Table 6]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12P 13/04 C12R 1:07) (C12P 13/04 C12R 1: 645) (58) Investigated field (Int.Cl. ⁷ , DB name) C12P 13/04 BIOSIS (DIALOG) WPI (DIALOG)

Claims (1)

(57) [Claims] 1. A compound represented by the following general formula (I): (Where R is an alkyl group or a benzyl group). When acted on a mixture of a D-form and an L-form of an amino acid protected with an amino group by a group represented by the formula A microorganism belonging to the genus Corynebacterium, the genus Microbacterium, the genus Bacillus, the genus Schizosaccharomyces, or a culture solution or treated cell thereof, which has the ability to hydrolyze to L-amino acids or salts thereof,
Under the conditions of a pH of 6 to 10 and a temperature of 10 to 50 ° C., the compound is allowed to act on a mixture of D-form and L-form of an amino acid or a salt thereof, in which the amino group is protected by the group represented by the general formula (1) And a method for producing an L-amino acid or a salt thereof, wherein the produced L-amino acid or a salt thereof is collected.