JP3018471B2 - Method for producing L-amino acid - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing a corresponding L-amino acid from a 5-substituted hydantoin using a microorganism.

(Prior Art) Conventionally, as a method for producing a corresponding L-amino acid from a 5-substituted hydantoin using the cells of a microorganism, an aromatic L-amino acid is produced using L-phenylalanine using a Flavobacterium genus strain. Production method (Japanese Patent Publication No. 54-227)
4. The maximum accumulation example is 4.26 g / dl) or the production method of L-tryptophan (Japanese Patent Publication No. 54-8749, the maximum accumulation example is 4.45 g / dl), L-phenylalanine, L-
Method for producing tryptophan or L-tyrosine (Japanese Patent Application Laid-Open No. 61-28)
5995, 3.83, 1.75 and 0.86 g / dl for each of the maximum accumulation examples). However, these methods use aromatic L-
Although it is an advantageous method for producing an amino acid from the corresponding 5-substituted hydantoin, the enzyme involved in the reaction has a narrow substrate specificity and does not act on the 5-substituted hydantoin corresponding to an aliphatic L-amino acid. It cannot be applied to the production of aliphatic L-amino acids.

As a method for producing an aliphatic L-amino acid from a 5-substituted hydantoin, a conventional method for producing L-leucine, L-isoleucine, L-methionine, etc. using a strain such as Bacillus (Japanese Patent Publication No. 42-13850; Is 0.81 g of L-isoleucine
/ dl. Agricultural journal 43 [8] 528 (1969), the largest accumulation example is L-menionin at 0.25 g / dl. ), A method for producing L-valine, L-leucine and L-methionine using a strain of the genus Arthrobacter (Japanese Patent Laid-Open No. 62-270, the maximum accumulation example being 0.77 g of L-methionine).
/ dl) and methods for producing L-leucine and L-methionine using Pseudomonas strains (Japanese Patent Application Laid-Open No. 64-71476, the maximum accumulation example of which is 3.36 g / dl of L-methionine). However, all of these methods have a low yield and / or accumulation amount, and require more purification steps than a method using cells isolated from the culture solution to produce L-amino acids in the culture solution. There are problems such as the narrow substrate specificity and the limited number of amino acids that can be produced.

Thus, the inventor has assigned the aliphatic L-amino acid the corresponding 5
As a result of intensive studies on a method for producing from a substituted hydantoin, a new method for causing a bacterium belonging to the genus Bacillus to act on a 5-substituted hydantoin was invented (Japanese Patent Laid-Open No.
63-24895, the maximum accumulation example was 0.51 g / dl of L-leucine).
This method uses L-valine, L-leucine, L
-Can be applied to the production of aromatic L-amino acids such as L-phenylalanine in addition to aliphatic L-amino acids such as methionine, but from the viewpoint of industrial production, the yield and / or Or the improvement of the accumulation amount was still desired.

(Problems to be Solved by the Present Invention) An object of the present invention is to provide L-valine, L-leucine, L-isoleucine, L-methionine, L-phenylalanine, which are L-amino acids directly corresponding to 5-substituted hydantoins. It is an object of the present invention to provide a method for producing L-tyrosine, L- (3'4'-methylenedioxyphenyl) alanine, L-alanine, L-cyanoethylglycine and the like in a high accumulation amount.

(Means for Solving the Problems) As a result of various studies to solve the above problems, the present inventors have found that cells of bacteria belonging to the genus Bacillus are L-form, D-form
ATP (adenosine triphosphate) is essential for the hydrolysis of the 5-substituted hydantoin when producing the corresponding L-amino acid by acting on the 5-substituted hydantoin in the isomer or DL form. Intracellular ATP is decomposed into ADP (adenosine diphosphate), and the shortage of ATP in the cell during the reaction is the cause of the low accumulation of L-amino acid from 5-substituted hydantoin. (Agr. Biol. Chem., 52 [11], 2857 (1987)).

Conventionally, when such a high-energy substance such as ATP is required for the progress of the reaction, a method of simultaneously performing the growth and the reaction of the bacterium by adding a substrate to the culture solution is often used and separated from the culture solution. There are almost no examples in which a reaction is carried out by using cells in combination with a regeneration system of an energy source, especially in bacteria. The present inventors believe that if the ATP can be regenerated from ADP in the cells during the reaction, the amount of L-amino acid accumulated from the 5-substituted hydantoin can be increased, and as a result of further research, such as glucose in the reaction solution, ATP regeneration proceeds at the same time as the reaction in the presence of a substance that is the energy source for the conversion of ADP to ATP and with the supply of an enzyme, resulting in the production of the desired amino acid in high accumulation. Have been found, and based on such findings, the present invention has been completed.

That is, the present invention provides a cell of a microorganism belonging to the genus Bacillus having the ability to convert a 5-substituted hydantoin into an L-amino acid or a treated product of the microorganism represented by the following general formula (I): (Wherein, R represents a substituent) L-form, D-form and / or 5-substituted hydantoin represented by the following formula:
Alternatively, the present invention relates to a method for producing an L-amino acid, which comprises causing an energy source substance to be present in a reaction mixture and supplying oxygen when producing a corresponding L-amino acid by acting on a DL form.

 Hereinafter, the present invention will be described sequentially.

The substituent R of the 5-substituted hydantoin of the general formula (I), which is a raw material of the method for producing an L-amino acid according to the present invention, includes, for example, CH ₃ − and NCCH ₂ CH ₂ − can be mentioned, and such a 5-substituted hydantoin may be any of L-form, D-form and DL-form. The corresponding L-amino acids produced from these 5-substituted hydantoins are L-valine, L-leucine, L-isoleucine, L-methionine, L-phenylalanine, L-tyrosine, L- (3'4 ' -Methylenedioxyphenyl) alanine, L-alanine and L-cyanoethylglycine.

Microorganisms that have the ability to hydrolyze 5-substituted hydantoins to the corresponding L-amino acids include, for example, Bacillus brevis AJ-12299 (FERM P-8837). It is a matter of course that the mutant may be a mutant induced by a method such as a mutation treatment with a chemical mutagen or ultraviolet irradiation.

In order to obtain cells of such a microorganism, the microorganism may be cultured and grown in an appropriate medium. There is no particular limitation on such a medium, and a normal medium containing a normal carbon source, a nitrogen source, inorganic ions, and if necessary, an organic nutrient source may be used.

As the carbon source, carbohydrates such as glucose, alcohols such as glycerol, organic acids and the like are appropriately used. As a carbon source, ammonia gas, aqueous ammonia,
Ammonium salts and others are used. As the inorganic ion, a magnesium ion, a phosphate ion, a potassium ion, an iron ion, a manganese ion and others are appropriately used as needed. As organic nutrients, vitamins, amino acids and the like and yeast extracts, peptone, meat extract, corn steep liquor, casein hydrolyzate and the like containing them are appropriately used.

If the medium is further added with a small amount of the 5-substituted hydantoin of the present invention or an N-carbamoylamino acid obtained by hydrolyzing the same, the 5-substituted hydantoin can be converted into L-substituted hydantoin.
In some cases, cells having high activity of converting to amino acids can be obtained (acclimation).

There is no particular limitation on the culturing conditions. For example, culturing may be carried out for about 12 to 48 hours under aerobic conditions while appropriately limiting pH and temperature within the range of pH 5 to 8 and temperature of 25 to 40 ° C.

As the cells to act on the 5-substituted hydantoin, the cells separated from the culture solution are used with or without washing. As the treated cells, freeze-dried cells, acetone-dried cells, surfactants, cells treated with toluene or the like, and the like are appropriately used. Furthermore, it can also be used as a treated bacterial body in which the bacterial body is entrapped and immobilized in a polymer substance such as carrageenan. The amount of the cells or the treated product of the cells may be an amount (effective amount) that exhibits a target effect in the case of a given reaction, and this effective amount can be easily determined by those skilled in the art by a simple preliminary experiment. For example, in the case of washed wet cells, the amount is 1 to 20 g per 1 dl of the reaction solution.

As an energy source substance, a substance such as carbohydrates, alcohols, and organic acids can be used as long as the microorganism can be used as an energy source and the ATP consumed during the reaction can be regenerated in the reaction solution including the inside of the cells. Any of glucose, glycerol, fructose, galactose,
Examples include xylose, mannitol, maltose, trehalose, succinic acid, fumaric acid, citric acid, acetic acid and mixtures thereof. The amount of the energy substance to be used may be an amount (effective amount) that exhibits a desired effect in the case of a given reaction, and this effective amount can be easily determined by those skilled in the art by simple preliminary experiments. However, for example, in the case of glucose, 0.1 to 10 g per 1 dl of the reaction solution
It is. The energy source substance may be added at the start of the reaction or may be added during the reaction.

Now, the cells or the treated cells and the 5-substituted hydantoin are suspended or dissolved in water, and the temperature is adjusted to 10 to 70 ° C., preferably 20 to 70 ° C.
If the pH is kept at 5050 ° C. and the pH is kept at 5-11, preferably 6.5-9, the corresponding L-amino acid is formed from the 5-substituted hydantoin, the inventor then has to add glucose and / or It has been found that, when other energy source substances are added and oxygen is further supplied, the accumulated amount of L-amino acids is greatly increased, and a desirable result is obtained from the viewpoint of industrial production.

The concentration of the 5-substituted hydantoin is 0.1 to 30%, preferably 0.5 to 10% of the total weight of the reaction mixture, but if necessary, for example, when a high concentration of the 5-substituted hydantoin inhibits the reaction. The 5-substituted hydantoins can be supplemented during the reaction.

Oxygen can be supplied, for example, by shaking under aerobic conditions or by aeration and stirring.

Addition of a surfactant, coenzyme, hydroxylamine, cobalt ion or other metal ions to the reaction mixture may improve the reaction yield in some cases.

Thus, after 5 to 100 hours, a large amount of L-amino acid is produced and accumulated in the reaction mixture.

In order to collect and separate the L-amino acid thus accumulated from the reaction-completed mixture, according to the method of the present invention, since the D-amino acid is not produced as a by-product, the L-amino acid is precipitated at the isoelectric point by using an ion exchange resin. An ordinary method for collecting and separating L-amino acids can be employed.

L-amino acid can be quantified by, for example, high performance liquid chromatography or a microorganism quantification method using Leuconostoc mesenteroides ATCC8042.

(Examples) Hereinafter, the present invention will be further described with reference to examples.

Example 1 (1) Maltose 2.0 g / dl, (NH ₄ ) ₂ SO ₄ 0.5 g / dl, KH
₂ PO ₄ 0.1g / dl, K ₂ HPO ₄ 0.3g / dl, MgSO ₄・ 7H ₂ O 0.05g / dl, F
_{eSO 4 · 7H 2 O 0.001g /} dl, MnSO 4 · 4H 2 O 0.001g / dl, yeast extract 1.0 g / dl, polypeptone 1.0 g / dl, an aqueous medium containing DL-5-isopropyl hydantoin 0.2 g / dl ( pH 7.0) 500m
The flask was sterilized by heating at 120 ° C. for 15 minutes, and then allowed to cool. Bacillus brevis AJ-1 cultured on bouillon agar medium at 30 ° C for 24 hours.
One loopful of 2299 (FERM P-8837) was inoculated and cultured at 30 ° C. for 16 hours. The cells were centrifuged from this culture, washed once with the same amount of physiological saline as the culture, and centrifuged again to collect the cells.

The cells were weighed with 1 g of DL-5-isopropylhydantoin.
to a test tube (16.5 mmφ, 25 ml capacity) containing 5 ml of 0.1 M Tris-HCl buffer (pH 7.0) containing 5 g / dl, and 0.5 g / dl of glucose as an energy source. The reaction was continued by shaking under aerobic conditions at 30 ° C. for 16 hours. During this time, the pH of the reaction mixture was maintained at 7 with an aqueous solution of hydrochloric acid or potassium hydroxide.

The cells were removed from the reaction-completed mixture by centrifugation, and the L-valine produced in the supernatant was measured by a microorganism assay. The results shown in Table 1 below were obtained.

(2) For comparison, the results for the case where the above reaction was carried out without adding glucose, the case where glucose was added but allowed to stand still, and the case where the above reaction was carried out without adding glucose and standing still Are also shown in Table 1.

Example 2 L-valine was produced by performing exactly the same reaction as in (1) of Example 1 except that glucose as an energy source was changed to an energy source material shown in Table 2.

 The results are shown in Table 2.

Example 3 Bacillus brevis AJ-12299 was used in Example 1 (1).
Using the same medium as in (1), culturing was performed at 30 ° C. for 16 hours in the same manner as in (1) of Example 1. The cells were centrifuged from the culture, washed once with the same amount of physiological saline as the culture, and centrifuged again to collect the cells.

The cells were weighed with 1 g of the 5-substituted hydantoin shown in Table 3.
5 g / dl was added to 5 ml of 0.1 M Tris-HCl buffer (pH 7.0) containing 0.5 g / dl and glucose at 0.5 g / dl, and the mixture was shaken under aerobic conditions at 30 ° C. for 48 hours.

During this period, 16 hours and 32 hours later, the same 5-substituted hydantoin was further added at 1 g / dl and glucose at 0.5 g / dl.

After completion of the reaction, the cells were removed from the reaction-completed mixture by centrifugation, and each amino acid present in the supernatant was assayed for microorganisms (L-valine, L-leucine, L-isoleucine, L-isoleucine, L-valine).
Methionine, L-phenylalanine and L-tyrosine)
Or high performance liquid chromatography (other amino acids)
Table 3 shows the results of the measurement.

(Effect of the Invention) In general, a 5-substituted hydantoin produced by an organic synthesis method is a DL form, but according to the method of the present invention, all of the 5-substituted hydantoin of the DL form can be converted to the corresponding L-amino acid,
Moreover, it can be obtained at a high concentration suitable for industrial production.

Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI C12P 13/12 C12P 13/12 A 13/22 13/22 BC (56) References JP-A-63-24895 (JP, A) (58) ) Fields surveyed (Int. Cl. ⁷ , DB name) C12P 13/00-13/22 C12P 41/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A microorganism or a treated product of a microorganism belonging to the genus Bacillus having the ability to convert a 5-substituted hydantoin into a corresponding L-amino acid is represented by the following general formula (I): (Wherein, R represents a substituent) L-form, D-form and / or 5-substituted hydantoin represented by the following formula:
Or a method for producing a corresponding L-amino acid by acting on a DL form, wherein the step of culturing the microorganism, the step of separating and collecting cells from a culture solution of the microorganism, the collected cells, Preparing an energy source substance capable of producing ATP by the action of the body, and a reaction solution containing at least the 5-substituted hydantoin; and a reaction for converting the 5-substituted hydantoin to a corresponding L-amino acid. The above method, comprising the step of supplying oxygen to the reaction solution. 2. The method according to claim 2, wherein the substituent R is CH ₃ - or method according to claim 1 NCCH is a group represented by ₂ CH _2. 3. The method according to claim 1, wherein the energy source substance is glucose, glycerol, fructose, galactose, xylose, mannitol, maltose, trehalose, succinic acid, fumaric acid, citric acid and / or acetic acid.
The method described in. 4. The method according to claim 1, further comprising the step of replenishing the reaction solution with the energy source substance and / or the 5-substituted hydantoin during the reaction for converting the 5-substituted hydantoin into the corresponding L-amino acid. The method according to any one of claims 1 to 3.