JPH04365492A - Production of l-alanine - Google Patents

Abstract

PURPOSE:To find out inhibition of L-aspartic beta-decarboxylase by sulfate ion derived from cells or raw material aspartic acid, to reduce influence of sulfate ion in a reaction solution and to stably and efficiently produce L-alanine. CONSTITUTION:An aqueous reaction solution containing L-aspartic acid or a salt thereof having sulfate ion concentration restricted to <=100ppm is treated with cells of a bacterium containing L-aspartic beta-decarboxylase or a treated material thereof to give L-alanine.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing L-alanine by an enzymatic method. L-alanine is an important amino acid as a raw material for medicine, food, or the chemical industry.

0002

[Prior Art] As an industrial method for producing L-alanine, L-aspartic acid is enzymatically decarboxylated to produce L-alanine using bacterial cells containing L-aspartate β-decarboxylase belonging to the genus Pseudomonas. Method of manufacturing
28951, Japanese Patent Publication No. 46-7560) are known.

0003

[Problems to be Solved by the Invention] In the above conventional method, the enzymatic reaction for producing L-alanine required a long time. As a result of intensive research into the cause, the present inventors found that it is because L-aspartate decarboxylase is inhibited by sulfate ions in the reaction solution. Such sulfate ions in the reaction solution are brought into the microbial cells from the culture solution, or mixed into the manufacturing process of L-aspartic acid as a raw material.

The present invention provides a method for stably and efficiently producing L-alanine by reducing the influence of sulfate ions in the reaction solution.

[0005]

[Means for Solving the Problems] The present invention allows microbial cells containing L-aspartate β-decarboxylase or a treated product thereof to act on an aqueous reaction solution containing L-aspartic acid or a salt thereof. In the method for producing L-alanine, the method is applied to the reaction solution in which the sulfate ion concentration is suppressed to 100 ppm or less, thereby reducing inhibition of enzyme action by sulfate ions.

[0006] The microorganism containing L-aspartate β-decarboxylase used in the present invention is a microorganism containing the enzyme belonging to the genus Pseudomonas, such as Pseudomonas dacu.
nhae) IAM1152, ATCC21192, Pseudomonas pu
ATCC21812, IAM1506, Pseudomonas fluorescens
as fluorescens) IFO3081, Pseudomonas aeruginosa (Pseudomonas
aeruginosa) IAM1054, and these bacterial cells are preferably used. Furthermore, a processed product of the above-mentioned microbial cells, such as a destroyed product of the microbial cells, can also be used. Destruction of bacterial cells can be performed using methods known per se, such as ultrasonication and squeezing.

[0007] L-aspartate β-decarboxylase-containing bacterial cells can be obtained by culturing the above-mentioned microorganisms by a conventional method. The carbon source used in the culture medium is not particularly limited, and examples thereof include fumaric acid, succinic acid, aspartic acid, and the like, with fumaric acid being preferred. As a nitrogen source for the culture medium, inorganic salts such as ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea, etc. can be used, and organic nitrogen sources such as peptone, yeast extract, corn steep liquor, casamino acids, etc. can also be used. . As the inorganic salt, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, etc. are used.

[0008] The microbial cells containing L-aspartate β-decarboxylase are cultured under aerobic conditions such as aeration and shaking, and the culture temperature is 20-40°C, preferably 28-30°C.
It is 2℃. pH during culture is 5-10, preferably 7-10
It is around 8, and its adjustment is performed using acid or alkali. The fumaric acid concentration at the start of culture is preferably 0.1-
It is 5% by weight, more preferably 0.5-2% by weight. The culture time is 8 hours to 4 days, preferably 10 hours to 3 days.

In the method of the present invention, an aqueous solution containing L-aspartic acid or a salt thereof is subjected to an enzymatic reaction in the presence of the microbial cells prepared above or a crushed product thereof. Here, the concentration of L-aspartic acid or its salt added to the aqueous solution is 0.5 to 50% by weight, preferably 3 to 3% by weight.
It is 0% by weight. Note that L-aspartic acid exists in powder form even when dissolved due to its solubility in the reaction solution (
There is no problem even if it is in an undissolved state. pH of reaction solution
For the adjustment, an alkaline solution, for example, an aqueous solution of aqueous ammonia, sodium hydroxide, potassium hydroxide, etc., is suitably used.

[0010] The aqueous solution further contains pyridoxal.
5'-phosphoric acid may be added in an amount of 0.0005 to 0.05% by weight, preferably 0.001 to 0.01% by weight. Furthermore, if necessary, nonionic surfactants such as polyoxyethylene octylphenyl ether, polyoxyethylene sorbitan monolaurate, etc.
．． It can be used in an amount of 0.01 to 0.5% by weight, preferably 0.03 to 0.2% by weight. If necessary, add 0.00% α-keto acid such as pyruvic acid or α-ketobutyric acid.
01 to 0.5% by weight, preferably 0.001 to 0.2% by weight. In the present invention,
pH during enzyme reaction is 4.3 to 5.0, preferably 4.5
-4.8, the reaction temperature is 40-47°C, preferably 42-45°C, and the reaction is usually carried out for about 3 to about 48 hours. Further, if necessary, sodium chloride can be added, and the concentration used is 0.02 to 1.0% by weight, preferably 0.03 to 0.5% by weight.

When acting on a reaction solution to which a water-soluble barium salt has been added, barium chloride, barium bromide, barium nitrate, barium acetate, etc. are used as the water-soluble barium salt. The concentration of barium to be added is 0.001 to 0.00, whether added alone or in combination.
5% by weight, preferably 0.01-0.1% by weight is used.

[0012] When cleaning microbial cells or their treated products with an aqueous solution containing an alkali metal chloride or an alkaline earth metal chloride, the chloride may be an alkali such as sodium chloride, potassium chloride, calcium chloride, etc. Metal chlorides or alkaline earth metal chlorides are preferred. Its concentration is 3-500mM, preferably 10-400mM.

[0013] For washing, the bacterial cells or their treated product are suspended in the chloride aqueous solution and then subjected to solid-liquid separation by centrifugation, ultrafiltration, etc., and the bacterial cells are collected. It is also possible to repeat the cleaning if necessary.

Separation and purification of L-alanine produced in the above reaction solution can be carried out by known ion exchange resin treatment or the like.

[0015]

[Examples] The present invention will be explained in detail with reference to Examples below. In addition, in the following reference examples and examples, % means weight % unless otherwise specified.

Reference Example 1 Medium 1 (sodium fumarate 0.5%, ammonium fumarate 1.0%, yeast extract 0.5%, KH2 PO4
0.1% and MgSO4..7H2O 0.05
%, pH 7.0) into a 500 ml Erlenmeyer flask, sterilized it at 120°C for 20 minutes, and then added Pseudomonas dacne to it.
Inoculate one platinum loop of IAM1152 (unhae) at 30°C.
Shaking culture was performed for 12 hours.

1,500ml of medium 1 to 3,000ml
30 ml of the Erlenmeyer flask culture solution was inoculated into a jar fermenter that had been sterilized at 120°C for 20 minutes, and cultured at 30°C with a rotation speed of 600 rpm and a ventilation of 1 vvm. Adjust the pH to 7 using concentrated sulfuric acid diluted 5 times with deionized water.
．． At the same time, the dissolved oxygen concentration during the culture was measured using a dissolved oxygen electrode (Model S-1, manufactured by Oriental Electric), and the increase in dissolved oxygen concentration due to consumption and depletion of fumaric acid, which is a carbon source, was measured. At the time of observation, the culture was continued while adding 100 ml of 20% ammonium fumarate aqueous solution (pH 6.5) twice in total. After 19 hours, the culture was terminated, and the bacterial cells were collected by centrifugation, and the wet bacterial cells were approximately 45g was obtained.

Reference Example 2 The bacterial cells obtained in Reference Example 1 were suspended in a 0.9% NaCl aqueous solution and washed by centrifugation three times.
．． 2g was added to an aqueous reaction solution (as shown in Table 1) with ammonium sulfate added so that the sulfate ions had a predetermined concentration.
L-aspartic acid 30%, polyoxyethylene octylphenyl ether 0.05%, pyridoxal 5'-phosphate 0.001% and sodium pyruvate 0.05%
Contains, adjusted to pH 4.7 with 28% ammonia water) 20
ml and reacted at 42°C. The reaction was stopped 2 hours after the start of the reaction, and the concentration of produced L-alanine in the liquid was determined by high performance liquid chromatography.

[0019] The sulfate ion concentration when no ammonium sulfate is added is 0, and the L-alanine concentration produced at that time is 100%, and the values are expressed as relative values. The L-aspartic acid used was a special reagent grade (Wako Pure Chemical Industries, Osaka, Japan), and the sulfuric acid content was "SO4 max 0.0".
3%" was displayed.

[0020]

[Table 1]

Even at the initial stage of 2 hours after the start of the reaction, 1
5% by 00ppm SO42-, 500ppm
It can be seen that about 20% inhibition is caused by SO42-.

Example 1 0.2 g of wet bacterial cells obtained in Reference Example 1 were subjected to an aqueous reaction in the experimental areas shown in Tables 2 to 4, with the addition of water-soluble barium salts as shown in each table. liquid (L-aspartic acid 30%, polyoxyethylene octylphenyl ether 0.05%,
Containing 0.001% of pyridoxal 5'-phosphate and 0.05% of sodium pyruvate, adjusted to pH 4.7 with aqueous ammonia containing 28% NH3), suspended in 20 ml and heated to 42°C.
I reacted with The results were expressed as the time required to complete the reaction.

[0023]

[Table 2]

[0024]

[Table 3]

[0025]

[Table 4]

Example 2 0.2 g of the wet bacterial cells obtained in Reference Example 1 washed three times with 0.9% NaCl aqueous solution was added to 20 ml of the reaction solution of Reference Example 2 in which sulfate ions were not added externally. Suspend at 42℃
I reacted with As a control, wet bacterial cells that were not washed at all were reacted in the same manner. The results were expressed as the time required to complete the reaction.

[0027]

[Table 5]

[0028]

[Effect of the invention] As described above, by keeping the sulfate ion concentration in the reaction solution low, inhibition of L-aspartate β-decarboxylase activity by sulfate ions was avoided, and the reaction completion time was significantly shortened. .

Claims (3)

[Claims] Claim 1: A microbial cell containing L-aspartate β-decarboxylase or a treated product thereof is allowed to act on an aqueous reaction solution containing L-aspartic acid or a salt thereof to produce L-aspartate.
In the method for producing alanine, the sulfate ion concentration is reduced to 1
A method for producing L-alanine, which is characterized in that the reaction is carried out in a reaction solution whose concentration is suppressed to 00 ppm or less. 2. The method according to claim 1, wherein the microorganism cells or a treated product thereof are allowed to act on the reaction solution to which a water-soluble barium salt has been added. 3. The method according to claim 1 or 2, wherein the microorganism cells or a treated product thereof washed with an aqueous solution containing an alkali metal chloride or an alkaline earth metal chloride act on the reaction solution.