JPH0347839B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-phenylalanine. L-phenylalanine is one of the essential amino acids and is a nutritionally or pharmaceutically useful substance. A method for producing L-phenylalanine from cinnamic acid and ammonia or an ammonia donor using enzymes produced by microorganisms is known (UK Patent No. 1489418, Japanese Patent Application Laid-Open No. 1983-96388, Publication No. 56-26197, etc.). However, the methods described in these publications are still not satisfactory. The present inventors have conducted various studies on methods for efficiently producing L-phenylalanine from cinnamic acid and ammonia. It has been found that L-phenylalanine can be efficiently produced by reacting cinnamic acid and ammonia or an ammonia donor in an aqueous medium in the presence of an alkylglycine-treated product containing enylalanine ammonia-lyase activity. . The present invention will be explained in detail below. As the microorganism used in the present invention, any microorganism can be used as long as it has L-phenylalanine ammonia thiase activity. Specific examples include Rhodotorula rubra ATCC20258, Rhodotorula texensis IFO932, Rhodotorula
Glytinis IFO0559, Sporobolomyces roseus IFO1040, etc. As a medium for culturing the above-mentioned microorganisms, either a synthetic medium or a natural medium can be used as long as it contains appropriate amounts of carbon sources, nitrogen sources, inorganic substances, etc.
Carbon sources include glucose, sheikrose,
Sugars such as blackstrap molasses, sugar alcohols such as glycerol, sorbitol, and mannitol sugars can be used. Nitrogen sources include aqueous ammonia, ammonium chloride, ammonium sulfate, ammonium carbonate,
Various inorganic and organic ammonium compounds such as ammonium acetate and ammonium phosphate, nitrogenous compounds such as urea, peptone, yeast extract, casein hydrolyzate, and nitrogenous organic substances such as defatted soybean or its digested sugar can be used. As the inorganic substance, salts of metals such as sodium, potassium, manganese, magnesium, calcium, and copper, and salts of phosphoric acid, sulfuric acid, nitric acid, and hydrochloric acid can be used. Cultivation is carried out, for example, at pH 5-8, 20-40°C, 1-5
Do it for days. Next, the obtained culture solution of the above-mentioned microorganism, bacterial cells (e.g., washed bacterial cells, dried bacterial cells), or processed products thereof (e.g., bacterial cell grinding product, bacterial autolysed product, microbial cell ultrasonication product) , bacterial cells immobilized with, for example, acrylamide gel or carrageenan gel, etc.) are treated with cinnamic acid and ammonia or an ammonia donor in the presence of alkylglycine, or
Alternatively, a culture solution, cells, or a treated product of the microorganisms that have been brought into contact with a surfactant are allowed to act on cinnamic acid and ammonia or an ammonia donor to produce L-phenylalanine. The concentration of cinnamic acid is in the range of 0.05 to 1 mol, and the concentration of ammonia or ammonia donor is in the range of 1 to 10 mol. The raw materials cinnamic acid, ammonia, or an ammonia donor are supplied all at once or intermittently.
As an ammonia donor, ammonium acetate,
Examples include ammonium salts such as ammonium chloride and ammonium sulfate. The alkylglycine used in the present invention is one of the amphoteric surfactants, and Anon LG (trade name, manufactured by NOF Corporation) is preferably used as the alkylglycine-containing material. The amount of alkylglycine added is based on the reaction solution.
0.05-10W/V%, preferably 0.5-10W/V%
It is. These can be added directly to the reaction solution or as an aqueous solution. The reaction is usually carried out at a temperature of 20-45°C, preferably 25-35°C.
C. and pH 8 to 11, preferably 9 to 10, for 5 to 70 hours. After the reaction, L-phenylalanine produced from the reaction solution can be separated by a conventional ion exchange resin method, precipitation method, or the like. Examples are shown below. Example 1 Rhodotorula rubra ATCC20258 with yeast extract
1.0 g of peptone, 0.5 g of salt, and 0.05 g of L-phenylalanine at 28°C.
It was cultured with shaking for 17 hours. The cells obtained by centrifugation from the culture solution were washed with 0.9% cold saline and centrifuged to obtain washed cells. Add 10 ml of a substrate solution (PH = 10) containing 2.8 M ammonia and 150 mM cinnamic acid prepared in advance to the bacterial cells, and then add a 10% aqueous solution of the surfactant shown in Table 1 as shown in Table 1. The mixture was reacted at 30°C for 5 hours. After the reaction, the reaction mixture was centrifuged, and the supernatant was analyzed by paper chromatography [developing solvent: n-butanol:acetic acid:water=5:2:2 (v/v)] to detect phenyl. The amount of alanine produced was measured. The results are shown in Table 1.

【represent.
Example 2 200 ml of the culture solution obtained by culturing in the same manner as in Example 1 was treated in the same manner as in Example 1 to obtain washed bacterial cells.
Add 10 ml of a previously prepared substrate solution (PH = 10) containing 8 M ammonia and 300 mM cinnamic acid to the bacterial cells, and perform the reaction at 30°C with or without the addition of Anone LG to determine the amount of phenylalanine produced. Examined over time. The results are shown in Table 2.

[Table] In this reaction, due to the inhibition of cinnamic acid, there is no significant change in the reaction at the initial stage compared to the case without addition, but a large amount of phenylalanine is produced in 24 to 48 hours by adding Anone LG. Example 3 Washed bacterial cells were obtained from 100 ml of the culture solution in the same manner as in Example 1, except that Rhodotorula glutinis IFO0559 was used as the bacterial cells. 10 ml of a substrate solution (PH = 10) containing 2.8 M ammonia and 230 mM cinnamic acid and 10% Anone LG were added to the bacterial cells.
Aqueous solutions were added as shown in Table 3 and reacted at 30°C.
The results are shown in Table 3.

[Table] The effect is remarkable even when the amount of Anone LG added is 0.1%, and if it is 0.5% or more, the amount of phenylalanine produced increases significantly. Example 4 The bacterial cells shown in Table 4 were incubated at 28°C using 100 mg of a medium (PH6.0) containing 0.5 g of NaCl, 0.05 g of L-phenylalanine, and 3.0 g of corn stave liquor.
After culturing with shaking for 17 hours, washed bacterial cells were obtained in the same manner as in Example 1. 10 ml of ammonia (PH=10) containing 2.9 M of ammonia and 150 mM of cinnamic acid was added to the bacterial cells, and a 10% aqueous solution of Anone LG was added as shown in Table 4, followed by reaction at 30° C. for 8 hours. The results are shown in Table 4.

[Table] Example 5 Washed bacterial cells obtained in the same manner as in Example 1 were treated with 2% Anon LG aqueous solution at room temperature for 30 minutes, centrifuged, and further washed with 10 ml of 0.9% cold saline. The cells were then centrifuged to obtain Anon LG treated bacterial cells.
Using this bacterial cell and Anon LG untreated bacterial cell,
The reaction was carried out at 30°C using a substrate solution (PH=10) containing 2.8M ammonia and 150mM cinnamic acid. The results are shown in Table 5.

[Table] The effect of Anon LG is not only achieved by adding it to the substrate solution, but also by contacting it with the bacterial cells in advance.
It can be effective. Example 6 In the same manner as in Example 1, washed bacterial cells of Rhodotorula rubra ATCC20258 were collected from 300 ml of the culture solution, and then added to 20 ml of Anon LG 2% aqueous solution at room temperature.
The contact treatment was carried out for 30 minutes. The Anon LG-treated cells were immobilized with κ-carrageenan in a conventional manner to obtain immobilized cells. Ammonia is added to the immobilized bacterial cells.
Add 30 ml of substrate solution (PH = 10) containing 2.8 M and 3.150 mM of cinnamic acid and react at 30°C for 48 hours.
The amount of L-phenylalanine produced was examined over time. For comparison, a reaction was carried out under the same conditions using washed bacterial cells obtained from the same culture solution as described above and immobilized bacterial cells that had not been treated with anon. The result is the 6th
Shown in the table.

【table】

Claims (1)

[Claims] 1. In the presence of a culture, microbial cells, or a treated product of a microorganism having L-phenylalanine ammonia-lyase activity (hereinafter referred to as "substance containing L-phenylalanine ammonia-lyase activity") and alkylglycine, or in the presence of an alkylglycine, or Cinnamic acid and ammonia or an ammonia donor are reacted in an aqueous medium in the presence of an alkylglycine-treated product containing enylalanine ammonia-lyase activity.
A method for producing L-phenylalanine, which comprises producing phenylalanine and collecting it.