JPS6144474B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-phenylalanine by an enzymatic method, and more specifically, using a culture solution of a microorganism containing L-phenylalanine ammonia-lyase, microbial cells collected from the culture solution, or a processed product of the microbial cells. The present invention relates to a method for efficiently producing L-phenylalanine from cinnamic acid and ammonia or an ammonia donor. A method for producing L-phenylalanine from cinnamic acid and ammonia or an ammonia donor using enzymes produced by microorganisms has already been patented in the UK.
It is reported in Publication No. 1489468. This British patented method involves the production of L-phenylalanine by allowing L-phenylalanine ammonia-lyase produced by microorganisms of the genus Rhodotorula or Fusarium to act on cinnamic acid and ammonia or an ammonia donor. more than twice the molar ratio of cinnamic acid,
It is characterized in that it is preferably used at 8 times the amount, and under such specific conditions, it enables the production of L-phenylalanine from cinnamic acid by L-phenylalanine ammonia lyase, which has not been recognized in the past. be. However, when using this method. (i) Although it is stated that up to 20% of the cinnamic acid used can be converted to L-phenylalanine, if such a conversion rate can be achieved, the concentration of cinnamic acid in the enzyme reaction solution is
Only at a very low concentration of 0.006 molar concentration (0.1%), and as shown in the example of the same publication, the concentration of cinnamic acid is 0.33 to 0.66 molar concentration (5 to 10%).
%), the conversion rate to L-phenylalanine decreases to 5% or less. And (ii) As mentioned above, since the conversion rate from cinnamic acid to L-phenylalanine is low, the amount of L-phenylalanine accumulated is also low (slightly less than 2.5 mg/ml). Unreacted cinnamic acid, which far exceeds L-phenylalanine, will be present in the reaction solution, which not only makes it extremely difficult to isolate L-phenylalanine, but also (iv) unreacted raw materials. There have been many problems, such as the recovery and reuse of carbon dioxide, which has become an industrial necessity. As a result of various studies conducted by the present inventors regarding the enzymatic reaction in the above-mentioned British patented method, the present inventors have found that (a) the ammonium ion concentration in the enzymatic reaction solution is significantly involved in the conversion of cinnamic acid to L-phenylalanine; , and (b) the relationship between ammonium concentration and reaction equilibrium, and the cinnamic acid concentration, such as the fact that when the cinnamic acid concentration in the enzyme reaction solution is high, L-phenylalanine ammonia-lyase, which catalyzes the reaction, is subject to substrate inhibition by cinnamic acid. We have obtained new knowledge about the relationship between fertilization and substrate inhibition. As a result of further research based on this new knowledge, the present inventors added ammonia or an ammonia donor to the reaction solution during the enzyme reaction so that the ammonium ion concentration was at least 3 molar or more. Cinnamic acid, whose concentration is approximately
If each is added at a concentration of 0.05 to 0.2 molar, L-phenylalanine can be produced at a conversion rate of about 50% or more, about 80% higher than that of cinnamic acid. It can be accumulated in the reaction solution at a practically high concentration (4 to 25 mg/ml), and the amount of unreacted cinnamic acid in the reaction solution is also reduced, making it easier to isolate the L-phenylalanine produced. They found that this was significantly improved and completed an industrially advantageous method for producing L-phenylalanine. That is, the present invention involves enzymatic reaction of cinnamic acid and ammonia or an ammonia donor in the presence of a culture solution of a microorganism containing L-phenylalanine ammonia-lyase, cells collected from the culture solution, or a treated product of the cells. When producing L-phenylalanine, the enzymatic reaction is carried out in such a manner that the concentration of ammonia or an ammonia donor is at least about 3 molar or higher, and the concentration of cinnamic acid is about 0.05 to 0.2 molar.
This is a method for producing L-phenylalanine using an enzymatic method, which is characterized in that it is carried out under conditions of molar concentration. The method of the present invention will be explained in detail below. In the present invention, the microorganism containing L-phenylalanine ammonia-lyase is a microorganism containing L-phenylalanine ammonia-lyase,
- Any microorganism that has the ability to produce phenylalanine may be used, but
Among others, e.g. Sporobolomyces roseus
227 (Feikoken Bacterium No. 3852), Sporobolomyces roseus 221 (Feikokuken Bacterial Reference No. 3853) [Mycological properties of these two strains are described in The East A Toxonomic Study, J.・Loder and N,
J. W. Creger Van Ritzge, 2nd edition, 348
~352 pages (1967), “North Holland Publishing Company, Amsterdam”], Rhodosporidium toruloides
Culture solution of microorganisms such as IF00559, Rhodotorula thezensis IF00932, Rhodotorula rubra OUT6158, bacterial cells collected from the culture solution by centrifugation, or processed products of the bacterial cells (e.g., washed bacterial cells, dried bacterial cells, bacterial cell polishing) crushed materials, autolysed bacterial cells, ultrasonicated bacterial cells, and immobilized bacterial cells using, for example, acrylamide gel method or carrageenan gel method)
etc. can be suitably used. The enzyme reaction of the present invention can be carried out, for example, by allowing a culture solution of the above-mentioned microorganisms, microbial cells collected from the culture solution by centrifugation, or a treated product of the microbial cells to react with cinnamic acid and ammonia or an ammonia donor. I can do it. Examples of ammonia or an ammonia donor used as one of the raw materials in the enzyme reaction include aqueous ammonia and organic or inorganic ammonium salts such as ammonium acetate, ammonium sulfate, ammonium chloride, and the like. It is necessary to add these ammonia or ammonia donors to the enzyme reaction solution so that the ammonium ion concentration is at least 3 molar.
It becomes possible to convert to L-phenylalanine at a high conversion rate of % or more. When the ammonium ion concentration is below 3 molar concentration, as shown in Experimental Example 2,
Cinnamic acid cannot be converted to L-phenylalanine at a high conversion rate. The other raw material, cinnamic acid, is added to the reaction solution at a concentration of about 0.05 to 0.2 molar (0.75 to 3%).
Avoid substrate inhibition of L-phenylalanine ammonia lyase activity as much as possible in the reaction system,
It is also necessary to accumulate L-phenylalanine at a practical concentration or higher. If the cinnamic acid concentration in the reaction solution is 0.2 molar or higher, as shown in Experimental Example 1 below, the enzyme will be inhibited by the substrate and its activity will be significantly reduced, so the reaction will hardly proceed or will not proceed at all. However, the speed decreases drastically, and
At concentrations below 0.05 molar, the enzyme activity is not inhibited by the substrate at all, but since the amount of cinnamic acid charged is small, even if the conversion rate of cinnamic acid to L-phenylalanine is high, L-phenyl The accumulated amount of alanine itself is too small to be obtained at a practical concentration, and in any of these cases, a decrease in reaction efficiency cannot be avoided. As described above, the ammonium ion concentration and the cinnamic acid concentration in the reaction solution are independently determined by the L-
Although they are largely involved in the conversion to phenylalanine, the preferred concentrations of ammonium ions and cinnamic acid in the reaction solution are within the above-mentioned practicable range, the conversion rate of cinnamic acid to L-phenylalanine, and the accumulation of L-phenylalanine. It may be determined experimentally by considering the concentration of L-phenylalanine, the isolation procedure of L-phenylalanine, etc. For example, in the case of Sporobolomyces roseus 221 (Fiber Science and Technology Research Institute No. 3853) and Rhodosporidium toruloides IF00559, which are shown in Experimental Examples 1 and 2 below, the ammonium ion concentration is approximately 8. It is preferable that the concentration of cinnamic acid is 0.15 to 0.2 molar, and in this case, L-phenylalanine is added to the reaction solution at a concentration of 20 molar.
Significant amounts of ~25 mg/ml can be accumulated. The enzymatic reaction of the present invention is typically carried out in an aqueous medium at a temperature of 20°C.
℃~60℃, preferably about 30℃~40℃, PH about 9~
Preferably, the test is carried out under 10 conditions. The reaction time varies depending on the method of standing, stirring, flowing down, etc., or the form and capacity of the enzyme, but it is usually about 1 to 72 hours in the batch method.
In addition, when using live bacterial cells as an enzyme source in this reaction, the reaction time may be significantly shortened by adding a surfactant. Separation and purification of L-phenylalanine produced and accumulated in the reaction solution can be easily carried out using a combination of conventional ion exchange resins and other known methods. According to the method of the present invention, the conversion reaction of cinnamic acid to L-phenylalanine can be carried out at a conversion rate of 50% or more to 80%, so the amount of cinnamic acid used as a raw material substrate can be reduced and L-phenylalanine can be converted into L-phenylalanine. Phenylalanine can be produced in high yield, and L-phenylalanine produced in the reaction solution can be isolated and obtained very easily, so the method of the present invention is more economical than the British patented method. This is a much more advantageous method for producing L-phenylalanine. Hereinafter, the method of the present invention will be explained in more detail with reference to experimental examples and examples. - It was carried out by bioassay method using Mesenteroides P-60. Experimental example 1 (Influence of cinnamic acid concentration on enzyme reaction) Cinnamic acid was changed as shown in Table 1, each of the cinnamic acids was dissolved in 55 ml of 28% ammonia water (commercially available), and an appropriate amount of hydrochloric acid was added to adjust the pH. After adjusting the amount, add distilled water to make a total volume of 80ml. To this, add 20 ml of bacterial cell suspension of Sporobolomyces roseus 221 (Feikoken Bibori No. 3853) or Rhodosporidium toruloides IF00559 as an enzyme preparation, and add 100 mL of bacterial cell suspension.
ml, a conversion reaction was carried out at 30°C, and the initial velocity was measured. The results are shown in Table 1, and it was found that the concentration of cinnamic acid was significantly involved in the enzyme activity of the conversion reaction of cinnamic acid to L-phenylalanine.

[Table] However, for the enzyme activity, the activity in the case of cinnamic acid 50mM was set as 100. In addition, S and R in the table represent the following. Same hereafter. S: Cell of Sporobolomyces roseus R: Cell of Rhodosporidium toruloides Experimental Example 2 (Effect of ammonium ion concentration on enzyme reaction) Add 1000 mg or 3000 mg of cinnamic acid to 28% ammonia water (commercially available). After adjusting the pH to 10.0 with hydrochloric acid, add distilled water to make a total volume of 80 ml. Add 20ml of the same bacterial cell suspension as in Experimental Example 1 to this.
The volume was adjusted to 100 ml, and the reaction was carried out at 30°C for 18 hours. After the reaction, the conversion rate of cinnamic acid to L-phenylalanine was calculated, and the influence of ammonium ion concentration on the conversion rate was investigated. The results are shown in Table 2, and it was found that the ammonium ion concentration was significantly involved in the conversion of cinnamic acid to L-phenylalanine.

[Table] However, A and B in the table represent the following. A: 1000 mg/100 ml of cinnamic acid B: 3000 mg/100 ml of cinnamic acid Example 1 (1) Put 100 ml of the culture medium (PH6.5) with the following composition into a 500 ml Sakaguchi flask, and add Sporobolomyces roseus 221 (Fiber Technology Research Institute) to a 500 ml Sakaguchi flask. Inoculate one platinum loop of Bacterium No. 3853). Incubate at 25℃ for 40 hours. After culturing, centrifuge 100ml of the culture solution and collect Sporobolomyces roseus 221 cells. Medium composition (in 100ml) Peptone 0.5g Yeast extract 2.5g Salt 0.5g (2) 3000g cinnamic acid and 28% ammonia water (commercially available)
After dissolving in 55 ml and adjusting the pH to 10.0 with hydrochloric acid, suspend the Sporobolomyces roseus 221 cells obtained in (1) above. Add water to this cell suspension to make a total volume of 100 ml, and react at 30°C for 18 hours. After the reaction, 80% of the cinnamic acid in the reaction solution is L.
- Converted to phenylalanine, approximately 2500 mg of L
- Accumulation of phenylalanine was observed. The bacterial cells were removed from this reaction solution by centrifugation, and the supernatant solution was adjusted to pH 2.0 with hydrochloric acid and cinnamic acid was removed by filtration. The filtrate was washed with ether to remove remaining cinnamic acid, and the supernatant was concentrated to a pH of 5.5.
to precipitate L-phenylalanine. 2325 mg (yield 75%) of L-phenylalanine was obtained. Example 2 (1) Put 100 ml of a medium with the same composition as in Example 1 into a 500 ml Sakaguchi flask, and add 1 portion of Sporobolomyces roseus 227 (Feikoken Bacterial Serial No. 3852) to it.
Inoculate with platinum ear. Thereafter, Sporobolomyces roseus 227 cells were collected by the same treatment as in Example 1. (2) 2220 mg of cinnamic acid in 28% ammonia water (commercially available)
Sporobolomyces roseus obtained in (1) above after dissolving in 55 ml and adjusting the pH to 10.0 with hydrochloric acid.
Suspends 227 bacterial cells. Add water to this cell suspension to make a total volume of 100 ml, and react at 30°C for 24 hours. After the reaction, 80% of the cinnamic acid in the reaction solution is L-
Converted to phenylalanine, approximately 2040mg of L-
Accumulation of phenylalanine was observed. Example 3 (1) Put 100 ml of a medium (PH6.5) with the following composition into a 500 ml Sakaguchi flask, and inoculate one platinum loop of Rhodosporidium toruloides IF00559. At 30℃
After incubating for 16 hours, the cells of Rhodosporidium toruloides are collected by centrifugation. Medium composition (in 100ml) Peptone 3g Yeast extract 0.5g Salt 0.5g (2) 2220mg of cinnamic acid in 28% ammonia water (commercially available)
After dissolving in 55ml and adjusting the pH to 10.0 with hydrochloric acid,
Suspend the Rhodosporidium toruloides cells obtained in (1) above. When the reaction solution was treated in the same manner as in Example 2, it was found that 80% of the cinnamic acid in the reaction solution was converted to L-phenylalanine, and about 2040 mg of L-phenylalanine was accumulated.

Claims (1)

[Claims] 1. Cinnamic acid and ammonia or an ammonia donor in the presence of a culture solution of a microorganism containing L-phenylalanine ammonia-lyase, or a microbial cell collected from the culture solution, or a treated product of the microbial cell. When producing L-phenylalanine by enzymatic reaction, the enzymatic reaction is carried out under conditions where the concentration of ammonia or an ammonia donor is at least 3 molar or more, and the cinnamic acid concentration is 0.05 to 0.2 molar. A method for producing L-phenylalanine using an enzymatic method, characterized by: 2. The production method according to claim 1, wherein the L-phenylalanine ammonia-lyase is L-phenylalanine ammonia-lyase produced by a microorganism of the genus Sporobolomyces or the genus Rhodotorula. 3. The production method according to claim 2, which uses a culture solution of a microorganism of the genus Sporobolomyces or Rhodotorula, or cells collected from the culture solution, or a processed product of the cells as a source of L-phenylalanine ammonia-lyase. . 4. The production method according to claim 2, wherein the microorganism of the genus Sporobolomyces is Sporobolomyces roseus 221 (Feikokenbokuyori No. 3853). 5. The production method according to claim 2, wherein the microorganism of the genus Sporobolomyces is Sporobolomyces roseus 227 (Feikokenbokuyori No. 3852). 6. The production method according to claim 3, wherein the Rhodotorula microorganism is Rhodotorula glutinis IF00599. 7. Claim 2, wherein the concentration of ammonia or ammonia donor is about 8 to 10 molar.
3. The manufacturing method described in Section 3, Section 3, Section 4, Section 5, or Section 6. 8 Claims 2, 3, 4, and 5, in which the cinnamic acid concentration is approximately 0.15 to 0.20 molar.
The manufacturing method according to item 6 or 7.