JPS60234591A - Production of l-phenylalanine - Google Patents

Abstract

PURPOSE:To obtain L-phenylalanine economically, by carrying out the enzymatic reaction of cinnamic acid with NH3 or an NH3 donor in the presence of an L- phenylalanine ammonia lyase produced by microorganisms and a surfactant, thereby enabling the reaction in a region of high cinnamic acid concentration which is advantageous for industrial practice. CONSTITUTION:The enzymatic reaction of cinnamic acid with NH3 or an NH3 donor is carried out in the presence of an L-phenylalanine ammonia lyase and a surfactant. The enzymatic activity can be improved remarkably at a cinnamic acid concentration of about 50-500m-mol, especially 220-300m-mol. The surfactant is e.g. a sorbitan alkyl ester nonionic surfactant. The enzymatic reaction of the present invention is carried out preferably at 20-60 deg.C and about 8.5-10.0pH. The L-phenylalanine produced in the reaction liquid can be recovered easily by a combination of conventional ion exchange resin treatment with other conventional separation techniques.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing L-phenylalanine by an enzymatic method.

According to the method of the present invention, the enzyme reaction can be carried out in an industrially advantageous high cinnamic acid concentration range.

- Phenylalanine, one of the essential amino acids,
It is an important substance used in medicine, etc.

Prior Art A method for producing L-phenylalanine by reacting cinnamic acid with ammonia or an ammonia donor using an enzyme produced by a microorganism is disclosed in British Patent No. 1,489,468.
No. specification, JP 53-96388, JP 56-2619
7 publications and the like.

However, in these known methods, reaction conditions with a high concentration of ammonium ions are specifically applied, and especially when considering industrial production, under reaction conditions with a high concentration of cinnamic acid, it becomes a reaction catalyst. There was a problem in that the enzyme L-phenylalanine ammonia-lyase was inhibited by the substrate by cinnamic acid, resulting in a rapid decrease in enzyme activity.

As a result, when a surfactant is added to the reaction system,
The present invention was completed by discovering that substrate inhibition by cinnamic acid is reduced and the activity of L-phenylalanine ammonia-lyase is significantly increased under conditions of high cinnamic acid concentration.

This effect of the present invention is remarkable because the enzyme source used in this reaction is not only a live bacterial cell, but also a crude enzyme, that is, an enzyme system in which the living bacterial cell is crushed and solubilized, and is a new finding that is completely different from known techniques. However, it cannot be easily achieved using known techniques.

SUMMARY OF THE INVENTION The present invention provides a method for producing L-phenylalanine by enzymatically reacting cinnamic acid and ammonia or an ammonia donor in the presence of L-phenylalanine ammonia lyase produced by a microorganism. The present invention provides a method for producing L-phenylalanine, which is characterized in that it is carried out in the presence of.

3. Detailed Description of the Invention The microorganism used in the method of the present invention produces l'i, 4 as phenylalanine ammonia lyse.
It is an enzyme that generates I-phenylalanine from skin RT and ammonia donor (I)-phenylalanine.
For example, culture fluids of microorganisms such as Rhodotorula glutinis (IFO (15591), Trichosporon, Cutaneum (IF'01198), Rhodotorula rubra (I F (') u 592), For example, bacterial cells collected from a culture solution by centrifugation or the like, treated bacterial cells that have been subjected to a crushing process such as ultrasonic disruption, freeze-dried bacterial cells, toluene-treated bacterial cells, and the bacterial cells and crushed cells. A is possible by using immobilized substances.

The enzyme reaction of the present invention is carried out, for example, by allowing a culture solution of the above microorganism, cells collected from the culture solution, or treated cells to react with cinnamic acid and ammonia or an ammonia donor in the presence of a surfactant. In this case, improvement in enzyme activity is observed when the cinnamic acid concentration ranges from about 50 to 5 mmol, preferably from 100 to 350 mmol, particularly preferably from 2211 to 3 mmol.
Enzyme activity is significantly improved in the range of 0.00 mmol.

The surfactant used is a sorbitan alkyl ester type nonionic surfactant, such as 'l'Ween.
4 U J Arike [Tween 60 J (Kao Atlas Co., Ltd. #1 etc., alkyltrimethylammonium salt type cationic surfactant, e.g. [cation PB-3
00J, alkylpyridinium salt type cationic surfactants such as "cation BBJ (NOF Corporation), cetyltrimethylammonium bromide", carboxylic acid type amphoteric surfactants such as "cetylpyridinium chloride",
For example, "Ryonon 13FJ (manufactured by Erbon Yushi) etc."
A combination of p4 or higher can be used. The concentration of the surfactant used varies depending on the type of surfactant, the *i of cinnamic acid, etc., but is generally 0.0 (14 (v/vl) or more, preferably o, o o o 4 (v/vl) to 20 (
Used in v/vl concentrations.

To give a specific example, when the surfactant is a sorbitan alkyl ester type nonionic surfactant φ, its concentration is 1. s (v/v) 1 or more, especially 0.5 (v/vl~
15 (VlVl ratio is preferred, and when the surfactant is an alkyltrimethylammonium salt type cationic surfactant, the concentration is tl, 01) 4 (v/v 14 or more -E, especially +1.1) l) 4(v/vl% ~2
(v/vl) is preferable, and when the surfactant is an alkylpyridinium salt type cationic surfactant, the concentration is (1.04 (v/vl) or higher, especially 0.04
(v/v) ratio is preferably ~2 (v/v)%, and when the surfactant is a carboxylic acid type amphoteric surfactant,
The *i is l), 004 (v/v) 4 or more, particularly preferably 0.0 (14 (%) to 2 (v/v)).

As one raw material ammonia or ammonia donor used in this enzyme reaction, in addition to ammonia double water,
Examples include ammonium salts such as ammonium chloride, ammonium sulfate and ammonium acetate. Generally, it is necessary to add ammonium ion so that the concentration is at least 3 molar.

The enzyme reaction in the present invention is usually carried out at a temperature of 20°C to 60°C.
, preferably about 30-45°C, pH about 8.5-10.
A condition of 0 is preferred. The reaction time varies depending on the strength of stirring, standing conditions, etc., and the form and amount of enzyme, but it is usually 3 to 30 minutes.
It takes about 90 hours.

Recovery of monophenylalanine produced in the reaction solution is as follows:
This can be easily carried out by combining ordinary ion exchange resins and other known methods.

EXPERIMENTAL EXAMPLE The present invention will be specifically described in the following experimental examples and examples. The qualitative and constant t of L-phenylalanine was determined using the ninhydrin coloring method using paper chromatography, an automatic amino acid analyzer, and Leuconostoc mesenteroides P-60.
The microbial quantification method was used.

Example 1 Polypeptone: 10f, yeast extract: 1 (1'.

NaCt: st, tap water: IL10117, PH6,
Contains 100- of a medium consisting of u! The mixture was dispensed into 501 Jm Erlenmeyer flasks and sterilized at 120°C for 15 minutes.

Oo 5591 was inoculated and cultured at 30°C for 24 hours. Fifty tons of this culture solution was inoculated into 1 ton of the above medium in a 5 ton Erlenmeyer flask, and incubated at 3 ()°C and pH 6.0 for 18 hours.
000 rpm, 15 minutes, 4°C), and the bacterial cells were collected.

After washing the cells once with 100mM + 77 acid buffer (pH 7,0) and suspending them in 1- of the same buffer, the cells were disrupted using an ultrasonic disrupter (BRANSON 200 TYPEI), and the treated product was Centrifugation (15U OU rpm,
(40ml, 0°C) and drain the supernatant as a crude enzyme solution.

The reaction solution was prepared by changing cinnamic acid as shown in Table 1,
The cinnamic acid is 28% ammonia water (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) 3.7
After adding the surfactant shown in Table 1, the pH was adjusted to 10.0 with hydrochloric acid, and water was added to make the total volume 10 ml.

For the reaction, the above crude enzyme solution (), l- was added to the reaction solution u, 9tnl.
was added to the solution, and then reacted at 30°C for 1 hour.

The reaction results are shown in Table 1 as a relative activity with the activity in the absence of surfactant (control) at a cinnamic acid concentration of 2 omM as 100.

Example 2 Cinnamic acid was dissolved in 3.3-liter ammonia water (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) at a concentration shown in Table 2, and 5.0 (v/vl) of each surfactant shown in Table 2 was added. After that, the pH was adjusted to 10.0 with hydrochloric acid, and water was added to make the total 98-O
For the 6° reaction with the reaction solution of d, 2.0 d of the crude enzyme solution prepared in the same manner as in Example 1 was added to 8.0 d of the reaction solution described above, and the mixture was reacted at 30° C. for 200 hours. After completion of the reaction, the amount of phenylalanine produced in the reaction solution was 9 as shown in Table 2.

When the L-phenylalanine produced in the reaction system of IfzoomM was precipitated and recovered, 35 μm of L-phenylalanine crystals were obtained from each reaction system to which a surfactant was added.

(Margin below) Table 2 Example 3 Rhodotorula glutinis (IF00559) was cultured in 1 t of a medium having the same composition as in Example 1 under the same culture conditions as in Example 1.

To prepare the reaction solution, change the cinnamic acid as shown in Table 3, and add the cinnamic acid to 2.8 r of aqueous ammonia (manufactured by Wako Tsumugi).
After dissolving in z/ and further adding the surfactant shown in Table 3,
After adjusting the pH to 10.01C with hydrochloric acid, add water to make a total of 5RI! However, it is used as the core liquid.

The reaction was carried out by centrifugation (SO
00 rpm% for 15 minutes at 4°C), the obtained bacterial cells were suspended in reaction solution No. 54, and then reacted at 30°C for 1 hour. L-
Phenylalanine ammonia lyase activity was measured in the same manner as in Example 1. The results are shown in Table 3, with cinnamic acid concentration somM
The relative activity is expressed as 100 relative to the activity in C control (without surfactant added).

(Left below) Example 4 Cinnamic acid was dissolved in 50 d of aqueous ammonia of Group 28 (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) at the concentrations shown in Table 4, and surfactants shown in Table 4 were dissolved either alone or in combination (mixing ratio 1:1). ) and 10 each
(add v/v 14 then p with hydrochloric acid)I to 10.0
After preparing the solution, water is added to make a total volume of 100 d of reaction solution.

The reaction was carried out in the same manner as in Example 1, and the cells obtained by centrifugation (SOOORPM,
The reaction was carried out for 20 hours. The amount of phenylalanine produced in the reaction solution after the completion of the reaction was as shown in Table 4.

When I,-phenylalanine produced in a reaction system containing 200 mM cinnamic acid was precipitated and collected, it was found that 98
0) η, Id 99 in a system with a combination of surfactants added
Crystals of L-phenylalanine of o trq were obtained respectively.

In addition, when L-phenylalanine produced in a reaction system with a cinnamic acid concentration of 330 mM was precipitated and collected, it was found that 1
400 ■, 148 in the system added in combination with surfactant
0.0 cm of Noshiichi phenylalanine crystals were obtained.

Table 4

Claims (1)

[Claims] (1) A method for producing phenylalanine by enzymatically reacting cinnamic acid and ammonia or an ammonia donor in the presence of L-phenylalanine ammonia lyase produced by a microorganism, in which the reaction is carried out in the presence of a surfactant. A method for producing L-phenylalanine, characterized by the following.