JPS6143993A - Production of alpha-amino acid - Google Patents

Abstract

PURPOSE:To produce the titled compound economically in an industrial scale, preventing the inactivation of enzyme, by reacting a 2-ene-carboxylic acid with excess ammonia in water in the presence of an ammonia-lyase or a microbial cell containing the same, preventing the contact with oxygen. CONSTITUTION:The objective compound can be produced by reacting (A) a 2- ene-carboxylic acid with (B) excess ammonia, in water in the presence of (C) an ammonia-lyase catalyzing the reaction to produce an alpha-amino acid from the corresponding 2-ene-carboxylic acid and ammonia, or microbial cell containing said lyase, or treated cell, under the condition kept from the contact with oxygen or air. The enzyme source is e.g. immobilized cell or immobilized enzyme, and the combination of the 2-ene-carboxylic acid and the corresponding ammonia-lyase is e.g. cinnamic acid and phenylalanine ammonia lyase, trans-p- cumalic acid and tyrosine ammonialyase, etc.

Description

[Detailed description of the invention] Producing soil benefits The present invention relates to a method for producing α-amino acids using an enzymatic method.

It has been known to produce phenylalanine from cinnamic acid and ammonia using phenylalanine ammonia lyase (British Patent No. 1489468, Japanese Patent Application Laid-open No. 56-26197, etc.).

It is also known that an immobilization method is used to produce aspartic acid from fumaric acid and ammonia using aspartate ammonia lyase (=aspartase) (Japanese Patent Application Laid-Open No. 80160/1983).

Problems to be Solved by the Invention However, α-amino acids can be produced by reacting the 2-enecarboxylic acid and ammonia as described above in the presence of ammonia lyase, which catalyzes the reaction that produces the corresponding α-amino acids from both compounds. It has been found that during production, the enzyme is easily deactivated by ammonia, and in particular, the enzyme strongly promotes deactivation.

Steps for Solving the Problems The present invention is directed to carrying out the enzyme reaction under conditions that prevent contact with oxygen or air in order to avoid such deactivation of ammonia lyase due to oxygen. More specifically, the present invention provides two
- The 2-enecarboxylic acid and excess ammonia are dissolved in water in the presence of an ammonia lyase that catalyzes the reaction of producing the corresponding α-amino acid from the enecarboxylic acid and ammonia, or a bacterial cell containing the same, or a processed product thereof. , relates to a method for producing α-amino acids, characterized in that the reaction is carried out under conditions that prevent contact with oxygen or air.

Next, the present invention will be explained in more detail.

The reaction system (in water) is the condition where oxygen or air contact is prevented.
This can also be achieved by replacing the oxygen in the gaseous portion of the reaction vessel with an oxygen-free gas, such as air excluding oxygen, nitrogen, or an inert gas such as helium.

In this case, it is appropriate to carry out gas replacement by blowing an inert gas into the water until no oxygen is detected in the water or in the gas portion. Further, gas replacement may be performed only before the start of the enzyme reaction, or may be performed before and throughout the start of the enzyme reaction. Conditions that prevent oxygen or air contact are also
It can also be achieved by dissolving antioxidants in water. Alkali metal salts of sulfite (sodium salts, potassium salts, etc.) are used as antioxidants.

Alkaline earth metal salts (such as magnesium salts) or ammonium salts; SH group-containing compounds such as mercaptoethanol, glutathione, cysteine, hydrogen sulfide, and dithiothreitol; hydroquinones such as hydroquinone; phenols such as butyl cresol and catechol; ascorbin Reductone compounds such as acids can be used.

The appropriate concentration of the antioxidant used is 0.1mM to 10mM. Conditions that prevent oxygen or air contact are also
When using immobilized bacterial cells/enzyme as an enzyme source, this can also be achieved by mixing an antioxidant together with the bacterial cells or enzyme during the preparation of the immobilized bacterial cells/enzyme. Such antioxidants include tocopherol, 2.6-tert
-Butyl-4-methyl.

Phenol, trisnonylphenyl phosphite.

Many amine-based, quinone-based, phenol-based, sulfur-based, and phosphorus-based compounds can be used, such as triphenyl phosphite and distearyl-3,3'-monothiodipropionic acid. In addition to the above compounds, compounds listed in the section of antioxidants in the Applied Chemical Handbook, page 938 (published in 1980) can be used for the same purpose. These antioxidants are added in amounts of 1 to 1 per kg of carrier gel.
Use 0g.

For preparation, the antioxidant and bacterial cells or enzymes are mechanically well dispersed in a gelling solution to form an emulsion, and then fixed by a conventional method.

The above-mentioned conditions for preventing contact with oxygen or air may be used not only individually but also in combination, and the effect is further enhanced when used in combination.

Combinations of 2-enecarboxylic acids and corresponding ammonia lyases include cinnamic acid and phenylalanine ammonia lyase, trans-p-coumaric acid and tyrosine ammonia lyase, fumaric acid and aspartate ammonia lyase, mesaconic acid and methylaspartate ammonia lyase, and transcaffeinate. Acid, dihydroxyphenylalanine ammonia lyase, etc. are used.

The concentration of 2-enecarboxylic acid is 0.02 to 3.0M
is appropriate. In the case of cinnamic acid, in order to avoid inhibition of phenylalanine ammonia lyase activity by this, the
．． 02-062M is suitable. The appropriate amount of ammonia to be used is 1.5 to 200 times the molar amount of the 2-enecarboxylic acid.

In the present invention, a processed product of bacterial cells (e.g. washed bacterial cells, dried bacterial cells) containing ammonia-lyase as an enzyme source (
For example, bacterial cell grinding, bacterial autolysis, ultrasonication of bacterial cells, immobilized bacterial cells, enzymes encapsulated in ultrafiltration membranes)
Or the enzyme (crude enzyme).

Purified enzymes) or immobilized enzymes can be used.

Also, ethanol to improve the membrane permeability of bacterial cells.

Treatment with solvents such as toluene and ethyl ether (Japanese Patent Publication No. 58-19275), treatment with surfactants (Japanese Patent Application No. 58-1927)
151415) etc., and these solvents and surfactants may be added to the enzyme reaction system. The type and amount of surfactant used may be the same as in the case of the above-mentioned patent application. Immobilized bacterial cells and enzymes can be prepared by any of the gel entrapment methods, adsorption methods, covalent bond immobilization methods, and the like. For example, in the gel entrapment method, it is included in polyacrylamide gel, polyvinyl alcohol gel, agar gel, carrageenan gel, alginate gel, etc. by a conventional method (Japanese Patent Publication No. 56-29517.56
-19994 etc.). The larger the amount of enzyme source, the more advantageous it is to the reaction, and the enzyme activity varies depending on the type of enzyme, so the amount of enzyme source to be used cannot be determined unconditionally, but usually when using bacterial cells. , substrate 0.008 per unit bacterial cell 1)
~40i mat is suitable. Various microorganisms containing each of the above-mentioned ammonia lyases within their cells are known. For example, for aspartase, strains belonging to Escherichia coli, Citrobacter freundii ATCC6750
, Rhodotorula rubra ATCC 20258 and Sporobolomyces roseus IFO 1040 for phenylalanine ammonia-lyase, Rhodotorula rubra ATCC 20258 and Sporobolomyces roseus IFO 1040 for tyrosine ammonia-lyase, and dihydroxyphenyl, a strain belonging to Clostridium titanomorphum for methylaspartate ammonia-lyase. alanine Rhodotorula rubra IFO9 for ammonia lyase
01, Sporobolomyces roseus IF01037.
Sporobolomyces salmonicara-IF01038.
Pichia membranaefaciens IFO460, Vitia farinosa IF0465. Satucharomyces lipolytica IFO717, Vitia Guillermondi IF
Examples include 0961.

During the enzyme reaction, Mn'' and Co2'' are present in the reaction solution.

The presence of divalent metal ions such as Fe22Ca'' and Mg22ln'', particularly Mn'' and Zn'' can enhance the stability of the enzyme and further enhance the enzyme removal effect.

Specifically, MnC1 is added to the reaction solution before and during the initiation of the enzyme reaction.
*, MnSO4, CoCJ2. Fe5Oa.

CaCj! z, MgSO3, ZTISO4, ZnCRw
etc. are added. These metal compounds are 1μM to 50mM
, preferably in the range of 3 μM to 5 mM.

Although the enzyme source may be used batchwise or immobilized and used at 6° C., a fixed column method is usually preferred because of the advantages of continuous use.

The pH during the enzyme reaction is usually pH 8-11. Particularly held at 8-10. Since this reaction is carried out in excess ammonia, the pH
If adjustment is required, it is carried out by adding an inorganic acid such as hydrochloric acid or sulfuric acid, or an organic acid such as acetic acid or propionic acid. Although the reaction temperature varies depending on the enzyme used, etc., 15-45°C is usually appropriate.

A significant amount of α-amino acid is produced 1, usually 1100 hours after the start of the reaction. In the case of a column method, the time is even longer (e.g. 1
000 hours) continuous reaction is possible.

The α-amino acid was purified from the reaction solution using silica gel.

Column chromatography using Sephadex or the like, ion exchange resin such as Duolite A7, etc. can be used.

Next, examples of the present invention will be shown.

Example 1 Sporobolomyces roseus IFO1040 was mixed with 15 g of cornstarch liquor and 2.5 g of L-phenylalanine.
2 in 509 ml of medium containing 12.5 g of NaCl and
Shaking culture was carried out at 8°C for 17 hours. The cells obtained by centrifugation were washed with 0.9% (W/V) cold saline, and then centrifuged to obtain washed cells. This was suspended in a 3% (w/v) carrageenan aqueous solution 5Qm+ and dropped into a 2% (w/v) KCl aqueous solution to kill the immobilized bacterial cells.

Each 10 g of the immobilized bacterial cells was fractionated into four columns, and the culture medium shown in Table 1 was passed through each column at a rate of lil/hr. The results are shown in Table 2. The column was kept at 28°C.

Table 1 - Table 2 Example 2 0. Dotorla rubra ΔTCC20258 with yeast extract 1
．． Medium 10 containing 0 g, peptone 1.0 g, sodium chloride 0.5 g and L-phenylalanine 0.05 g
Shaking culture was carried out in 0ml at 28°C for 17 hours. The culture solution 50
0.9% (W/V) of bacterial cells obtained by centrifuging 0 ml
After washing with cold saline, centrifugation was performed to obtain washed bacterial cells. Add this to Anon LG (trade name of amphoteric active agent alkylglycine, manufactured by NOF Corporation) 2% (W/V) aqueous solution 201+
11 at room temperature for 30 minutes. This anon LG
Treated bacterial cells with 3% (W/V) kappa carrageenan solution 50
The cells were suspended in ml and added dropwise to a 2% (W/V) KCl aqueous solution to obtain immobilized bacterial cells.

10 g each of these immobilized bacterial cells were fractionated into four test tubes A to D. The substrate solutions shown in Table 3 were added to each of A-D,
The reaction was carried out at 28°C for 48 hours. The reaction solution was removed and the reaction was repeated using each substrate solution. Table 4 shows the results of analyzing the amount of phenylalanine produced in the reaction solution each time.

Table 3 Table 4 Example 3 25 g of N. coli cells were autolysed in a pH 7 buffer, and the filtered filtrate was buffered to pu 8.0. Suspend resin IQml, p
After adjusting to H8.0, the mixture was gently stirred for about 18 hours. Divide the resin into two columns.

B was filled. The substrate liquid shown in Table 5 was passed through each column at a rate of 5V-1 (passing capacity/resin capacity/hr).

Table 6 shows the amount of aspartic acid in the effluent over time. The column was kept at 40°C.

Table 6 Example 4 Each 11 immobilized cells were prepared in the same manner as in Example 1 except that L-phenylalanine in the medium was replaced with L-tyrosine.
1 g was fractionated into three columns A, B, and C, and passed through the media shown in Table 7 at a rate of 0.5 ml/hr, respectively. The results are shown in Table 8.

Example 5 40g of Anon LG treated orchard prepared in the same manner as in Example 2
Weigh it in a wet state and place 10g of each on the following method (A
-D). In A and B, each sample was mixed with 40 g of a 10% solution of polyvinyl alcohol (degree of saponification 99.5 mol%, degree of polymerization 1700) and frozen at -25°C. In C, trisnonylphenyl phosphite (dermal chemical Sumilizer TP) was added to 40 g of 10% (w/v) polyvinyl alcohol solution.
S) After 100 mg was well dispersed and mixed, it was mixed well with 10 g of bacterial cells in the same manner as A and B, and then frozen. 5 frozen items
It was cut into +nm square pieces, and each of A to C was packed into a column using 1001111. Example 2 was applied to columns B and C while keeping the column warm at 25°C. Add liquid D from Table 3 to the cam Δ at 25%.
While keeping the column warm at °C, solution A shown in the same table was passed through the column at a rate of 4 ml/hr.

Table 9 shows the composition of the distillate from each column.

Table 9 Effects of the Invention According to the present invention, enzyme activity can be sustained during the production of α-amino acids from 2-enecarboxylic acids using ammonia lyase.

Procedural amendment dated February 26, 1980 1, Indication of the case 1982 Patent Case No. 165998 2, Name of the invention Process for producing α-amino acids 3, Person making the amendment Relationship to the case Patent applicant postal code 100 Address: 6-1 Otemachi-chome, Chiyoda-ku, Tokyo Name
(102) Column 5 of the detailed description of the invention in the Kyowa Hakko Kogyo Co., Ltd. specification, content of amendments

Claims (6)

[Claims] (1) In the presence of ammonia lyase that catalyzes the reaction of producing the corresponding α-amino acid from 2-enecarboxylic acid and ammonia, or a bacterial cell containing this, or a processed product thereof, the 2-enecarboxylic acid and excess 1. A method for producing an α-amino acid, which comprises reacting with ammonia in water under conditions that prevent contact with oxygen or air. (2) The production method according to claim 1, wherein the conditions preventing contact with oxygen or air are achieved by replacing oxygen in the reaction system with a gas that does not contain oxygen. (3) The production method according to claim 1, wherein conditions preventing contact with oxygen or air are achieved by dissolving an antioxidant in water. (4) A patent in which the enzyme source used is immobilized bacterial cells or an immobilized enzyme, and the conditions that prevent contact with oxygen or air are achieved by incorporating an antioxidant into the immobilized bacterial cells or immobilized enzyme. The manufacturing method according to claim 1. (5) Combinations of 2-enecarboxylic acids and corresponding ammonia lyases include cinnamic acid and phenylalanine ammonia lyase, trans-p-coumaric acid and tyrosine ammonia lyase, fumaric acid and aspartate ammonia lyase, and mesaconic acid and methylaspartate ammonia lyase. , or transcaffeic acid and dihydroxyphenylalanine ammonia lyase. (6) The manufacturing method according to claim 1, wherein divalent metal ions are present in water.