JPS61265096A - Production of l-malic acid - Google Patents

Abstract

PURPOSE:To obtain in high yield L-malic acid, by reacting fumaric acid (salt) in a solvent of water by the use of a culture mixture obtained by cultivating a specific bacterium aerobically or a treated material of it. CONSTITUTION:Fumaric acid or its salt is reacted in a solvent of water in the presence of a culture mixture obtained by cultivating aerobically a bacterium belonging to the genus Brevibacterium, having resistance to alpha-aminobutyric acid, especially a bacterium belonging to the genus Brevibacterium having positively provided resistance to alpha-aminobutyric acid and/or raised resistance to alpha-aminobutyric acid or of a treated material of it, to form L-malic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field The present invention relates to a method for producing L-IJ malic acid.

According to the method of the present invention, L-malic acid can be produced in high yield.

L-malic acid is currently used for medical purposes, and is also expected to be used for food purposes in the future.

Prior art ■ - As a method for producing malic acid, there is a fermentation method (for example, Japanese Patent Publication No. 41-16547 ), and enzymatic methods for producing L-malic acid from fumaric acid or its salts using the enzyme fumarase in microorganisms having fumarase activity [e.g., The JournFLtof GenerFLtan
d Apgied Microbio/igy, ヱ, 1
08-116 (1960), European Jou
rnatofApptiedMj crobiol
ogyX3.169-183 (1976), etc.] are known.

However, in the production of L-malic acid by fermentation method,
It requires large-scale fermentation equipment, and the production yield of L-'J malic acid is extremely low, resulting in problems such as high production.

The known enzymatic method for producing monomalic acid uses microbial fumarase to obtain monomalic acid from fumaric acid. Therefore, it is important to obtain microorganisms with strong fumarase activity for industrial production. Although this has become an important element, isolating microorganisms that meet these purposes from the natural world has been accompanied by many difficulties and has been an obstacle to industrialization.

SUMMARY OF THE INVENTION The present inventors conducted intensive studies to solve the above problems and completed the present invention.

That is, the present invention provides fumaric acid or a salt thereof in the presence of a culture obtained by aerobically cultivating a microorganism belonging to the genus Prepibacterium and having resistance to α-aminobutyric acid, or a processed product thereof, in an aqueous solvent. The present invention provides a method for producing monomalic acid, which is characterized in that L-malic acid is produced by reacting with L-malic acid.

The microorganism used in the method of the present invention belongs to the genus Prepibacterium and is resistant to α-aminobutyric acid. This microorganism is not limited to a microorganism that has been artificially affected with the resistance mechanism, but may be a microorganism that has acquired the resistance mechanism through accidental mutation in nature. Examples of such microorganisms include, for example,
Prepibacterium disclosed in Publication No. 6755
There are Flavum MJ-233 (Feikoken Bibori No. 3068), etc.

The microorganism having α-aminobutyric acid resistance used in the method of the present invention may be the above-mentioned naturally obtained microorganism, but preferably belongs to the genus Prepibacterium and is actively resistant to α-aminobutyric acid. Microorganisms with endogenous and/or enriched microorganisms are used. An example of such a microorganism is Prepibacterium flavum MJ-, which is disclosed in Japanese Patent Publication No. 59-28398.
233-AB-41 (Feikoken Bibori No. 3812), etc.

The above-mentioned microorganism belonging to the genus Prepibacterium and having α-aminobutyric acid resistance actively imparted and/or increased, which is preferably used in the method of the present invention, belongs to the genus Prepibacterium and has α-aminobutyric acid resistance. A microorganism resistant to aminobutyric acid can be induced by a known method, for example, by the following procedure. That is, for example, Prepibacterium flavum M is treated by ultraviolet irradiation or chemical agent treatment (for example, N-methyl-N'-nitro-N-nitrosoguanidine, etc.).
After inducing mutations in J-233, this bacterial suspension was
-Amino-n-butyric acid 1011 Jf/me gold-containing plate medium (urea 0.2%, ammonium sulfate 0.7%, K2HO4o,
05%, K2HPO40.05%, MgSO4・7H2
00,05%, Nact2W/L, Cact, +・
2H20219/l, FeSO4・7H202m! /
L, Mn504m4~6T (zoXZn SO2・7
H202"t/tN biotin 200μy7t, thiamine hydrochloride 100μy/l, α-amino-n-butyric acid 1.0%, agar 2.0%, ethanol 3% by volume [added after sterilization]) at 30°C. A resistant mutant strain can be obtained by culturing for days and separating the resulting large colonies.

Here, the α-aminobutyric acid resistance of the microorganism used in the present invention is defined as the relative growth rate when 2% α-aminobutyric acid is added to the medium (Note-1) and the culture is incubated at 30°C with shaking for 3 days. and the relative growth rate of microorganisms that have been actively conferred and/or enhanced with α-aminobutyric acid resistance is 15
It is preferably 30 or more.

Note that the relative growth rate is expressed by the 11th-order equation.

(In the above formula, α-ABV! is an abbreviation for DL-α-aminobutyric acid) (Note-1) Composition of the medium used and culture method Urea 0.2%, ammonium sulfate 0.7%, KH2PO40.05%
, K2HPO40,05%, MgSαC7H2O0,0
5%, yeast extract 0.01%, casamino acid 0.01%,
FeSO4・7HzO24/t, MnSO4・4~
6HzO2”%F/l, NaC12q/l, Ca
Cjzφ2Hz0 2m1j'/L, ZnSO4・
7H202”f/ Lz Bichiy!r>200p?/
A medium (D
For L-α-aminobutyric acid, add the ingredients shown in Table 1) 10d
Dispense into large test tubes with a diameter of 24 mm, sterilize at 120°C for 10 minutes, and remove microorganisms such as Prepibacterium flavum.
Each MJ-233-AB-41 strain was inoculated, 0.3 d (3% each) of ethanol was added under aseptic conditions, and 3
Shaking culture was performed at 0°C for 3 days.

(Note-2) Growth rate 0. Da 10% Relative Viability Viability 0. Ds+o indicates absorbance at the immediate wavelength of 61O, and this absorbance was measured according to Experimental Agricultural Chemistry, Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Volume 1, p. 212, Asakura Shoten (1975). In addition, the growth rate when DL-α-aminobutyric acid was not added was 0.
．． D61G is set as 100 and expressed as relative growth rate.

Prepibacterium flavum MJ-2a3 and Prepibacterium flavum M measured according to the above definitions
The relative growth rate of J-233-AB-41 to DL-α-aminobutyric acid was as shown in Table 1.

(The following are blank spaces) Table 1 The above-mentioned microorganisms belonging to the genus Prepibacterium and having resistance to α-aminobutyric acid are cultured aerobically.

The composition of the medium, including carbon sources, nitrogen sources, inorganic salts, etc., used in the culture is not particularly limited. For example, carbon sources include ethanol, methanol, n-paraffin, molasses, etc., and nitrogen sources include ammonia, molasses, etc. ammonium sulfate,
Ammonium chloride, ammonium nitrate, urea, etc. are used, and as the inorganic salt, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, etc. are used. These carbon sources, nitrogen sources and inorganic salts can be used alone or in combination.

Furthermore, in addition to these nutrients, nutrients such as hefton, meat extract, yeast extract, cornstarch, casamino acid, and various vitamins can be added to the medium if necessary for the growth of the bacteria.

The culture is carried out under aerobic conditions such as aeration, stirring, and shaking, and the culture temperature is 20 to 40°C, preferably 25 to 35°C. The pH during the cultivation is kept at around 5-101, preferably around 7-8, and the pH in the culture solution is adjusted by adding acid or alkali.

The concentration of the carbon source, such as ethanol, at the start of the culture is suitably 1 to 5% by volume, preferably 2 to 3% by volume.

The culture period is 2 to 8 days, preferably 4 to 5 days.

The culture thus obtained contains highly active fumarase, so fumaric acid or its sodium salt,
Using fumaric acid such as calcium salt as a raw material, L-malic acid can be produced in good yield by enzymatic reaction.

Here, the culture obtained as described above or its treated product is used for the enzyme reaction, but these include the culture solution itself, the bacterial cells contained therein, and the bacterial cells contained therein. It includes everything obtained by culturing, such as destroyed matter, ground matter, and autolyzed fluid, and also includes immobilized bacterial cells, disrupted bacterial bodies, ground matter, and the like.

The enzyme reaction in the present invention is carried out by reacting fumaric acid or its salt in an aqueous solvent in the presence of the culture or its treated product.

The enzyme reaction is carried out in an aqueous medium at a pH of 4 to 10 and a reaction temperature of approximately 15
to about 60°C, preferably about 20 to about 50°C, typically 0
．． It is carried out for 5 to about 48 hours.

This enzymatic reaction is carried out in an aqueous solvent, but in addition to water, a solvent such as phosphate buffer or Tris-HCl buffer is added at 10 to 500 m
M (hardness) can also be used.Also, in order to adjust the pH of the reaction solution to 4 to 10, alkalis such as sodium hydroxide, potassium hydroxide, and ammonium hydroxide, and inorganic acids such as hydrochloric acid and sulfuric acid are added. You can also do that.

There is no particular restriction on the number of fumaric acid or its salt used in the reaction, but it is generally appropriate to use it in the range of 0.5 to 30% (WVvol). Further, the amount of the culture or the processed material to be used is not particularly limited, but it can generally be used in the range of 0.5 to 10% (wt/vol).

Separation and scouring of monomalic acid produced in the reaction solution obtained by reacting fumaric acid and water using a culture or its processed product can be carried out by adsorption with ion exchange resin, activated carbon, etc.
This can be done by a known method such as desorption treatment.

Experimental Examples Examples 1 and 2 Urea4. Of, ammonium sulfate 14. Of, K1-1zPO40,
5t.

KzHPOa O, 5f, WIgF304・7H20
0.5f.

FeSO4 fist 7Hz06q, Mn5O4s4~6Hz
0 6gIf.

Yeast extract 1. Of, Casamino Acid 1.1llIF, Biotin 200μ? , thiamine hydrochloride 100 μv 1 tap water 1
10 d of a medium consisting of 1 was dispensed into large test tubes with a diameter of 24, sterilized under pressure at 120° C. for 1°, and 0.3 d of ethanol was added aseptically to prepare a preculture medium. In this medium, Prepibacterium flavum AMJ-233 (Example 2) and Prepibacterium flavum MJ-233-A were added.
B-41 (Example 1) was inoculated with 1 platinum Jflt each, and 3
Shaking culture was performed at 0°C for 2 days.

Next, a medium 100 having the same preculture medium composition as described in -1
m/m each into two square colbens of 500 t/volume, sterilized under pressure at 120°C for 10 minutes, and aseptically added ethanol 3- to each to prepare the main culture medium. 1.0 ml of the preculture solution of each of the two types of microorganisms was inoculated and cultured with shaking at 30° C. for 3 days.

In order to collect a constant number of bacterial cells from each culture obtained in this way, 0.0
The a1o value was adjusted to 10.0 with water, and bacteria were collected from each of the prepared solutions by centrifugation (4000 rpm for 15 minutes) from 100 m/min.

On the other hand, fumaric acid 10? was added to 70 d of water, and 5N-Na
After adjusting the pH to 6.0 with OH solution, the total pH was adjusted to 100 m/s with water.
After cooling, 5° of the mixture was dispensed into a 100 m Erlenmeyer flask. Such raw material solutions were prepared for 2 hours, and each of the aggregates described in F was added thereto, followed by shaking at 45° C. for 2 hours. After the reaction was completed, microbial cells were removed by centrifugation (4000 rpm, 15 minutes), and monomalic acid needles produced in the supernatant were measured using a high-performance liquid chromatograph. As a result, the parent strain Prepibacterium spp. When using a bacterial collection of Prepibacterium flavum MJ-233, 40 μ/tie (Example 2) was used. 233-AB
-41, the result was 82'If/d (Example 1).

In addition, 6N-HCtm solution was added to 50 liters of each reaction completed solution to adjust pi to about 2, and then a strong basic resin (Amberlite IRA-400J, R manufactured by Rohm and Haas) was added.
L-malic acid was adsorbed onto the resin. Next, the mixture was eluted with IN-ammonium carbonate and concentrated to precipitate crude crystals of L-malic acid. This was washed with acetone and dried. Prepibacterium
Reaction solution using Flavum MJ-233 bacterial collection 50m/
1.2f of Karake L-malic acid crystals were added, while 2. I got 6f.

Effects of the Invention Fumaric acid or its salt is produced by enzymatic reaction in an aqueous solvent.
17 In the method for producing malic acid, microorganisms of the genus Prepibacterium are used as the enzyme source, and microorganisms having α-aminobutyric acid resistance, especially those that have been actively imparted with α-aminobutyric acid resistance and/or α-aminobutyric acid It has become possible to produce LIJ malic acid at a high yield by using a culture obtained by aerobically cultivating a microorganism belonging to the genus Prepibacterium with increased resistance or a processed product thereof.

Patent applicant: Mitsubishi Yuka Co., Ltd. Agent: Patent Attorney Hidetoshi Furukawa Agent: Patent Attorney Masahisa Nagatani −14=

Claims (1)

[Claims] (1) Fumaric acid or its salt is reacted in an aqueous solvent in the presence of a culture obtained by aerobically cultivating a microorganism belonging to the genus Prepibacterium and having resistance to α-aminobutyric acid, or a processed product thereof. A method for producing L-malic acid, which comprises producing L-malic acid.