JPH03290194A - Production of d-isocitric acid by fermentation - Google Patents

Abstract

PURPOSE:To obtain d-isocitric acid useful as a food additive, etc., at a low cost in high yield by fermenting with a yeast in a medium containing fumaric acids or maleic acids. CONSTITUTION:The objective acid can be produced by using a medium containing preferably 0.01-0.3mol of fumaric acids or maleic acid and fermenting with a yeast (e.g. Candida lipolytica).

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for producing d-isocitric acid by fermentation.

d-isocitric acid is an organic acid useful as a pharmaceutical or food additive.

Conventional technology Conventionally, fermentation using yeast has been known as a method for producing d-isocitric acid [Journal of the Japanese Society of Agricultural Chemistry, 44
(11), 493-498 (1970), British Patent No. 1199700, French Patent No. 1596056].

For the purpose of obtaining mutant strains with high d-isocitric acid production, Cantheida lipolyteica (Candi
Attempts were made to induce a fluoroacetic acid-resistant mutant strain from A. da 1ipolytica), and a strain with increased activity of aconitase, which is an inhibition site for fluoroacetic acid, was obtained, but this strain was not a strain with an improved ratio of d-isocitric acid.

[Japan Agricultural Chemistry, 4B(10), 543548 5
49-554 (1974)].

Furthermore, in the production of d-isocitric acid by a fermentation method using yeast, a method of adding itaconic acid to improve the production ratio of d-isocitric acid [Japan Society of Agricultural Chemistry Abstracts, 348 (1976), special Kosho 56-391
Publication No. 94] Candida zeylanoides (Candida 1ey
A method of adding yellow blood salt in the production of d-isocitric acid by fermentation using lanoides [Journal of Fermentation Technology (J,
Ferment Technol), 52(8),
542-550 (1974)] is known.

Problems to be Solved by the Invention There is a need for a method for industrially producing 1-isocitric acid, which is useful as a pharmaceutical or food additive, more efficiently and at lower cost.

Means for Solving the Problems The present invention provides a method for producing d-isocitric acid, which is characterized in that fumaric acids or maleic acids are present in the medium in the production of d-isocitric acid by a fermentation method using yeast. do.

Here, fumaric acids refer to fumaric acid or its salts, or derivatives that can be easily converted to fumaric acid by hydrolysis, and maleic acids refer to maleic acid or its salts, or derivatives that can be easily converted to fumaric acid by hydrolysis. means a derivative that can be converted into

The yeast used in the present invention may be any strain as long as it has the ability to produce 1-isocitric acid. (d) There are many yeasts that have the ability to produce isocitrate, regardless of whether they are spore yeast or non-spore yeast. However, among them, the genus Candida and the genus Pichia.
, ) RuT) Torulopsis, SaccharomyceS, ZygosaccharomyceS
, Debaryomyces ,
Leucosporidium spp.
), Brettanomyces
), yeasts belonging to the genus Hansenula (order ansenul'a) are effectively used. Typical yeast strains include the strains shown below.

Candida lipolyteica (Candida 1ipo)
lytica) Candida seiranoides (c, z
eylanoides), Candida gearmandei (
C. guilliermondii), C. albicans, Candida albicans
Yeasts of the genus Candida, such as C. humicola, C. parapsilosis, and C. brumpti, Pichia farinot (Pic)
hia farinosa), Pichia ohmeri (p.
[51,) of the genus Pihy'r, such as Toruloρ5ISxyli,
nus), Torlopuns famata (T, fama
Yeasts of the genus Torulopuns such as ta), Thesocalomyces
cerevisiae (Saccharomyccs cerc)
Yeasts of the genus Duccharomyces, such as Zygosaccharomyces
Yeasts of the genus Chigosaccharomyces, such as S. romyces tikumaensis),
Yeasts of the genus Debaryomyces such as Debaryomyces hansenii, Leucosporidium cap
Yeasts of the genus Leucosporidium such as P. suligenum, Brettanomyces errambicus
yeasts of the genus Pletanomycetes, such as Yces ambicus;
Yeasts of the genus Hansenula such as Anomala).

Of course, any strain selected from mutant strains derived from these yeasts may also be used as long as it has the ability to produce d-isocitric acid.

The mutation may be caused naturally or by commonly used mutation processing methods such as ultraviolet irradiation or chemical treatments such as N-methyl-N'-21-Tl1-N nitrosocanidine treatment. It may also be something you have.

The production of d-isocitric acid according to the present invention can be carried out using conventional culture methods for producing organic acids, except that fumaric acids or maleic acids are present in the culture solution.

The medium to be used is selected as appropriate depending on the properties of the yeast used, but either synthetic or natural media can be used as long as it contains adequate amounts of carbon sources, nitrogen sources, inorganic substances, and other nutrients required by the strain used. It is possible.

Carbon sources include crucose, sucrose, molasses,
Carbohydrates such as starch hydrolysates, alcohols such as ethanol and chrycerol, organic acids such as acetic acid, fatty acids such as oleic acid, fats and oils such as soybean oil and fish oil, or paraffinic hydrocarbons such as butrabucane and hexadecane,
A wide range of other organic compounds that can be assimilated by yeast are used and can be used alone or in mixtures.

As a nitrogen source, ammonia or ammonium chloride,
Various inorganic and organic substances such as ammonium sulfate, ammonium carbonate, ammonium acetate, salts or urea and other nitrogen-containing substances and nitrogen-containing organic substances such as peptones, meat extracts, yeast extracts, corn steep liquor, casein hydrolysates, etc. Various types such as the following can be used. Depending on the yeast used, nitrates are also used as a nitrogen source. The various nitrogen sources mentioned above can be used alone or in combination of two or more.

Furthermore, as inorganic substances, potassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, ammonium chloride, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, and the like are used.

The vitamins and amino acid sources necessary for yeast growth are
It does not need to be added as long as it is supplied to the medium by other medium components as described above.

In carrying out the present invention, fumaric acids or maleic acids are added to the medium as described in 1.

The concentration of 7 malic acids or maleic acids in the medium ranges from 0.005 mol to 0.5 mol in terms of free fumaric acid and maleic acid.
It is used in moles, preferably from 0.01 to 0.3 moles. Even when fumaric acids or maleic acids are used in the form of their salts or derivatives that can be easily converted into fumaric acid or maleic acid by hydrolysis, it is necessary to provide the above concentration as free 7-maric acid or free maleic acid. The amount added can be adjusted accordingly. Furthermore, it may be added at any time during the entire culture period, but it is preferably added to the medium before the start of culture, or by the middle stage of culture.

Cultivation is performed under aerobic conditions such as shaking culture or aerated agitation culture. Although the culture temperature varies depending on the yeast used, 20 to 35°C is generally suitable. The pH of the medium is
It generally ranges from 2.5 to 10, preferably from 3 to 8.

The culture period is usually 2 to 8 days.

The method for collecting d-isocitric acid from the culture solution is carried out by a known method such as removing the bacterial cells and concentrating and crystallizing after the completion of the culture, treatment with activated carbon, or treatment with an ion exchange resin.

The present invention will be specifically described below with reference to Examples.

Example 1 Candida zeylanoides having the ability to produce d-isocitric acid
Using AT CC20347, agar slant medium (10 g of malt extract, 4 g of yeast extract, 4 g of glucose, 2 g of agar)
The bacterial cells obtained by culturing at 30°C for 36 hours in a medium containing 0 g in 1 fl of water and adjusted to pH 6.0 were cultured in a 40-
Composition Nigelcose 5%, NH, [:ffl 0.2
%, KH2PO40.05%, Mg5O+・7H2[]
0.05%, yeast extract 01%, corn steep liquor 0.1%, CaCO33.3%, PII5.5
) was inoculated into a 250 mff Erlenmeyer flask and heated at 30°C.
The cells were incubated for 24 hours with shaking on a 210mm rotary shaker.

This seed culture solution 4- was inoculated into a 250-capacity Erlenmeyer flask containing a fermentation medium with the following composition supplemented with fumaric acid at the concentration shown in Table 1, and cultured for 7 days under the same conditions as the seed culture. . At this time, fumaric acid is hydroxylated due to its low solubility)
After dissolving in IJum solution, it was added to the fermentation medium.

Composition of fermentation medium: n-paraffin (carbon number 12 to 15
Mixture mainly composed of Nippon Mining Co., Ltd.) 5%, NH, C
ff1 0.4%, KH2PO40.05%, MgSO
4.7H200.05%, ZnSOs'7H202mg
/ R, Mn5O+・4~68202 mg/fl,
Fe50<・7) IJ 5 mg/R1CuSO+・
5) 120150μg/R, biotin 100μg/
j2.

Zyamine hydrochloride 5 mg/R, CaCO33% (p
H5,5) After completion of the fermentation, the amount of d-isocitric acid produced, the amount of by-product citric acid produced, and the amount of remaining fumaric acid were determined by high performance liquid chromatography. In addition, since citric acid was precipitated as calcilyl salt, the precipitate was dissolved by adding 0.5N hydrochloric acid solution, and then the supernatant obtained by centrifugation was used as a sample.

The results are shown in Table 1.

Of these, d- obtained by adding 0.043M fumaric acid
The isocitric acid-containing culture solution 21 was centrifuged to remove the bacterial cells, and the clear solution was purified by Diaion 5KlllB (H” type) (
Decationization was performed using resin 0.51 (manufactured by Mitsubishi Kasei Corporation),
After adjusting the pH to 3.6 with IO normal potassium hydroxide solution,
It was decolorized by activated carbon treatment. When this was condensed under reduced pressure to about 100, d-isocitric acid monopotassium salt was precipitated. Its weight was 46g and its purity was 95%.

Furthermore, after dissolving 10g of the crystals in 250mf of water,
After decationization using 40 ml of Diaion S K'' I B (H+ type) (manufactured by Mitsubishi Kasei Corporation) resin and concentrating at room temperature, a paste-like preparation containing the calculated amount of d-isocitric acid was obtained. 8.35g was obtained in terms of pure product.This was dissolved again in 50°C of water, heated at 100°C for 30 minutes, and then dried to 11w6. tma 17i7.57g is 15
) was given.

Example 2 In the method of Example 1, maleic acid at the concentration shown in Table 2 was added to the fermentation medium instead of fumaric acid, and cultured for 7 days. The amount of citric acid produced and the remaining amount of maleic acid were determined.

The results are shown in Table 2.

Table 2 Among these, the one obtained by adding 0.043M of maleic acid (
j The isocitric acid-containing culture solution 21 was treated in the same manner as in the example, and d-isocitric acid monopotassium salt was precipitated. Its weight was 44g and its purity was 95%.

In addition, 250m1 of that l f + g item! After dissolving it in water, it was decationized using Resin 40me of Diaion SK"lB (H" type) (manufactured by Mitsubishi Kasei Corporation) and concentrated at room temperature, resulting in a paste containing the calculated amount of d-isocitric acid. The standard sample weighed 8.35 g (in terms of pure product).

This was dissolved again in 50°C of water, heated at 100°C for 30 minutes, and then concentrated to dryness. As a result, 7.57 g of d-isocitric acid was obtained as a lactone.

Effects of the Invention According to the present invention, d-isocitric acid can be fermented and produced with good yield and at low cost.

2

Claims (1)

[Claims] 1. A method for producing d-isocitric acid by a fermentation method using yeast, characterized in that fumaric acids or maleic acids are present in the medium.