JPS5953035B2 - Method for producing itaconic acid by fermentation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing itaconic acid by a fermentation method.

That is, yeast belonging to the genus Rhodotorula that has the ability to produce itaconic acid is inoculated into a nutrient medium containing a carbon source that can be assimilated by the yeast and cultured aerobically. This is a method for producing itaconic acid, which is characterized by collecting .

Up until now, the methods for producing itaconic acid using microorganisms have been as follows:
Methods using filamentous fungi Aspergillus itaconicus and Aspergillus terreus and carbohydrates as raw materials are known.

Furthermore, there has recently been a report that itaconic acid could be accumulated in the culture of Cantada sp., which is a yeast.

The present inventors have isolated a wide range of yeasts from the natural world that grow using glucose as the main carbon source, and investigated the itaconic acid producing ability of these yeasts.

As a result, they discovered that yeast belonging to the genus Lido1herula produces itaconic acid from glucose, and completed the present invention.

The bacterial strain used in the present invention, Rhodotorula sp.
Rhodotorula sp, ) 5Y-668 (
FERM-P No, 5318) and SY'-
718 (FER:M-PNo. 5319) is a strain that the present inventors isolated from a flower in 1979, and its mycological properties are as follows.

(a) Growth status in each medium (1) Growth status in MY liquid medium (a) The growth is slightly cloudy.

(b) Cells range from about 2.0 to 2.2×3.9 to 5.6μ to 2.9 to 4. IX4. IX7, 8-8.6μ elliptical to elongated shapes are distributed.

(c) Cell arrangement is independent.

) The mode of proliferation is multipolar budding.

(E) There is sediment.

(f) A film is formed.

(g) No gas is generated.

(2) Growth status on MY agar medium (b) Growth is good.

(b) Cells are the same as in MY liquefied medium.

(c) The periphery of the colony is gold-rimmed.

) The colony ridge is flat.

(e) The gloss of the colony is semi-gloss.

(f) The hardness of the colony is uniform.

(1-) Colony color is pearl pink.

(3) Slide culture on potato extract agar medium (a) Form pseudohyphae and true hyphae.

(b) Form blastospores.

(c) Theriospores do not form gazing connections.

(b> Formation of ascospores No spore formation was observed on Gorodkova medium, Crane medium, and vegetable juice agar medium.

(C) Formation of extruded spores Does not form extruded spores on MY agar plate culture.

(d) Physiological properties (1) Optimal growth conditions: pH 5.0-6.0 Temperature 30-
31℃ (2) Growth range: pH 3.0-7.5 Temperature 8-33
℃ (3) Nitrate assimilation: Yes.

(4) Fat decomposition: None.

(5) Urea decomposition: Yes.

(6) Liquefaction of gelatin: Yes.

(7) Osmotic: Does not grow on 8% (W/V) sodium chloride.

No growth at 40% glucose (W/V). (8) Carotenoid production: Yes.

As a result of measuring the absorption spectrum of the n-hexane extract of bacterial cells, there is a maximum absorption at 450 and 480 mμ.

(9) Significant production of organic acid: Itaconic acid is produced in a significant amount.

(10) Formation of starch-like substances: None.

01) Vitamin requirement: None.

α2) Other physiological characteristics (a) Formation of ester: None.

(b) Reaction in lidmus milk medium: turns blue but does not coagulate.

(c) Riboflavin production: None.

(e) Assimilation of each carbon source (1) D-arabinose: Yes.

(2) L-arabinose: Yes.

(3) D-ribose: Yes.

(4) D-xylose: Yes.

(5) D-glucose: Yes.

(6) D-galactose: Yes.

(7) L-rhamnose: None.

(8) L-sorbose: Yes.

(9) Maltose: Yes.

(io) Cane sugar: Yes.

01) Lactose: Yes.

(12) Melibiose: None.

(13) Cerbiose: Yes.

(God Trehalose: Yes.

(15)’ Raffinose: Yes.

(16) Melezitose: Yes.

0'7) α-Methyl-D-glucoside; present.

(18) Arbutin: Yes.

(1g) Soluble starch: Yes.

(20) Inulin: None.

(21) Ethanol: Yes.

c22) Erythrite: Yes.

(23) Inojit: Yes.

(24)・D-Mannit: Yes.

(25) D-sorbitol: Yes.

:26) Atonito: Yes.

n Zursit: None.

(28) Glycerin: Yes.

(29) Salicin: Yes.

(30) DL-sodium lactate: Yes.

(31) Sodium succinate: Yes.

(32) Sodium citrate: Yes.

(33) Sodium D-gluconate: Yes.

(34) Calcium 2keto-D-gluconate: Yes.

(f) Fermentability of each carbon source , (1) D-glucose: None.

(2) D-galactose: None.

(3) Maltose: None.

(4) Cane sugar: None.

(5) Lactose: None.

(6) Raffinose: None.

(7) Melibiose: None.

(8) Trehalose: None.

(g) Quinone type: Q10° (h) Mating test: Does not conjugate with any of the test bacterial strains.

The test strain is Rhodotorula sp. 5Y-
668 (FERM-P No, 5318) and 5Y
-718 (FERM-P No, 5319), Filobasidium capsurigenum
IFO1119 and 1185, Leucosporidium scottii
IFOO736, Rhodosporidium toruroides (
Rhodospridium toruloides)
IFOO413,0559,0880 and 1638
, Rhodotorula glicinis rufusa (Rhoditor)
ula glutinis rufusa)IF015
38. Rhodotorula glytinis
a glitinis) IFOO688 and 038
It is 9.

Based on the above mycological properties, J.
, Lodder), The East a Tafsonomic Study, 2nd edition (1970), Classification of microorganisms based on molecular species of quinones involved in the respiratory chain by Yamada [
According to a search using Fermentation and Industry, 37.940 (1979), etc., this strain does not form ascospores or extrusive spores, so it is not an ascospore yeast or an extrusive yeast.

In addition, this strain forms true hyphae and pseudohyphae, and the quinone type is Q10, so it seems to be a yeast derived from basidiomycelia. However, when we conducted a conjugation test with a known mating type strain, no conjugation occurred. I got a lot of power.

On the other hand, since this strain does not form theriospores or interlocking connections, it does not belong to Leucospororhizoium or Rhodosporolidium.

Furthermore, this strain was identified as a Rhodotorula genus because it produces carotenoid pigments, does not ferment carbon sources, decomposes urea, and does not produce starch-like substances.

A comparison was made with the mycological properties of known bacterial species belonging to the genus Rhodotorula.
la 5inensis) [Microbiological Journal, 14 (2)
), 143 (1974)) have similar properties, but the isolate used in the present invention and Rhodotorula
sinensis CB56962 in the following points.

(1) Rhodotorula sinensis contains lactose, soluble starch, and D
- Ribose, erythritol, atonite and DL--lactose is not assimilated, but this strain is.

(2) Rhodotorula sinensis produces starch-like substances, but this strain does not.

(3) Rhodotorula sinensis forms chlamydospores, but this strain does not.

Identification at the species level requires further investigation, so this strain was identified as Rhodotorula sp.
odotorula sp, ).

In the culture medium used in the present invention, glucose, sucrose, and all other carbohydrates that can be utilized by this strain are generally used as carbon sources, either alone or in a mixed state.

Also, substances containing carbohydrates that can be used by bacteria, such as blackstrap molasses, starchy hydrolysates, or wood saccharide liquor, can be used as carbon sources as well.

Examples of nitrogen sources include inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium nitrate, sodium nitrate, and ammonium phosphorus, as well as urea, ammonium acetate, peptone, yeast extract, corn steep leaker (C,
S, L, ), amino acid solution, meat extract, etc. are used.

These nitrogen sources can be used alone or in combination.

This strain can utilize nitrate, and nitrate is used as a good nitrogen source.

In any case, it is desirable to use the nitrogen source in an appropriate quantitative ratio to the carbon source.

In order to accumulate a large amount of itaconic acid in the culture, a medium containing a small amount of nitrogen source relative to the amount of carbon source, in other words, a medium with a relatively high C/N ratio is used. This is desirable.

Here, glucose is used as a carbon source, and ammonium chloride, ammonium sulfate, sodium nitrate, ammonium nitrate, ammonium phosphate and C, S,
Table 1 shows examples of experiments conducted using L.

Table 1 Effect of nitrogen source type and concentration Basal medium Niglucose 10%, KH2PO40.05%,
Mg504-7H200,01%, C, S, L, 1m
Ca in an aqueous solution with a composition of 1/l and 3 drops/l of Adekanol.
Medium supplemented with CO3 at a rate of 1%.

Test strain: Rhodotorula sp. SY-668 (
FERM-P No. 5318) Culture conditions: 50 ml medium in a 500 ml sakaro flask.
After dispensing and sterilizing, the test bacterial strain was inoculated and cultured with shaking at 26° C. for 5 days at 120 cycles per minute.

Quantification of itaconic acid: According to the quantitative method described in Kyoritsu Publishing ■ Microbial Engineering Course 5 Mold Utilization Industries, pp. 72-74, published in 1955.

That is, in this example, the nitrogen source is 0.0% nitrogen in the medium.
Itaconic acid accumulated the most in the medium containing it at a concentration of 1 to 0.02%.

The medium used in the present invention must contain various inorganic nutrients in addition to carbon sources and nitrogen sources, and addition of potassium phosphate, sodium phosphate, magnesium sulfate, etc. will promote suitable itaconic acid production. can get.

As for the pH of the next medium, a wide range in which the yeast used can propagate is applied.

However, since the production of itaconic acid is affected by pH,
Using sodium hydroxide and hydrochloric acid as pH adjusters,
Experimental examples regarding the itaconic acid productivity of yeast are shown in Table 2.

Table 2 Productivity of itaconic acid by pH Basal medium Niglucose 10%, Sodium nitrate 0.1%,
KH2PO40.05%, MgSO4・7H20o, 0
1%, C, S, L, 1ml/1 and 3 drops of Adecanil/
An aqueous solution having the composition of 1.

Test pH adjustment method: pH adjustment was performed twice daily using sodium hydroxide and hydrochloric acid solutions.

Test strain: Rhodotorula sp. SY-668 (
FERM-P No. 5318) Culture period: 5
After dispensing and sterilizing 50 ml of culture medium into a 00 ml Yosaka flask, inoculating the co-translated bacterial strain and incubating at 120 cycles per minute at 26°C for 50 minutes.
The cells were cultured with shaking for days.

That is, in this example, it was revealed that pH around 6.5 is most suitable for itaconic acid production.

In the present invention, the yeast is cultured under aerobic conditions.

Ordinary shaking culture methods, aeration stirring culture methods using tanks, etc. meet these conditions.

In this case, when an antifoaming agent is required, silicone resin or the like is used.

The culture temperature is preferably in the range of about 25 to 30°C.

In the present invention, itaconic acid is separated and collected from the culture by a conventional method.

For example, when sodium itaconate is present in the medium, insoluble matter such as bacterial cells is removed by centrifugation.

After desalting the obtained supernatant by passing it through cation-exchanged citrus,
Concentrate under reduced pressure to precipitate crystals, and filter and dry to obtain itaconic acid crystals.

In the case of recrystallization if necessary, water is added to the crude crystals, activated carbon is added, the mixture is filtered and cooled, recrystallized, separated and dried to obtain itaconic acid crystals.

Next, the present invention will be specifically explained with reference to Examples.

Example 1 Glucose (1st grade reagent) 10%, sodium nitrate 0.1
5%, KH2PO40.05%, MgSO4・7H20
0.01%, and C, S, L, 0.2 ml (p
After dispensing and sterilizing 50 ml of a medium consisting of H5.
Rhodotorula sp. SY cultured at 30°C for 4 hours
'-668 (FERM-P No. 5318) was inoculated and cultured with shaking at 30°C for 7 days.

During the culture, the pH was adjusted to 6.0 with sodium hydroxide twice a day.

After the completion of the culture, itaconic acid in the medium was quantified.14
．． It was 9 mg/ml.

Example 2 In the method of Example 1, Rhodotorula sp. 5Y-668 (FERM-PNo, 5318) was grown using a medium to which 10% sucrose was added instead of glucose in the medium.
was inoculated and cultured aerobically at 26°C for 7 days.

After completion of the culture, production of 12.5 mg/ml of itaconic acid was observed in the culture solution.

Example 3 In the method of Example 1, Rhodo 1 to Lula species 5Y-7 were grown using a medium to which 10% beet molasses (6.2% as glucose) was added instead of glucose in the medium.
18 (FERM-P No. 5319), separately sterilized calcium carbonate was added, and cultured aerobically at 30°C for 5 days.

After completion of the culture, production of 5.2 mg/ml itaconic acid was observed in the medium.

Example 4 The same medium 16.61 as in Example 1 was dispensed into a 301-volume jar fermenter, and after sterilization, 900 ml of the same composition medium as above (900 ml of medium with the same composition as above (9 500 ml Erlenmeyer flasks with 1 volume)
Rhodotorula sp. 5Y-668 (FERM-P No, 53
18) was inoculated at 26°C, aeration rate 91/min, stirring number 35 Or, p, m.

The cells were cultured for 7 days.

During cultivation, pH 5.8-6 with 10% sodium hydroxide solution
．． Controlled to 0.

After completion of the culture, itaconic acid in the culture solution was 21 mg/ml.

This culture solution 17.51 was heated to 80°C, the bacterial cells were removed using a centrifuge, the supernatant was passed through a Diaion 5KIB, desalted, and concentrated under reduced pressure (60 mmHg, 50-60 mmHg).
℃) and the volume was approximately 780 m1.

This was crystallized in the refrigerator (7℃), filtered and dried, and the
80 g of crude crystals were obtained.

Add 500 ml of water to the crude crystals, heat to dissolve at 80°C, add 5 g of activated carbon, and then heat the crystals. It was filtered and recrystallized, and the resulting crystals were filtered and dried to obtain about 224 g of itaconic acid crystals (purity 99.96%).

Claims (1)

[Claims] 1 A yeast belonging to the genus Rhodotorula having the ability to produce itaconic acid is cultured aerobically in a nutrient medium containing a carbon source that can be assimilated by the yeast, and itaconic acid produced in the culture is collected. A method for producing itaconic acid using a fermentation method.