KR101428508B1 - Aspergillus terreus and manufacturing method of itaconic acid using the same - Google Patents

Abstract

The present invention relates to novel strains, such as Aspergillus terreus ) KCTC12234BP. Phosphoric acid can be used for the production of itaconic acid, not for cell growth, even in a culture medium containing a high concentration of phosphoric acid. Thus, itaconic acid is excellent in productivity and production efficiency of itaconic acid .

Description

TECHNICAL FIELD The present invention relates to a method for producing aspartic acid and a method for producing itaconic acid using the same,

The invention teriwooseu Aspergillus (Aspergillus terreus ) and a process for preparing itaconic acid.

Itaconic acid has a methylene group and two carboxyl groups. Itaconic acid is added as an intermediate for synthesizing polyester resin and various polymers by using the bond between these two groups. Itaconic acid is currently used as a fiber, a coloring agent, a cleaning agent, Herbicides, and the pharmaceutical industry. Since itaconic acid has been reported for the first time in 1931, the production scale has been steadily growing since the fermentation method has been established through liquid culture which shows high productivity with cheap carbon source.

On the other hand, the production of itaconic acid can be largely divided into a chemical method and a biological method. As a chemical method, citric acid or cis-aconitic acid, which is a precursor of itaconic acid, is heated and decomposed to obtain itaconic acid. However, due to various side effects accompanying chemical reactions, Industrial production of the konnan is more preferably carried out by a biological culture process by a microorganism according to a biological method. A variety of biological species have been reviewed by the US Department of Agriculture (USDA) for the production of itaconic acid since the mid-1940s on biologic methods for the production of itaconic acid, with about 11 species producing over 45% of theoretical yield Respectively. Therefore, the Aspergillus terreus strain NRRL1960, which is still widely used, has been developed.

In the 1960s, companies such as Pfizer developed molasses-based fermentation technology to produce itaconic acid more inexpensively. Nowadays, the use of raw starch as a carbon source Studies have also been conducted, and a culture engineering approach to mass production of itaconic acid using an air-lift reactor has also been attempted.

However, despite these attempts, there have been limitations in the development of high productivity of itaconic acid. Especially, as the concentration of phosphoric acid contained in the culture medium is increased, the production of itaconic acid is rapidly increased There is a problem of lowering. In addition, in a large-scale culture which is difficult to precisely control the concentration, productivity of itaconic acid is remarkably lowered, and there is a problem that it is not suitable for a continuous culture method.

Japanese Patent Application No. 1992-196906 U.S. Patent Application No. 1992-855470 U.S. Patent Application No. 2000-510449

An object of the present invention to through the chemical mutagenesis of a novel strain selected cells, as well as in the concentration of the phosphate medium high productivity of itaconic acid can be increased large-scale culture, new teriwooseu Aspergillus (Aspergillus terreus ).

It is another object of the present invention to provide a method for producing itaconic acid in high yield through cultivation of a new strain of Aspergillus terreus .

In order to achieve the above object, the present invention provides a novel strain, Aspergillus terreus KCTC 12234BP.

According to one embodiment of the present invention, the Aspergillus terreus KCTC12234BP may be one which produces itaconic acid.

According to another embodiment of the present invention, the Aspergillus terreus KCTC12234BP may be arsenate or phosphate-resistant.

The present invention also relates to a method for the production of A) a culture of Aspergillus terreus KCTC12234BP; And B) obtaining itaconic acid from the culture broth of step A).

According to one embodiment of the present invention, the Aspergillus terreus ) KCTC12234BP may contain a phosphoric acid at a concentration of 0.02 g / L to 0.2 g / L, based on the total weight of the culture liquid.

According to another embodiment of the present invention, the Aspergillus teriwooseu (Aspergillus terreus ) KCTC12234BP was isolated from a) known Aspergillus terreus ) to ultraviolet or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to generate Aspergillus terreus ) R104; b) Aspergillus ( Aspergillus < RTI ID = 0.0 > terreus ) R104 as a chemical mutagen; And c) culturing the Aspergillus terreus R104 treated in b) above in a medium containing arsenate and selecting an arsenate resistant strain. .

According to another embodiment of the present invention, the chemical mutagen of step b) may be N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

The invention new Aspergillus teriwooseu selected through a chemical mutation (Aspergillus terreus ), phosphoric acid can be used for the production of itaconic acid, rather than cell growth, even in a culture medium containing a high concentration of phosphoric acid, so that the productivity of itaconic acid is excellent.

The present invention also relates to a novel Aspergillus It is possible to produce itaconic acid in high yield through cultivation of terreus , and it shows excellent efficiency even in large scale production facilities.

FIG. 1 is a graph showing the number of colonies of the strain of Example 1 and the strain of Comparative Example 1 according to the arsenate concentration. FIG.
FIG. 2 is a graph showing the amount of itaconic acid produced in the strains of Example 1 and Comparative Example 1 when the culture was produced in a conventional production medium (phosphate 0.02 g / L).
FIG. 3 is a graph showing the weight of dry cells in Example 1 and Comparative Example 1 when the culture was produced in a production medium (phosphate 0.02 g / L).
4 is a graph showing the amount of itaconic acid produced in the strains of Example 1 and Comparative Example 1 when the production culture was carried out in a production medium containing high concentration of phosphoric acid (phosphate 0.2 g / L).
FIG. 5 is a graph showing the production of itaconic acid in the strains of Example 1 and Comparative Example 1 when a 5 L fermenter was used to produce and cultivate the high-concentration phosphoric acid-containing production medium (phosphate 0.2 g / L).

The present invention is directed to a new strain, Aspergillus terreus KCTC 12234BP. Since one of the organic acids secreted by Aspergillus terreus KCTC 12234BP of the present invention from the cell is itaconic acid, itaconic acid can be used for production. In particular, the Aspergillus terreus KCTC 12234BP of the present invention is expected to produce itaconic acid at a high efficiency even in the production medium of itaconic acid to which phosphoric acid is added at a high concentration.

Aspergillus terreus KCTC 12234BP of the present invention can be produced by modifying a known strain of Aspergillus terreus .

Specifically, a mutant is induced by using MNNG in a spore of a known Aspergillus terreus strain, and the mutant is cultured in a solid medium (PDA: potato-dextrose agar) to select strains with high resistance to arsenate. Thus, the strain of the present invention, Aspergillus ( Aspergillus) terreus KCTC 12234BP.

More specifically, the present invention teriwooseu Aspergillus (Aspergillus terreus ) KCTC12234BP,

a) a known Aspergillus terreus ) is treated with UV or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as a mutagenic source to produce Aspergillus terreus ) R104;

b) Aspergillus ( Aspergillus < RTI ID = 0.0 > terreus ) R104 as a chemical mutagen; And

c) the Aspergillus teriwooseu processed in the b) (Aspergillus terreus R104 is cultured in a medium containing arsenate and then arsenate-resistant strains are selected.

Wherein a) the stage known teriwooseu Aspergillus (Aspergillus terreus may be Aspergillus terreus (ATCC 10020).

The chemical mutagenic source of step b) may be N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

In addition, the novel strain of the present invention teriwooseu Aspergillus (Aspergillus terreus ) KCTC 12234BP is able to produce itaconic acid from its culture solution through flask culture with high efficiency and can be used for mass production using 5L fermenter culture.

In particular, the novel strain of the present invention teriwooseu Aspergillus (Aspergillus terreus ) KCTC 12234BP exhibits arsenate resistance and exhibits remarkably superior itaconic acid production even in media containing high concentrations of phosphoric acid. Exhibits excellent arsenate resistance even in a culture solution containing phosphoric acid at a concentration of 0.02 g / L to 0.2 g / L based on the total weight of the culture liquid. In fact, the present inventors have found that even in the case of a poor production medium in which the content of phosphoric acid is 10 times higher than that of the conventional production medium, the inventive novel strain Aspergillus terreus ) KCTC 12234BP showed 36% increase in the production of itaconic acid and 40% increase in the 5L fermenter compared to the parent strain. This is a significant improvement in the production of itaconic acid in the case of the existing Aspergillus sterius strain by inducing consumption of nutrients to the cell growth in a medium having a high phosphoric acid concentration, ( Aspergillus terreus ) KCTC 12234BP.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Reference Example  1: Storing the strain>

In this experiment, Aspergillus The strains were inoculated on a PDA (potato dextrose agar) plate and incubated for 7 days at 28 ° C. After incubation for 20 days at 20 ° C, Suspended in glycerol, stored at -80 ° C, taken out whenever necessary, and plated on PDA medium in a volume of 100 μl.

< Reference Example  2: Medium and culture condition>

The medium used in the present invention was divided into a spore-forming medium, a liquid-phase growth medium and a liquid-phase medium according to the purpose of use.

Specifically, 20 g / l of PDA (potato dextrose agar) was used as the spore forming medium, and 55 g / l of glucose, 2 g / l of yeast extract, ammonium nitrate, A medium containing 6 g / l potassium sulfate, 0.02 g / l potassium phosphate dibasic and 2 g / l magnesium sulfate was used. The liquid production medium contained glucose 112 g / l, calcium chloride calcium sulfate, 3.75 g / l ammonium sulfate, 0.06 g / l potassium phosphate dibasic, 0.5 g / l magnesium sulfate, and 0.00085 mg CuSO ₄ 7H ₂ O / L, and MnCl24H2O 0.0000219 mg / L and a pH of 2.1.

In order to prevent sedimentation and browning during the wet sterilization, the medium of the present invention was prepared by using a concentrated solution of glucose and inorganic salts of the medium, sterilized, and mixed with the rest of the medium in a sterile condition. In the production culture using the flask, spores formed by culturing the cells at 28 ° C. for 7 days were inoculated on the PDA as the spore forming medium, and the spores were recovered by distilled water containing 20% glycerol to grow 40 ml In a volume of 5% (v / v) in a 250 ml flask containing the medium. The cells were cultured in a shaking incubator at 37 ° C and 220 rpm for 2 days. Production culture was carried out by inoculating the mycelia obtained in the growth culture into a 250 ml flask containing 40 ml of the production medium at a volume of 5% (v / v) at 37 ° C and 230 rpm for 3 to 4 days.

< Reference Example  3: Itaconic acid  Analysis method>

For the measurement of itaconic acid in the culture, shake cultured mixture was centrifuged at 12,000 rpm for 10 minutes, and 600 μl of the supernatant, which had been removed from the foreign material, was passed through a 0.45 μm microfilter and used as HPLC quantitative analysis sample. The analytical conditions for itaconic acid using HPLC are as follows:

Column: Mightysil RP-18 GP 250-4.6 (5 탆) (Kanto chemical Co. INC)

Mobile phase: 20 mM sulfuric acid

Temperature: 40 ° C

Detector: M720 absorbance detector (Younglin Co. Korea)

Detection wavelength: 210 nm

Mobile phase velocity: 1.2 ml / min

Sample assay volume: 20 μl

&Lt; Example 1: Aspergillus  Terius ( Aspergillus terreus ) KCTC12234BP ) &Gt;

(1) parent strain ( Aspergillus  Terius ( Aspergillus terreus ) R104 ) Preparation

In this experiment, Aspergillus The strains were inoculated on a PDA (potato dextrose agar) plate and incubated for 7 days at 28 ° C. After incubation for 20 days at 20 ° C, Suspended in glycerol, stored at -80 ° C, taken out whenever necessary, and plated on PDA medium in a volume of 100 μl.

Specifically, the R104 strain is obtained by mutagenizing Aspergillus terreus (ATCC10020) (KTCC) using MNNG and isolating strains producing a large amount of itaconic acid through random screening by HPLC analysis Were repeatedly obtained in sequence.

That is, the spore of the Aspergillus terreus (ATCC10020) (KTCC) grown in the spore-forming medium PDA (potato dextrose agar) was collected with distilled water containing 20% glycerol and then filtered to remove the residue The removed pure spore suspension was prepared. The collected spores were washed twice with 0.1 M TM buffer (pH 6.8) and diluted to 1 × 10 ⁶ (spores / ml) using a hemocytometer. MNNG was added to the spore suspension of 0.1 M TM buffer at a total concentration of 1,000 ppm and then treated at 28 ° C for 15 minutes. Alternatively, the concentration of spores was diluted to 1 x 10 ⁶ (spores / ml) in a pure spore suspension from which the residue was removed, and then treated with UV light (UV treatment to kill 99% of spores). After treatment, spores were collected at 12,000 rpm using a centrifuge and washed twice with 0.1 M TM buffer. The spore concentration of the recovered spore suspension was diluted to 1 × 10 ^{4 to 6} (spores / ml) using TM buffer, and 0.1 ml of the diluted spore suspension was applied to 25 ml of PDA medium. After incubation at 37 ° C for 24 hours, grown colonies were selected. The grown colonies were cultured in a liquid production medium for the production of itaconic acid, respectively, and analyzed by HPLC. Aspergillus ( Aspergillus than terreus) (ATCC10020) (KTCC) were selected for the strain R104 productivity is increased.

(2) Parental  Mutation induction

A mutation was introduced into the parent strain obtained in the above (1) by the following method using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, Respectively.

The spore of Aspergillus terreus R104 of the above (1), which was grown in a sporangia forming medium, PDA (potato dextrose agar), was collected by distilled water containing 20% of glycerol and then filtered to remove pure water from which the residue was removed A spore suspension was prepared. The collected spores were washed twice with 0.1 M TM buffer (pH 6.8) and diluted to 1 × 10 ⁶ (spores / ml) using a hemocytometer. MNNG was added to the spore suspension of 0.1 M TM buffer at a total concentration of 1,000 ppm and then treated at 28 ° C for 15 minutes. After treatment, spores were collected at 12,000 rpm using a centrifuge and washed twice with 0.1 M TM buffer.

(3) Arsenate ( arsenate ) Selection of resistant strains

Arsenate is a structurally very similar material to phosphate. If there is a strain that easily absorbs phosphate from the culture, this strain tends to be used only for growth of cells, not phosphate, after the phosphate is absorbed in the culture medium and then used to produce itaconic acid. Therefore, the productivity of itaconic acid is low in these strains. Therefore, in the selection of strains, when phosphate is added, phosphate - like strains are absorbed by confusing phosphate with phosphate. However, arsenate is structurally similar to ginseng salt, but it is highly toxic. Therefore, strains absorbing a lot of arsenate are killed, whereas strains of arsenate which do not use it are resistant to arsenate And these strains are slow to absorb even phosphate in the culture medium or use phosphate in other ways, which slows cell growth and increases the production of itaconic acid. Therefore, in order to find a phosphate - resistant strain with high phytotoxic acid phosporic acid, a non - acid resistant strain was selected as follows.

The spore concentration of the spore suspension recovered in (2) above was diluted to 1 × 10 ⁶ (spores / ml) using TM buffer, and 0.1 ml of the diluted spore suspension was added to PDA (minimal medium + arsenate To 300 ppm)) medium. After incubation at 37 ° C for 24 hours, grown colonies were selected. When the cells were observed with the naked eye, they were transferred to agar slant (PDA), which had the highest growth rate of colony in the upper 10%, and spores were formed. The resulting spores were used as spore suspension for the production of itaconic acid .

< Comparative Example  One: Aspergillus  Terius ( Aspergillus terreus ) R104 Gt;

In the same manner as in (1) of Example 1, Aspergillus terreus ) R104.

< Experimental Example  1: New strain  Dissolve resistance test>

AAS in Example 1 Aspergillus's teriwooseu (Aspergillus terreus) KCTC12234BP and Aspergillus teriwooseu of Comparative Example 1 (Aspergillus In order to test the arsenate resistance sensitivity of terreus R104, spore suspension solutions of Example 1 and Comparative Example 1 were prepared, and then PDA solid medium containing 0 to 300 ppm of arsenate was added to the PDA medium 0.1 ml of spore suspension diluted to 1 x 10 ⁶ (spores / ml) was plated on PDA medium containing arsenate. The number of colonies by the addition of arsenate was measured and shown in Fig.

Referring to Figure 1, of the Comparative Example 1 Aspergillus's teriwooseu (Aspergillus In the case of the terreus strain R104, the number of colonies decreased drastically due to the addition of arsenate and the number of colonies was not observed when the arsenate was 80 ppm or higher (minimum inhibitory concentration was 80 ppm), while the new strain Aspergillus Aspergillus In the case of the terreus KCTC12234BP strain, colony formation was still observed even when the concentration of arsenate increased by 300 ppm or more.

Thus Aspergillus of the present invention's teriwooseu (Aspergillus terreus ) KCTC12234BP strains, conventional Aspergillus Terreus R104, which is highly resistant to arsenate, and it can be shown that itaconic acid production ability is high even in production medium containing high concentration of phosphoric acid.

< Experimental Example  2: According to the phosphoric acid concentration of the production medium, New strain Itaconic acid  Productivity test (flask culture)>

AAS in Example 1 Aspergillus's teriwooseu (Aspergillus terreus) KCTC12234BP and Aspergillus teriwooseu of Comparative Example 1 (Aspergillus In order to confirm the productivity of itaconic acid according to the concentration of phosphoric acid in the production medium, the following experiment was carried out.

In the production culture using the flask, the strains of Example 1 and Comparative Example 1 were inoculated to a potato-dextrose agar (PDA), which was a spore forming medium, and cultured at 37 DEG C for 7 days. The spores formed were cultured in a medium containing 20% glycerol (V / v) volume in a 250 ml flask containing 40 ml of growth medium, and then cultured in a shaking incubator at 37 ° C and 200 rpm for 2 days.

In the production culture, the mycelia obtained from the growth culture were inoculated in a volume of 5% (v / v) in a 250 ml flask containing 40 ml of production medium, and cultured at 37 ° C and 220 rpm for 3 to 4 days. At this time, the production medium (phosphoric acid 0.02 g / L) and the high concentration phosphoric acid-containing production medium (phosphate 0.2 g / L) in which the concentration of phosphoric acid was increased 10 times Lt; / RTI &gt; The production of itaconic acid and the dry cell weight of each strain over time are shown in FIGS. 2 to 4.

FIG. 2 is a graph showing the amount of itaconic acid produced in the strains of Example 1 and Comparative Example 1 when the culture was produced in a conventional production medium (phosphate 0.02 g / L). 3 is a graph showing the weight of dry cells in Example 1 and Comparative Example 1 when the culture was produced in a production medium (phosphate 0.02 g / L).

When Fig. 2, the in existing production medium a lower concentration of acid, the first embodiment of the Aspergillus teriwooseu (Aspergillus terreus) about 6g compared to Aspergillus teriwooseu (Aspergillus terreus) R104 strain in Comparative Example 1 For KCTC12234BP strain / L high itaconic acid. This is because, in the case of Example 1, which is a novel strain of the present invention, phosphoric acid added to the medium is not used for cell growth but used for the production of itaconic acid. 3, the amount of dried cells of Example 1 and Comparative Example 1 was measured. As a result, the Aspergillus terreus ) R104 strain of Example 1 compared to Aspergillus Terreus KCTC12234BP strains have a low value of about 5 g / L.

4 is a graph showing the amount of itaconic acid produced in the strains of Example 1 and Comparative Example 1 when the culture was produced in a production medium containing high concentration of phosphoric acid (phosphate 0.2 g / L). 4, the case of high-concentration phosphoric acid-containing production medium, increase the amount of the phosphoric acid 10-fold compared to the conventional production medium, the Aspergillus, the Comparative Examples 1's teriwooseu (Aspergillus terreus strain R104 showed a decrease of about 19 g / L compared to the condition in which phosphoric acid was added at 0.02 g / L, whereas in the case of Aspergillus terreus KCTC12234BP strain of Example 1, the concentration of phosphoric acid , Itaconic acid productivity was almost similar regardless of the concentration of phosphoric acid. Thus Aspergillus of the embodiment 1 through the screening mutant's teriwooseu (Aspergillus terreus ) KCTC12234BP strain, which is a strain capable of greatly restricting the decrease in the productivity of itaconic acid due to the addition of a high concentration of phosphoric acid.

< Experimental Example  3: New strain  Mass culture compatibility test>

AAS in Example 1 Aspergillus's teriwooseu (Aspergillus terreus) KCTC12234BP and Aspergillus teriwooseu of Comparative Example 1 (Aspergillus terreus ) R104 was performed as follows.

The 5L fermenter culture was performed using a top driven incubator. The 5L fermenter (Best Korea) is equipped with two turbine impellers (D = 85mm), three baffles (width = 14mm) and a 12 hole sparifier (D = 78mm) (DO) probe (Mettler-Toledo, Switzerland) was connected to a computer and monitored online.

For the inoculation of seeds, the spores cultured for 7 days from slope agar were recovered with 20% glycerol, followed by 5% inoculation in a 250 ml working volume (40 ml), followed by primary culturing at 37 ° C for 2 days. 5% in a 500 ml flask (working volume: 200 ml), and cultured in the same manner as the growth culture of Experimental Example 2. This was cultured as a seed culture for the fermenter and 4.5% in the fermenter. The incubation temperature of the fermenter was 37 캜. At this time, production culture was carried out using the production medium containing high concentration of phosphoric acid (phosphate 0.2 g / L) of Experimental Example 2. The production of itaconic acid and the culture parameters of the strains of Example 1 and Comparative Example 1 are shown in Fig. 5 and Table 1, respectively.

FIG. 5 is a graph showing the production of itaconic acid in the strains of Example 1 and Comparative Example 1 when a 5 L fermenter was used to produce and cultivate the high-concentration phosphoric acid-containing production medium (phosphate 0.2 g / L). Diagram of the first embodiment as shown in Fig. 5 teriwooseu Aspergillus (Aspergillus terreus strain KCTC12234BP showed about 40% (111.5 hours) increased production of itaconic acid compared to Aspergillus terreus strain R104 of Comparative Example 1 in the production medium containing 0.2 g / L of phosphoric acid. . I.e. Aspergillus according to the first embodiment of phosphoric acid or a non-resistant strain's teriwooseu (Aspergillus terreus) For KCTC12234BP strain, by acid itaconic acid of Comparative Example 1 according to the significantly lowered the production inhibition teriwooseu Aspergillus (Aspergillus terreus ) R104 strain in the production of itaconic acid.

That is, when the concentration of phosphoric acid is increased in an unbalanced production medium in which phosphoric acid is generally added as a limiting component, the culture medium is used for growth and growth of cells by the added phosphoric acid. In contrast, Aspergillus In the case of KCTC12234BP strain, it was shown that the use of phosphoric acid induces metabolism by itaconic acid biosynthesis rather than cell growth.

In addition to, as shown in Table 1, Example 1 and Comparative Example of the strains of the first comparison the culture parameters Aspergillus embodiment of the first embodiment's teriwooseu (Aspergillus terreus ) KCTC12234BP strain showed 40% increase in maximum itaconic acid productivity, 70% increase in average specific production rate, 57% increase in itaconic acid production per cell, 33% reduction in maximum dry cell weight and 33% .

Example 1 Comparative Example 1 P _f (maximum itaconic acid productivity, g / L) 44.95 26.17 X _f (maximum dry cell weight, g / L) 13.79 20.56 r _p (average production rate, g / L / hr) 0.403 0.287 q _p (average rate of production, g / g / hr) 0.12 (72h) 0.035 (64h) r _x (average growth rate, g / L / hr) 0.123 (111.5h) 0.184 (111.5h) μx (non-growth rate, 1 / hr) 0.037 (24h) 0.042 (91h) Y ' _{p / x} (production rate of itaconic acid per cell, g / g) 4.33 1.98

Korea Research Institute of Bioscience and Biotechnology KCTC12234BP 20120703

Claims (7)

A known Aspergillus terreus is treated with a mutagenic agent of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), then cultured in a medium containing arsenate, Characterized in that it is produced through the step of selecting an arsenate-resistant strain.
Process for the preparation of Aspergillus terreus KCTC12234BP.
The method of claim 1, wherein the Aspergillus terreus KCTC12234BP produces itaconic acid.
The method according to claim 1, wherein the Aspergillus terreus KCTC12234BP has arsenate or phosphate resistance.
A) culturing Aspergillus terreus KCTC12234BP; And
B) obtaining the itaconic acid from the culture in step A)
Aspergillus terreus KCTC12234BP,
a) the known Aspergillus terreus is irradiated with ultraviolet light or Treatment with a mutagenic agent of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to obtain Aspergillus terreus R104;
b) treating said Aspergillus terreus R104 as a chemical mutagen; And
c) culturing the Aspergillus terreus R104 treated in b) above in a medium containing arsenate, and then selecting an arsenate resistant strain. , Itaconic acid.
The method according to claim 4, wherein the Aspergillus terreus ) KCTC12234BP is cultured in a concentration of 0.02 g / L to 0.2 g / L of phosphoric acid based on the total weight of the culture liquid.
delete 5. The process according to claim 4, wherein the chemical mutagen of step b) is N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

Images