KR20140046931A - Actinobacillus succinogenes and manufacturing method of succinic acid using the same - Google Patents

Abstract

The present invention relates to actinobacillus succinogenes and a succinic acid manufacturing method using the same and, more specifically, to actinobacillus succinogenes (KCTC 12233BP) which is a novel strain and has excellent productivity, yield and process stability of succinic acid, is applicable in the process of being mass produced, and a succinic acid manufacturing method using the same. [Reference numerals] (AA) Yield rate (%); (BB) Example 1; (CC) Comparative example 1

Description

TECHNICAL FIELD The present invention relates to Actinobacillus succinogenes and a method for producing succinic acid using the same,

The present invention relates to Actinobacillus < RTI ID = 0.0 > succinogenes KCTC 12233BP and a process for preparing succinic acid using the same.

Succinic acid is a C4-based diacid that has been used for a variety of purposes such as food, clothing, medicines, and plastics. Recently, it has attracted attention as one of the representative compounds that can be produced from biomass (Technology development for The production of biobased products from the biorefinery carbohydrates-the US Department of Energy's Top 10 revisited. Green Chemistry, (2010), 12, 539-554). In addition, it is possible to produce succinic acid through fermentation of biomass-derived sugars using microorganisms.

Actinobacillus succinogenes ) are microorganisms known to produce succinic acid at high concentrations. In the microorganism, the C4 metabolites such as succinic acid are produced through the CO ₂ fixation pathway of phosphoenolpyruvic acid (C3), which is a result of the action of the carbon central metabolism, The same number of carbon dioxide as the C4 metabolite can be consumed as a reactant to promote the carbon dioxide consumption industrially.

Meanwhile, Actinobacillus succinogenes sp. (1999), 49, 207-216) discloses a microorganism for the production of succinic acid, which is a microorganism belonging to the genus Actinobacillus suisinoge Actinobacillus succinogenes ). However, there is a problem in that productivity and yield of succinic acid are low, and the yield of succinic acid is reduced during mass production.

Korean Patent Application No. 10-2011-0030899 Korean Patent Application No. 10-2011-0033456

Actinobacillus succinogenes sp. nov., a novel succinic acid-producing strain from the bovine rumen (International Journal of Systematic and Evolutionary Microbiology, (1999), 49, 207-216)

An object of the present invention is to provide a novel strain excellent in productivity of succinic acid compared with a strain producing succinic acid in the past.

It is another object of the present invention to provide a method for producing succinic acid at a high yield using a strain producing succinic acid with high productivity.

In order to achieve the above object, the present invention provides Actinobacillus succinogenes KCTC 12233BP.

The present invention also relates to a process for the preparation of A) Actinobacillus culturing succinogenes KCTC 12233BP; And B) obtaining succinic acid from the culture broth.

The present invention relates to a process for producing actinobacillus ( Actinobacillus) which is excellent in the productivity, yield and process stability of succinic acid succinogenes KCTC 12233BP.

The succinic acid production method of the present invention shows excellent succinic acid productivity, yield and stability not only in the production process of succinic acid on the laboratory scale but also in the mass production for industrialization and can be applied to various fields such as petrochemical derivatives, have.

Figure 1 is a graph showing the effect of Actinobacillus < RTI ID = 0.0 > ( Actinobacillus < / RTI > Actinobacillus < / RTI > derived from succinogenes ATCC 55618 succinogenes ) WD27. < tb >< TABLE > Columns = 2 < tb >
2 is a graph showing cell growth of Comparative Example 1 (red control line graph) and Example 1 and Comparative Examples 2 to 48 (bar graph).
3 is a graph showing the productivity distribution chart of Comparative Example 1 (red control line graph) and Example 1 and Comparative Examples 2 to 48 (bar graph).
4 is a graph showing the production of succinic acid by the strain of Comparative Example 1 and the strain of Example 1 during fermentation.
5 is a graph showing production of succinic acid per hour of the strain of Comparative Example 1 and the strain of Example 1. Fig.
6 is a graph showing the percentage of succinic acid produced by the strain of Comparative Example 1 and the strain of Example 1 in the amount of succinic acid of the theoretical yield.
7 is a histogram showing the distributions according to succinic acid productivity of the strains of Example 1 and Comparative Examples 2 to 48. Fig.

Hereinafter, the present invention will be described in detail.

The present invention relates to Actinobacillus < RTI ID = 0.0 > succinogenes KCTC 12233BP. Actinobacillus succinogenes KCTC 12233BP is a strain that produces succinic acid and is excellent in succinic acid productivity and stability and can be usefully used for producing succinic acid. Further, in the production of succinic acid, it can be applied not only to a small scale production facility but also to a large scale production facility, and can be used in various industrial fields for producing succinic acid.

The Actinobacillus < RTI ID = 0.0 > succinogenes KCTC 12233BP was prepared by using Actinobacillus succinogenes WD27 derived from Actinobacillus succinogenes ATCC 55618, which was previously developed, as a host strain and transforming the microbial cells into a new chemical mutant ethyl Methylsulfonate (EMS). ≪ / RTI >

It is possible to obtain a strain having a high productivity of succinic acid by selecting strains showing a relatively high growth rate after the mutagen treatment because the strains with high cell growth are highly productive due to the production characteristics of succinic acid. The relative growth rate of the strain can be compared and analyzed by measuring the change in turbidity over time in liquid culture.

Actinobacillus succinogenes KCTC 12233BP obtained by the above method has a higher growth rate than other mutant strains and also produces succinic acid up to about 107 g / L in a 30 mL serum bottle culture, It was confirmed that the yield was 75%.

The Actinobacillus < RTI ID = 0.0 > succinogenes ) KCTC 12233BP was deposited with the Center for Microbial Resources (KCTC) of the Korea Biotechnology Research Institute.

The present invention relates to A) Actinobacillus ( Actinobacillus < RTI ID = 0.0 > culturing succinogenes KCTC 12233BP; And B) obtaining succinic acid from the culture broth.

Actinobacillus succinogenes ATCC ( Actinobacillus succinogenes ), which is known as a strain producing conventional succinic acid, is prepared by using Actinobacillus succinogenes KCTC 12233BP. Actinobacillus succinogenes WD27, which is derived from the strain 55618, and exhibits high productivity in the mass production of succinic acid.

The Actinobacillus < RTI ID = 0.0 > succinogenes) KCTC 12233BP Evil Tino Bacillus succinonitrile's Ness (Actinobacillus succinogenes ATCC 55618 as a chemical mutagen. Preferably, the chemical mutagen is ethyl methanesulfonate, but is not limited thereto. The ethyl methanesulfonate acts as a chemical mutagen that induces a mutation in the protein sequence by inducing a random alkylation reaction on the base sequence in the gene base sequence.

In the method for producing succinic acid according to the present invention, the step of culturing Actinobacillus succinogenes KCTC 12233BP comprises culturing glucose, yeast extract, corn steep liquor, sodium hydrogencarbonate (NaHCO ₃ ) and magnesium carbonate MgCO ₃ ), but the present invention is not limited thereto and can be appropriately selected according to need.

The liquid medium contains 90 to 120 g / L of glucose, 10 to 30 g / L of yeast extract, 15 to 30 g / L of corn steep liquor, 5 to 15 g / L of sodium hydrogencarbonate and 50 to 120 g / , More preferably 100 to 110 g / L of glucose, 10 to 15 g / L of yeast extract, 15 to 20 g / L of corn steep liquor, 7 to 12 g / l of sodium hydrogencarbonate, L and 90 to 110 g / L of magnesium carbonate and most preferably 109 g / L of glucose, 13 g / L of yeast extract, 19 g / L of corn steep liquor solid, 10 g / L of sodium hydrogencarbonate, 100 g / L. When the composition of the liquid medium satisfies the above-mentioned range, the actinobacillus succinogenes ) The productivity of succinic acid by KCTC 12233BP is increased, yield is improved, and it is advantageous from the economical point of view.

The Actinobacillus < RTI ID = 0.0 > succinogenes ) In the step of culturing KCTC 12233BP, the culture method is not particularly limited and can be suitably selected according to need by a person skilled in the art.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  One: Actino Bacillus Succinogenes  ( Actinobacillus succinogenes ) KCTC 12233 BP >

Actinobacillus Actinobacillus Actinobacillus &lt; / RTI &gt; derived from succinogenes ATCC 55618 succinogenes ) WD27 was used as a host strain to obtain mutant strains with improved productivity of succinic acid.

The Actinobacillus &lt; RTI ID = 0.0 &gt; succinogenes) After a treatment 0 ~ 240ppm ethyl methane sulfonate (Ethyl methanesulfonate, the EMS) on WD27 was determined concentration of 160ppm represents the death rate of 90% as a mutagen concentration (Fig. 1).

Washing after the EMS for the mutation induced in the strain for 10 minutes at a concentration of 160ppm, and serially diluted (serial dilution) to about 1 × 10 ² gae colonies (colony) growth is the possible conditions of 10 agar plates (agar per plate over plate. At 38 ℃ 10 ^Three single colonies from 10 agar plates after 24 hours incubation (single colony) a were obtained, of which are clearly marked as a single colony types were selected the 144 growth rapid colony 24-well microplate ( 24 well microplates). The degree of growth of the colonies was determined by the size of a single colony. For liquid culture, one colony was inoculated per well, and the volume of culture in each well was 1.25 mL. Liquid - phase culture was carried out twice, and the effect of the inoculation amount difference was excluded by adjusting the colony - specific inoculation amount in the second culture. For the screening of strains with rapid growth, the amount of bacteria was measured for each colony during culture. The turbidity (absorbance at a wavelength of 660 nm) of the culture was measured to determine the relative amount of the cells. The turbidity was measured once at 12 hours after the start of the first and second growth cultures. Based on these measurements, 48 strains with fast growing rates were selected and designated as UK1 ~ 48, respectively. Among the above UK1 to 48, Actinobacillus ( Actinobacillus ), which is the 13th most mutant strain (UK13) succinogenes KCTC 12233BP.

< Comparative Example  One: Actino Bacillus Succinogenes  ( Actinobacillus succinogenes ) WD27 >

Actinobacillus Actinobacillus Actinobacillus &lt; / RTI &gt; derived from succinogenes ATCC 55618 succinogenes ) WD27 was used as the strain of Comparative Example 1. To obtain the WD27 strain, ATCC 55618 strain stock was appropriately diluted with distilled water and 20% glycerol solution, and was plated on an agar plate to form about 100 single colonies per plate. A single colony was randomly obtained from 5 to 10 agar plates and inoculated with one colony per well at a constant temperature of 38 DEG C and a working volume of 1.25 mL to perform growth culture on a 24 well microplate and then cultured under the same culture conditions And the culture was continued for 48 hours using the culture medium. [0060] The strain selection process described above was repeated with the strain having the highest productivity of succinic acid selected as the host strain, The highest strain was named WD27 strain.

< Comparative Example  2 to 48>

In Example 1, the colonies grown on the agar plates were collected to select 48 strains of 144 cells, which were excellent in growth. The strains of Actino (SEQ ID NO: 13), which is the 13th mutant strain (UK13, Example 1) The remaining 47 strains (UK1-12, UK14-48) were used as Comparative Examples 2 to 48, respectively, except for Actinobacillus succinogenes KCTC 12233BP.

< Reference Example  1: Storing the strain>

For the storage of the strains of Example 1 and Comparative Examples 1 to 48, the cells obtained from the liquid culture were stored at 4 ° C or 20% glycerol stock and stored at -80 ° C. When necessary, the stored stocks were taken out, And cultured after inoculation on a medium.

< Reference Example  2: medium and culture conditions>

The solid culture was carried out using TSA (tryptic soy agar) medium prepared by dissolving 15 g / L of pancreatic digest of casein, 5 g / L of papaic digest of soybean, 5 g / L of NaCl and 15 g / L of agar in distilled water Respectively. A single colony was allowed to grow through serial dilution and 30 ml of TSA agar medium was inoculated and 100 μl of the diluted bacterial solution was inoculated. The inoculated agar plates were incubated at 38 ° C for 24 hours.

The growth culture for the growth of the cells prior to the culture was pancreatic digest of casein 17 g / L, papaic digest of soybean 3 g / L, dextrose 2.5 g / L, NaCl 5 g / L, K ₂ HPO ₄ (potassium phosphate dibasic ) 2.5 g / L was dissolved in distilled water and TSB (tryptic soy broth) medium was used.

For the production of succinic acid, 10 g / L of glucose, 13 g / L of yeast extract, 19 g / L of corn steep liquor, 10 g / L of sodium hydrogencarbonate (NaHCO ₃ ) and 100 g / L of magnesium carbonate (MgCO ₃ ) A liquid medium of pH 7.0 prepared by dissolving in distilled water was used.

The cultures were cultured in a 24-well microplate at a culture volume of 1.25 mL, or in a 100 mL serum bottle at a volume of 30 mL. Liquid culture of the cells was incubated for 1 to 2 days at 38 DEG C and 200 rpm in a shaking incubator, or cultured for 9 hours and subcultured in another flask.

The fermenter was cultivated in accordance with the production medium, aerated with carbon dioxide at 0.6 vvm, and stirred at a speed of 300 rpm. The pH was kept constant at 6.8 to 7.2 and cultured.

< Reference Example  3: Measurement of cell density>

The dry weight (D.C.W) of the cells was used to determine the concentration of the cells. After uniformly recovering the cells from the culture, 1 mL of cells were centrifuged at 10,000 rpm for 10 minutes. The supernatant was recovered and transferred to a 1.5 mL microtube for use in the determination of the remaining sugars. The remaining supernatant was removed and the remaining sugars, insoluble matter and salts were removed through three washes.

Thereafter, the resultant was dried in a weight measuring dish at 90 ° C for 12 hours to confirm that there was no change in the weight of the dried cell mass, and the dry weight was measured and expressed in terms of the cell concentration per 1 L.

< Reference Example  4: Quantitative analysis of succinic acid>

For the quantitative analysis of succinic acid in the cultures of Actinobacillus succinogenes , samples were taken in 1.5 ml microtube, diluted by serial dilution, and then filtered twice using 0.45 ㎛ filter paper. And analyzed by HPLC under the following conditions.

Analysis temperature: 25 ° C

Flow rate: 0.8 ml / min

Mobile phase: 0.01NH ₂ SO ₄

Analysis time: 20 minutes

Sample injection volume: 20 μl

Column: organic acid column (Bio-Rad, Aminex HPX 87H, 125-0140)

Detector: UV detector

< Reference Example  5: sugar analysis>

The supernatant was centrifuged at 12,000 rpm for 10 minutes, centrifuged three times for 10 minutes at 12,000 rpm, and the supernatant was collected, followed by filtration using a 0.45 쨉 m filter paper for HPLC . The sugar assay conditions using HPLC were as follows:

Analysis temperature: 40 ° C

Flow rate: 1.2 ml / min

Mobile phase: 75: 25 (acetonitrile: water)

Analysis time: 15 minutes

Sample injection volume: 20 μl

Column: Amine column (RS tech, NH2 column (250 mm x 46 mm))

Detector: RI detector

< Experimental Example  1: Succinic acid productivity and yield test>

To bad Martino Bacillus succinonitrile in Example 1 Ness (Actinobacillus succinogenes) KCTC 12233BP and evil Martino Bacillus of Comparative Example 1 succinonitrile to Ness (Actinobacillus succinogenes) an evil Martino Bacillus succinonitrile to Ness (Actinobacillus derived from ATCC 55618 succinogenes WD27 and Actinobacillus strains of the above Comparative Examples 2 to 48 succinogenes) mutants (UK1 ~ 12, UK14 ~ 48 ) each were in the serum bottle subjected to the production culture in a volume of 30ml, No. 13 mutant of Example 1 over, cell growth is the most excellent, as shown in Figure 3 weeks (UK13 Actinobacillus &lt; / RTI &gt; ( Actinobacillus &lt; RTI ID = 0.0 & succinogenes KCTC 12233BP showed succinic acid productivity of about 107 g / L.

Succinic acid productivity (g / L) Relative yield (%) Example 1 106.8225 g / L 75% Comparative Example 1 79.6353 56% Comparative Example 2 67.2453 47% Comparative Example 3 80.4214 56% Comparative Example 4 75.8323 53% Comparative Example 5 67.1053 47% Comparative Example 6 77.8265 54% Comparative Example 7 81.9228 57% Comparative Example 8 67.7712 47% Comparative Example 9 69.5949 49% Comparative Example 10 82.8527 58% Comparative Example 11 76.9134 54% Comparative Example 12 69.2613 48% Comparative Example 13 76.3091 53% Comparative Example 14 98.0063 69% Comparative Example 15 72.6218 51% Comparative Example 16 79.9393 56% Comparative Example 17 77.8034 54% Comparative Example 18 72.0934 50% Comparative Example 19 70.1087 49% Comparative Example 20 92.5367 65% Comparative Example 21 88.4928 62% Comparative Example 22 89.2608 62% Comparative Example 23 72.5574 51% Comparative Example 24 73.9429 52% Comparative Example 25 68.7727 48% Comparative Example 26 73.8727 52% Comparative Example 27 61.9758 43% Comparative Example 28 85.8435 60% Comparative Example 29 82.9998 58% Comparative Example 30 68.5798 48% Comparative Example 31 71.5274 50% Comparative Example 32 68.5300 48% Comparative Example 33 61.9052 43% Comparative Example 34 50.7948 36% Comparative Example 35 49.7083 35% Comparative Example 36 63.7884 46% Comparative Example 37 96.3477 67% Comparative Example 38 75.5087 53% Comparative Example 39 90.9904 64% Comparative Example 40 64.8441 45% Comparative Example 41 71.8989 50% Comparative Example 42 71.8480 50% Comparative Example 43 61.5886 43% Comparative Example 44 63.3099 44% Comparative Example 45 71.8480 50% Comparative Example 46 75.5294 53% Comparative Example 47 63.5522 44% Comparative Example 48 87.6719 61% Relative yield (%) = (succinimide yield / succinic acid theoretical yield) x 100

As can be seen from Table 1, the parent strain Bacillus evil Martino succinonitrile to Ness (Actinobacillus succinogenes) an evil Martino Bacillus succinonitrile to Ness (Actinobacillus derived from ATCC 55618 succinogenes ) In comparison with WD27 (Comparative Example 1), Actinobacillus strain &lt; RTI ID = 0.0 &gt; succinic acid productivity succinogenes) KCTC 12233BP is approximately 1.34 times, the yield could be confirmed that increased 1.34 times. In addition, Actinobacillus succinogenes KCTC 12233BP of Example 1 produced 75% of succinic acid at a given yield in a given glucose.

On the other hand, strains of Comparative Examples 2, 6, 9, 14, 16, 20, 21, 22, 29, 37, 38, 39 and 48 exhibited higher succinic acid productivity than those of Comparative Example 1 (FIG. 3) When the cell growth was compared with that of Actinobacillus &lt; RTI ID = 0.0 &gt; ( Actinobacillus &lt; succinogenes ) KCTC 12233BP (FIG. 2). In the case of the strains of the comparative examples, the productivity of the strains of Comparative Example 1 is lower than that of the strains of Comparative Example 1.

7 showing a histogram showing the degree of distribution according to the productivity of succinic acid, Actinobacillus &lt; RTI ID = 0.0 &gt; ( Actinobacillus &lt; succinogenes KCTC 12233BP showed significantly higher succinic acid productivity than the strains of Comparative Examples 2 to 48. [

< Experimental Example  2: Mass production possibility experiment>

Actinobacillus &lt; RTI ID = 0.0 &gt; ( Actinobacillus &lt; / RTI & the succinogenes) KCTC 12233BP strain was tested whether the strains suitable for scale-up through the fermentor culture.

The culture medium of Actinobacillus strain 1 of Example 1 was cultured in the same manner as in Example 1 using the medium components of 109 g / L of glucose, 13 g / L of yeast extract, 19 g / L of corn steep liquor, 10 g / L of sodium hydrogencarbonate and 100 g / Actinobacillus succinogenes ) KCTC 12233BP were cultured and the volume of 3 L, which was 100 times higher than that of the serum bottle, was determined as the operation volume and cultured at a stirring speed of 300 rpm.

Ill Martino Bacillus in Example 1 through the 5L fermenter succinonitrile to Ness (Actinobacillus succinogenes) KCTC 12233BP and Ill to Martino Bacillus succinonitrile in Comparative Example 1 Ness (Actinobacillus Actinobacillus &lt; / RTI &gt; derived from succinogenes ATCC 55618 succinogenes ) WD27 was cultured for 48 hours. As a result, the succinic acid production amount in Comparative Example 1 was about 60 g / L, and the succinic acid production amount in Example 1 was about 110 g / L (FIG.

As a result, when Actinobacillus succinogenes KCTC 12233BP of Example 1 was used, it was found that the fermentation was excellently performed even at a scale of 100 times, and it was confirmed that the strain had excellent success potential in industrial application Respectively.

FIG. 5 shows changes in productivity per hour. It can be seen that succinic acid was intensively produced within 10 to 30 hours, and the production amount of succinic acid to the theoretical yield was as high as 76.4% 6).

Korea Research Institute of Bioscience and Biotechnology KCTC12233BP 20120703

Claims (7)

Actinobacillus Actinobacillus succinogenes ) KCTC 12233BP. The method according to claim 1, the Ill Martino Bacillus succinonitrile to Ness (Actinobacillus succinogenes) KCTC 12233BP is bad Martino Bacillus, characterized in that to produce the succinic acid succinonitrile to Ness (Actinobacillus succinogenes ) KCTC 12233BP. A) Actinobacillus culturing succinogenes KCTC 12233BP; And
B) A step of obtaining succinic acid from the culture solution. The method according to claim 3, the method for producing the bad Martino Bacillus succinonitrile to Ness acid, characterized in that (Actinobacillus succinogenes) KCTC 12233BP is prepared by treating a bad Martino Bacillus succinonitrile to Ness (Actinobacillus succinogenes) ATCC 55618 by a chemical mutagen . The method of claim 4, wherein the chemical mutagen is ethyl methanesulfonate. The method of claim 3, wherein the culturing of Actinobacillus succinogenes KCTC 12233BP comprises culturing in a liquid medium comprising glucose, yeast extract, corn steep solids, sodium bicarbonate, and magnesium carbonate. The manufacturing method of succinic acid characterized by the above-mentioned. 7. The method of claim 6, wherein the liquid medium comprises 90 to 120 g / L of glucose, 10 to 30 g / L of yeast extract, 15 to 30 g / L of corn steep liquor, 15 to 15 g / L of sodium hydrogen carbonate, To 120 g / L. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;

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