KR100641281B1 - Production of 3-hydroxypropionic acid by Rhodococcus erythropolis LG12 - Google Patents

Abstract

The present invention is the Rhodococcus Rhodococcus Rhodococcus sp. erythropolis LG12) provides a method for producing 3-hydroxypropionic acid (3-HP) using accession number KCTC 18102P.

Description

Production method of 3-hydroxypropionic acid using Rhodococcus erythropolis L12 [Production of 3-hydroxypropionic acid by Rhodococcus erythropolis LG12}

1A to 1C are HPLC chromatograms showing that acrylic acid is converted into 3-HP by Rhodococcus erythropolis LG12 accession No. KCTC 18102P strain according to the change over time.

Figure 2, Rhodococcus Rhodococcus erythropolis LG12) GC-MS spectrum which can identify 3-HP produced by Accession No. KCTC 18102P strain.

The present invention is the Rhodococcus Rhodococcus Rhodococcus sp. erythropolis LG12) relates to the production of 3-hydroxypropionic acid (3-HP) using accession number KCTC 18102P.

3-Hydroxypropionic acid (3-HP) is an organic acid, and calcium salt of 3-HP is about 100 times higher solubility in water than citric acid or malic acid calcium salt. to be.                         

3-HP is also an important synthetic intermediate in some chemical processes. In particular, it is important for the production of some chemicals and polymers, including the production of malonic acid by oxidation, the production of specific polyesters by esterification with alcohols, and the production of 1,3-propanediol by reduction. .

The method of preparing 3-HP can be divided into chemical synthesis and biological methods. As a chemical method, a method of preparing 3-HP using a palladium catalyst using 1,3-propanediol as a starting material (US5321156), to prepare 3-HP in the presence of palladium and platinum catalyst from 3-hydroxypropionaldehyde Method (US5831121), A method of obtaining 3-HP with 91% selectivity and 34% yield by reacting acrylic acid with a starting material for 100 hours in a high pressure reactor using ion exchange resin (Amberlyst 15) as a solid acid catalyst (JP2000) -159724).

The biological method is a method of synthesizing 3-HP by microorganisms under conditions of room temperature and atmospheric pressure. The method of synthesizing 3-HP from acrylic acid, propionic acid and propionaldehyde using Candida rugosa KT8201 (JP58). -158189), Synthesis of 3-HP from acrylic acid using Alcaligenes faecalis M3A (Ansede et. al ., Appl. Environ. Microbiol. 1999, 65 (11) 5075-5081), a method for synthesizing 3-HP from glucose using metabolic engineering of Escherichia coli has been reported (WO0116346, WO0242418).

However, no method of producing 3-HP from acrylic acid using a strain of Rhodococcus sp. Has not been reported.

Bacteria of the genus Rhodococcus sp. Are Gram-positive and are microorganisms belonging to Nocardioform Actinomycetes. The inventors of the present invention, Rhodococcus Rhodococcus Rhodococcus Rhodococcus Rhodococcus is a growth condition is simple and rapid growth, yet resistant to high concentration of acrylic acid and can convert acrylic acid quickly erythropolis LG12) found accession number KCTC 18102P. Furthermore, while continuing to study the decomposition of acrylic acid using this, Rhodococcus Rhodococcus erythropolis LG12) It was found that 3-HP can be produced using a culture solution of accession number KCTC 18102P and completed the present invention.

Therefore, the object of the present invention is to Rhodococcus Rhodococcus erythropolis LG12) using the Accession No. KCTC 18102P strain to provide a method for producing 3-HP from acrylic acid.

In order to achieve the above object, the present invention is characterized in that Rhodococcus erythropolis LG12 accession No. KCTC 18102P is contacted with acrylic acid to produce 3-hydroxypropionic acid (3-HP) from acrylic acid. To provide a production method of 3-HP.

According to one embodiment of the invention, the production method, preferably, Rhodococcus Rhodococcus erythropolis LG12) Accession No. KCTC 18102P is cultured in a nutrient medium to acrylic acid with the above microorganisms in the growth stage, and contacted with acrylic acid.

According to one embodiment of the invention, the production method, the contact with acrylic acid, Rhodococcus Rhodococcus erythropolis LG12) Accession No. KCTC 18102P was cultured in a culture medium to separate the microorganisms from the culture solution, and a cell suspension was added thereto, followed by contact with acrylic acid by adding acrylic acid.

According to one embodiment of the invention, Rhodococcus Rhodococcus Rhodococcus erythropolis LG12 for producing 3-HP from the acrylic acid erythropolis LG12 ) Accession No. KCTC 18102P can be used in the form of free cells or in the form of cells immobilized on a carrier.

In the present invention, the Rhodococcus Rhodococcus, which produces 3-HP from acrylic acid. Culture medium and culture conditions of erythropolis LG12) accession number KCTC 18102P can be used as known in the art. The culture medium is not particularly limited, but a medium containing a carbon source, a nitrogen source, an inorganic salt, and a suitable nutrient source can usually be used. In general, any medium can be used as long as it is an industrially inexpensive saccharide raw material, a nitrogen source (organic and inorganic nitrogen source), and a medium suitably added with inorganic salts, trace elements, vitamins, and the like necessary for the growth of microorganisms. In addition, the culture conditions, using the culture conditions of the genus Rhodococcus, preferably, inoculate the bacteria in the sterilized medium, shaking culture at 20-45 (C, pH 6-9, and perform aerobic.

In the present invention, the acrylic acid to be contacted includes not only acrylic acid itself, but also all substrates including acrylic acid, and all substrates that can be converted into acrylic acid.

 In addition, in the present invention, the process of separating the microorganisms from the culture to make a cell suspension, can be used a method commonly known in the art. That is, a cell suspension is prepared by collecting only cells from the culture and suspending them in a solution containing a suitable inorganic salt.

In the present invention, the method of contacting acrylic acid directly to the culture medium or to the cell suspension prepared as described above includes a method of aerobic contact, and through such aerobic contact, Rhodococcus LG12 ( Rhodococcus) erythropolis LG12) Accumulate 3-HP produced in accession number KCTC 18102P. In order to accumulate a large amount of 3-HP, it is preferable to add a suitable carbon source such as glucose or glycerol as an energy source at the end of the culture or at the time of contact reaction with the cells. Accumulated 3-HP can be separated and recovered by conventional methods known in the art.

In the present invention, the time of contact with acrylic acid is preferably in the middle of the culture. If acrylic acid is added from the beginning of the culture of the microorganism, Rhodococcus Rhodococcus erythropolis LG12) Even if Accession No. KCTC 18102P is resistant to acrylic acid, the growth of the cells is slow. Therefore, it is preferable to contact acrylic acid in the middle of the culture without adding acrylic acid so that the growth of the cells occurs. . Preferably, contacting acrylic acid with a culture solution after 20 to 48 hours, more preferably after 24 to 48 hours, or a microbial cell suspension separated from the culture solution is excellent in yield.

In the present invention, Rhodococcus Rhodococcus to produce 3-HP from the acrylic acid erythropolis LG12 ) Accession No. KCTC 18102P can be used in the form of free cells or in the form of cells immobilized on a carrier. That is, it is also possible to use the immobilized cells of the microorganisms cultured above, or to use immobilized cells using various polymers. The immobilization method is a conventional method known in the art, such as a method of immobilizing in a gel form to a natural polymer such as alginate, carrageenan and the like, and to a synthetic polymer such as acrylamide, polyvinyl alcohol And a method of immobilization on the polymer resin.

Rhodococcus of the present invention Rhodococcus erythropolis LG12 strain was deposited on May 19, 2004 with the Korea Biotechnology Research Institute Gene Bank, an international depository institution (Accession No. KCTC 18102P).

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

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example  One

Rhodococcus erythromycin Indianapolis LG12 (Rhodococcus erythropolis LG12) Accession No. KCTC 18102P Production of 3-HP from acrylic acid using strain resting cells.

Rhodococcus Rhodococcus in 5 mL of sterile YEPD medium consisting of 20 g / L glucose, 20 g / L peptone and 10 g / L yeast extract erythropolis LG12) inoculated with Accession No. KCTC 18102P strain and incubated at 30 ° C. and 200 rpm for 24 hours, inoculated into a 250 mL flask containing 50 mL of the same composition, incubated at 30 ° C. and 200 rpm for 24 hours, and then, acrylic acid (final concentration 0.1). % wt / vol) was added, and after 8 hours of incubation, acrylic acid concentration in the culture was analyzed by HPLC to confirm that all of them were removed.

The cells were recovered by centrifugation and then suspended in 10 mL of 0.1 M phosphate buffer (pH 7.0). Recovered Rhodococcus erythropolis LG12 erythropolis LG12) Accession No. KCTC 18102P Strain Resting cell suspension 3mL (dry cell weight 15.6mg / mL) was added with 1% acrylic acid concentration (wt / vol, 10g / L) and the sample was collected at 28 ℃ and 170rpm. Analyzed using chromatography (HPLC). The mobile phase used for the analysis was prepared by using 0.01NH ₂ SO ₄ , the flow rate of the solvent was 0.6 ml / min and Aminex87H column of Bio-Rad company was used. At 24 hours, acrylic acid showed a conversion of 76%, and the resulting 3-HP was obtained in 53.9% yield (5.1 g / L concentration).

It was confirmed by the HPLC chromatogram of FIG. 1 that acrylic acid is converted into 3-HP by the Rhodococcus erythropolis LG12 Accession No. KCTC 18102P strain according to the change over time.

In addition, 3-HP produced by the Rhodococcus erythropolis LG12 (Accessory No. KCTC 18102P strain) was confirmed through the GC-MS spectrum of FIG.

Example  2

Rhodococcus Rhodococcus erythropolis LG12 ) Synthesis of 3-HP from acrylic acid using growing cell of Accession No. KCTC 18102P.

First, colonies of each microorganism cultured in YEPD plate medium were inoculated and incubated in 3 ml of YEPD liquid medium (10 g / L yeast extract, 20 g / L bactopeptone, 20 g / L glucose). Addition of 0.3 ml the resulting culture solution to 3 ml of a YEPD a disposable tube 15 ml containing a liquid medium (Falcon Co.) and cells were grown at 200 rpm, 30 ℃ in a shaking incubator (J. ohtek Inc.), this yi OD ₆₀₀ value of more than 16 Incubation was performed until acrylic acid was administered so that the final concentration of acrylic acid was 0.05% (wt / vol) to induce related gene expression. After that, the culture was continued until the cell growth reached OD ₆₀₀ 30. At this time, acrylic acid was administered so that the final concentration of acrylic acid was 0.05% (wt / vol), and the concentration of residual acrylic acid was measured by liquid chromatography (HPLC) ( Shimadzu Co.). The mobile phase used for liquid chromatography was prepared by using 0.01 N H2SO4, the solvent flow rate was 0.6 ml / min and Biomine-Aminex87H column was used. Detection was performed using a reflective index detector. 94% of acrylic acid was converted and the produced 3-HP was obtained at a yield of 27.3% at 3.2 g / L concentration.

Example  3

Rhodococcus Rhodococcus erythropolis LG12) Conversion of Acrylate to 3-HP Salt of Accession No. KCTC 18102P

Rhodococcus Rhodococcus erythropolis LG12) Accession No. KCTC 18102P strain was incubated in 100 mL of YEPD medium, and the cells were recovered by centrifugation and suspended in 10 mL of 0.1 M phosphate buffer (pH 7.0). Recovered Rhodococcus erythropolis LG12 erythropolis LG12) Accession No. KCTC 18102P Strain Resting cell suspension 3 mL (dry cell weight 18.4 mg / mL) was added with various concentrations of 1% (wt / vol, 10 g / L) of various acrylic acid salts and reacted at 30 ° C. Collected and analyzed by the same method as in Example 1 using liquid chromatography (HPLC). The results according to the various acrylic acid salts are shown in Table 1 below.

Acrylic acid salt % Conversion yield(%) Acrylic acid sodium salt 87.6 15.6 Acrylic acid-potassium salt 70.6 21.1 Acrylic acid-ammonium salt 86.3 18.7

Example  4

Rhodococcus Rhodococcus erythropolis LG12) Accession number KCTC 18102P strain Conversion reaction according to cell concentration.

Rhodococcus Rhodococcus erythropolis LG12) Accession No. KCTC 18102P strain was incubated in 100 mL of YEPD medium, and the cells were recovered by centrifugation and suspended in 10 mL of 0.1 M phosphate buffer (pH 7.0). Recovered Rhodococcus The erythropolis LG12 strain was examined to investigate the reactivity according to the cell concentration by changing the suspension cell suspension. Various acrylic acid salts (sodium) were fixed at a concentration of 1% (wt / vol, 10 g / L), and the samples were collected while reacting at 28 ° C. and 170 rpm for 15 hours, using liquid chromatography (HPLC). The method was analyzed. The results are shown in Table 2 below.

Rhodococcus erythropolis LG12 strain concentration (cell dry weight, mg / mL) % Conversion yield(%) 4.1mg / mL 24% 31.5 15.6 mg / mL 48% 48.6 31.2 mg / mL 52% 70.3

Example  5

Conversion of Rhodococcus erythropolis LG12 Accession No. KCTC 18102P Strains According to Acrylic Acid Concentration

The cells cultured in the same manner as in Example 4 were fixed at 15.6 mg / mL of the cell dry weight, and sampled while reacting for 15 hours at 170 ° C. and 170 rpm while changing the acrylic acid salt (HPLC). It was analyzed in the same manner as in Example 1. The results are shown in Table 3 below.

Acrylic acid concentration (wt% / vol) % Conversion yield(%) 1.0 48% 48.6 2.0 28% 37.8 4.0 26.3% 18.3

Rhodococcus Rhodococcus invention strain Rhodococcus erythropolis as seen through the above embodiment erythropolis LG12 ) was able to produce 3-HP from acrylic acid under various culture and reaction conditions.

Claims (4)

Rhodococcus Rhodococcus erythropolis LG12 ) Process for producing 3-HP, characterized by contacting accession no. KCTC 18102P with acrylic acid to produce 3-hydroxypropionic acid (3-HP) from acrylic acid. The method of claim 1, wherein the contact with acrylic acid is characterized in that acrylic acid is added to the culture medium obtained by culturing Rhodococcus erythropolis LG12 accession No. KCTC 18102P in a nutrient medium while the microorganism is in the growth stage. 3-HP production method. The method according to claim 1, wherein the contact with acrylic acid is made by separating microorganisms from a culture medium obtained by culturing Rhodococcus erythropolis LG12 accession number KCTC 18102P in a nutrient medium to make a cell suspension, and then adding acrylic acid thereto. 3-HP production method characterized in that. Rhodococcus of claim 1 Rhodococcus erythropolis LG12 ) Accession No. KCTC 18102P is a production method of 3-HP, characterized in that it is used in the form of free cells or in the form of cells immobilized on a carrier.

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