KR20210050479A - Process for preparing ursodeoxycholic acid using deep eutectic solvent - Google Patents

Abstract

The present invention relates to: a composition for producing ursodeoxycholic acid (UDCA), containing a deep eutectic solvent, a 7 alpha-hydroxy steroid dehydrogenase (7αHSDH), and a 7 beta-hydroxy steroid dehydrogenase (7βHSDH); a kit for producing the UDCA including the composition; and a method for producing the UDCA from chenodeoxycholic acid (CDCA) by using 7αHSDH and 7βHSDH in the presence of a reaction solvent including the deep eutectic solvent. A composition for producing the UDCA according to the present invention can be used to efficiently produce the UDCA from the CDCA by using an enzymatic reaction in which a reverse reaction does not occur, and thus can be widely used in the production of various products using the UDCA.

Description

Method for preparing ursodeoxycholic acid using a eutectic solvent {Process for preparing ursodeoxycholic acid using deep eutectic solvent}

The present invention relates to a method for preparing ursodeoxycholic acid using a eutectic solvent, and more specifically, the present invention relates to a eutectic solvent, 7αHSDH (7 alpha-hydroxy steroid dehydrogenase), and 7βHSDH (7 beta-hydroxy steroid dehydrogenase) containing UDCA. A composition for producing (ursodeoxycholic acid), a kit for producing ursodeoxycholic acid (UDCA) containing the composition, and a method for producing UDCA from CDCA (Chenodeoxycholic acid) using 7αHSDH and 7βHSDH under the conditions of a reaction solvent including a eutectic solvent will be.

Bile acids are biomolecules necessary for digestion and absorption of fats, fatty acids and hydrophobic vitamins. The bile acid found only in small amounts in humans is ursodeoxycholic acid (UDCA). It has recently gained great therapeutic importance in the dissolution of cholesterol-containing gallstones. These compounds are produced industrially in tonnage quantities in chemical or enzymatic steps. An important precursor for the synthesis of UDCA is 12-ketoursodeoxycholic acid, which can be converted to UDCA by Wolf-Kishner reduction. The synthetic pathway of 12-ketoursodeoxycholic acid described in the literature begins with cholic acid (3α,7α,12α-trihydroxy-5β-cholanic acid), which is catalyzed by 7α-HSDH and 12α-HSDH. It can be prepared by two oxidation steps, and one reduction step catalyzed by 7β-HSDH. An additional pathway begins with 7-ketocholic acid, which can be converted to UDCA by stereoselective reduction of the 7-keto group; This step is also advantageously carried out using enzymatic catalysis catalyzed by 7β-HSDH. An additional advantageous synthetic route begins with dehydrocholic acid (DHCA), which can be converted to 12-ketoursodeoxycholic acid by two reduction steps; These two steps can be catalyzed by two stereoselective HSDHs (3α-HSDH and 7β-HSDH).

Various studies are being conducted to improve the synthesis pathway of UDCA. For example, Korean Patent No. 10-2023208 discloses a method for synthesizing UDCA using a 7β-hydroxysteroid dehydrogenase mutant, and Korean Patent Publication No. 10-2017-0036797 discloses a new 3α -A method of synthesizing UDCA using a hydroxysteroid dehydrogenase mutant is disclosed.

Under this background, the present inventors have made intensive research efforts to develop a method for producing UDCA more easily. As a result, when using a eutectic solvent, it has been confirmed that UDCA can be effectively produced by a one-pot reaction. The invention was completed.

The main object of the present invention is to provide a composition for producing ursodeoxycholic acid (UDCA) comprising a eutectic solvent, 7αHSDH (7 alpha-hydroxy steroid dehydrogenase) and 7βHSDH (7 beta-hydroxy steroid dehydrogenase).

Another object of the present invention is to provide a kit for producing UDCA (ursodeoxycholic acid) comprising the composition.

Another object of the present invention is to provide a method for producing UDCA from CDCA (Chenodeoxycholic acid) using 7αHSDH and 7βHSDH under the conditions of a reaction solvent containing a eutectic solvent.

One embodiment of the present invention for achieving the above object provides a composition for producing UDCA (ursodeoxycholic acid) comprising a eutectic solvent, 7αHSDH (7 alpha-hydroxy steroid dehydrogenase) and 7βHSDH (7 beta-hydroxy steroid dehydrogenase). .

The term "deep eutectic solvent" of the present invention means a substance that becomes liquid at a temperature lower than the melting point of each compound when a solid or liquid compound existing in nature is mixed in an appropriate ratio at room temperature. However, since it is cheaper than organic solvents, is easy to produce, and is composed of biodegradable materials, it is used for various materials development.

In the present invention, the eutectic solvent may be one containing HBA (Hydrogen bonding acceptors) and HBD (Hydrogen Bonding Donor), and as the HBA, betaine monohydrate may be used, and as the HBD, propylene glycol Can be used.

The term "7αHSDH (7 alpha-hydroxy steroid dehydrogenase)" of the present invention means an enzyme exhibiting the activity of converting the CDCA into 7-keto LCA (7-Ketolithocholic acid).

In the present invention, the 7αHSDH is not particularly limited thereto, but as an example, it may be derived from the Escherichia coli strain, and as another example, GenBank No. It could be of SMB28026.1.

In order to perform the reaction of converting CDCA to 7-keto LCA using the 7αHSDH, in addition to 7αHSDH and CDCA, NAD ⁺ for activating 7αHSDH, LDH involved in the production of NAD ^{+ and sodium for activating the LDH} Needs pyruvate.

The term "7βHSDH (7 beta-hydroxy steroid dehydrogenase)" of the present invention means an enzyme exhibiting the activity of converting 7-keto LCA into UDCA/

In the present invention, the 7βHSDH is not particularly limited thereto, but as an example, it may be derived from the Ruminococcus gnavus strain, and as another example, GenBank No. It could be of SMB28026.1.

In order to perform the reaction of converting 7-keto LCA to UDCA using the 7βHSDH, in addition to 7βHSDH and 7-keto LCA, NADP ⁺ for activating 7βHSDH, GDH involved in the production of the NADP ^{+ and the GDH are activated.} It needs glucose to make it.

As described above, since 7αHSDH converts CDCA to 7-keto LCA, and 7βHSDH converts the 7-keto LCA to UDCA, the composition of the present invention comprising the 7αHSDH and 7βHSDH is used to convert CDCA to UDCA. You can (Fig. 1).

1 is a schematic diagram showing a process for producing UDCA from CDCA using 7αHSDH and 7βHSDH.

The 7αHSDH and 7βHSDH exhibit enzymatic activity even under general buffer conditions such as phosphate buffer, so that CDCA can be converted to UDCA, but when a eutectic solvent is used, the conversion rate to UDCA can be improved.

Another embodiment of the present invention provides a kit for producing ursodeoxycholic acid (UDCA) comprising the composition.

The kit for producing UDCA of the present invention may be used to produce UDCA from CDCA, including the composition for producing UDCA, but is not particularly limited thereto, but one or more other constituent compositions, solutions, or devices suitable for the reaction are Can be included.

^{As a specific example, NAD +} , NADP ⁺ , LDH, GDH, sodium pyruvate, glucose, etc. required for 7αHSDH and 7βHSDH reactions included in the composition may be additionally included.

In addition, it may further include a phosphate buffer solution required for the reaction, a reaction vessel for performing the reaction, a timer for performing the reaction, and the like.

Another embodiment of the present invention provides a method of producing UDCA from CDCA by a two-step reaction using the composition.

Specifically, the method of producing UDCA from CDCA by a two-step reaction is (a) 7-keto by reacting CDCA (Chenodeoxycholic acid) and 7αHSDH (7 alpha-hydroxy steroid dehydrogenase) under the conditions of a reaction solvent containing a eutectic solvent. Obtaining LCA (7-Ketolithocholic acid); And, (b) reacting the obtained 7-keto LCA with 7βHSDH (7 beta-hydroxy steroid dehydrogenase) under conditions of a reaction solvent including a eutectic solvent, thereby obtaining UDCA (ursodeoxycholic acid).

The eutectic solvent used in step (a) includes betaine hydrate (HBA) and propylene glycol (HBD), but rather than using the eutectic solvent alone, a reaction solvent including the eutectic solvent and phosphate buffer can be used. have.

The content of the eutectic solvent contained in the reaction solvent is not particularly limited thereto, but as an example, it may be 1 to 40% (v/v), and as another example, it may be 10 to 30% (v/v). And, as another example, it may be 20% (v/v).

In addition, in order to perform the 7αHSDH enzymatic reaction, in addition to CDCA used as a substrate, NAD ⁺ , LDH and sodium pyruvate may be used.

The eutectic solvent used in step (b) includes betaine hydrate (HBA) and propylene glycol (HBD), but rather than using the eutectic solvent alone, a reaction solvent including the eutectic solvent and phosphate buffer can be used. have.

The content of the eutectic solvent contained in the reaction solvent is not particularly limited thereto, but as an example, it may be 1 to 50% (v/v), and as another example, it may be 10 to 40% (v/v). And, as another example, it may be 30% (v/v).

In addition, in order to perform the 7βHSDH enzymatic reaction, in addition to 7-keto LCA used as a substrate, NADP ⁺ , GDH and glucose may be used under conditions containing a eutectic solvent.

On the other hand, when the step (b) is performed, gelation is performed by gluconic acid generated from glucose, so that the final yield of UDCA may be reduced.

In order to prevent this, it may further include a step of titration so that the pH of the reactant maintains pH 8.0 while performing the reaction.

Another embodiment of the present invention provides a method of producing UDCA from CDCA by a one-pot reaction using the composition.

Specifically, the method of producing UDCA from CDCA by a one-pot reaction is in CDCA (Chenodeoxycholic acid), 7αHSDH (7 alpha-hydroxy steroid dehydrogenase) and 7βHSDH ( 7 beta-hydroxy steroid dehydrogenase) is added together to react.

The eutectic solvent used to perform the one-pot reaction includes betaine hydrate (HBA) and propylene glycol (HBD). Rather than using the eutectic solvent alone, the eutectic solvent and the phosphate buffer solution are used. It is possible to use a reaction solvent containing.

The content of the eutectic solvent contained in the reaction solvent is not particularly limited thereto, but as an example, it may be 1 to 40% (v/v), and as another example, it may be 10 to 30% (v/v). And, as another example, it may be 20% (v/v).

In addition, in addition to CDCA used as a substrate, NAD ⁺ , NADP ⁺ , LDH, GDH, sodium pyruvate and glucose can be used under conditions containing a eutectic solvent.

On the other hand, as described above, since gelation is performed by gluconic acid generated from glucose, the final yield of UDCA can be reduced. To prevent this, pH 8.0 is maintained while performing the reaction. It may further include a step of titration to do so.

If the composition for producing UDCA provided by the present invention is used, since UDCA can be produced in high yield from CDCA using an enzymatic reaction in which the reverse reaction is not performed, it will be widely used in the production of various products using UDCA.

1 is a schematic diagram showing a process for producing UDCA from CDCA using 7αHSDH and 7βHSDH.
2A is a graph showing the result of comparing the change in the production yield of 7-keto LCA according to the treatment concentration of 7αHSDH when 7αHSDH and LDH are used to generate 7-keto LCA from CDCA.
2B is a graph showing the result of comparing the change in the production yield of 7-keto LCA according to the treatment concentration of LDH when 7αHSDH and LDH are used to generate 7-keto LCA from CDCA.
3A is a graph comparing the change in concentration of CDCA and 7-keto LCA over time of a reaction for generating 7-keto LCA from CDCA using 7αHSDH and LDH in phosphate buffer conditions.
3B is a graph comparing the change in concentration of 7-keto LCA and UDCA over time of the reaction for generating UDCA from 7-keto LCA using 7βHSDH and GDH in a phosphate buffer condition.
Figure 3c is a graph comparing the concentration change of CDCA, 7-keto LCA and UDCA over time of the reaction to generate UDCA from CDCA by a stepwise reaction using 7αHSDH, LDH, 7βHSDH and GDH in a phosphate buffer condition.
Figure 3d shows the change in the concentration of CDCA, 7-keto LCA and UDCA over time of the reaction to generate UDCA from CDCA by a one-pot reaction using 7αHSDH, LDH, 7βHSDH and GDH in phosphate buffer conditions. This is a comparison graph.
3E is a photograph showing the result of confirming whether or not a white precipitate is generated by adding gluconic acid to 7-keto LCA.
3F is a photograph showing the result of confirming whether or not a white precipitate is generated by adding gluconic acid to UDCA.
3G is a graph showing the result of measuring the pH change of the reaction solutions of Experimental Groups 1 and 2 as the reaction time elapsed.
3H is a graph showing the level of change in the UDCA conversion rate measured in the reaction solutions of Experimental Groups 1, 2 and 3 over the course of the reaction time.
3i is a graph showing the results of comparing the UDCA conversion rates measured in the reaction solutions of Experimental Groups 1, 2 and 3 after 9 hours elapsed.
4 is a photograph and graph showing the experimental process and results of selecting a eutectic solvent for producing UDCA from CDCA by a one-pot reaction.
5 is a graph showing the result of comparing the conversion rate of 7-keto LCA converted by the 7αHSDH enzymatic reaction using a eutectic solvent of various concentrations (20, 40 or 60%) over the course of the reaction time.
6A is a graph showing the result of comparing the conversion rate of UDCA converted by the 7βHSDH enzymatic reaction using a eutectic solvent of various concentrations (20, 40 or 60%) over the course of the reaction time.
6B is a photograph showing the result of photographing the reaction product obtained by performing the 7βHSDH enzymatic reaction using 0 to 20% eutectic solvent.
Figure 7a is the concentration of CDCA, 7-keto LCA and UDCA over time of the reaction to generate UDCA from CDCA by a one-pot reaction using 7αHSDH, LDH, 7βHSDH and GDH in a 20% eutectic solvent condition. This is a graph comparing changes.
7B is a graph showing the result of comparing the final conversion (molar ratio) of CDCA, 7-keto LCA, and UDCA when performing a one-pot reaction using a eutectic solvent of various concentrations.
Figure 7c is a case of performing a single reaction (one-pot reaction) using 0, 20, 40 or 60% eutectic solvent, the conversion rate (molar ratio) of CDCA, 7-keto LCA and UDCA according to the lapse of reaction time It is a graph showing the result of comparing the change.
Figure 7d is a graph showing the result of comparing the final conversion (molar ratio) of UDCA in the case of performing a one-pot reaction using a eutectic solvent of various concentrations under the condition of increasing the concentration of 7βHSDH and GDH. .
Figure 7e is a single reaction (one-pot reaction) using a eutectic solvent of 0, 20 or 40% under the condition of increasing the concentration of 7βHSDH and GDH, the conversion rate of UDCA (molar ratio) according to the course of the reaction time ) Is a graph showing the result of comparing the change.
Figure 7f is a single reaction (one-pot reaction) using a eutectic solvent of 20% under the condition of increasing the concentration of each component involved in the single reaction (one-pot reaction), the elapse of the reaction time It is a graph showing the result of comparing the change in the conversion rate (molar ratio) of CDCA, 7-keto LCA and UDCA according to this.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Enzymatic reaction of 7αHSDH (7 alpha-hydroxy steroid dehydrogenase) using LDH

When 7αHSDH and LDH were used in a buffer solution (50mM potassium phosphate, pH 8) to generate 7-keto LCA from CDCA, we tried to calculate the optimal concentrations of 7αHSDH and LDH, which can represent a high production yield.

First, CDCA 50 mM, NAD ⁺ 5 mM, sodium pyruvate 50 mM and LDH 0.1 unit / buffer containing mL various concentrations (0.5, 1, 2, 4 or 8 unit / mL) to (50mM potassium phosphate, pH 8) 7αHSDH was added to react, and the yield of 7-keto LCA produced by the reaction was compared (FIG. 2A).

2A is a graph showing the result of comparing the change in the production yield of 7-keto LCA according to the treatment concentration of 7αHSDH when 7αHSDH and LDH are used to generate 7-keto LCA from CDCA.

As shown in Figure 2a, when 0.5 to 2 unit/mL of 7αHSDH was added, the production yield of 7-keto LCA increased in proportion to the treatment concentration of 7αHSDH, but when 7αHSDH of 4 unit/mL or more was added, the 7αHSDH was no longer 7- It was confirmed that the production yield of keto LCA did not increase.

Therefore, when generating 7-keto LCA from CDCA, it was found that the optimal treatment concentration of 7αHSDH was 2 unit/mL.

Next, in a buffer solution (50mM potassium phosphate, pH 8) containing 50 mM CDCA, NAD ⁺ 5 mM, 50 mM pyruvate, and 2 unit/mL of 7αHSDH, various concentrations (0, 0.01, 0.05, 0.1, 0.5 or 1 unit/mL) mL) of LDH was added to react, and the yield of 7-keto LCA produced by the reaction was compared (Fig. 2b).

2B is a graph showing the result of comparing the change in the production yield of 7-keto LCA according to the treatment concentration of LDH when 7αHSDH and LDH are used to generate 7-keto LCA from CDCA.

As shown in Fig. 2b, when 0 to 0.1 unit/mL of LDH was added, the production yield of 7-keto LCA increased in proportion to the treatment concentration of LDH, but when more than 0.5 unit/mL LDH was added, it was no longer 7- It was confirmed that the production yield of keto LCA did not increase.

Therefore, when generating 7-keto LCA from CDCA, it was found that the optimal treatment concentration of LDH was 0.1 unit/mL.

Example 2: Generation of UDCA using 7αHSDH and 7βHSDH enzymatic reaction

Example 2-1: 7αHSDH enzyme reaction

^{CDCA 20 g/L, NAD +} 5 mM, sodium pyruvate 50 mM, LDH 0.1 unit/mL, and 7αHSDH 2 unit/mL were added to a buffer solution (50 mM potassium phosphate, pH 8), and reacted for 14 hours, and then the reaction time Changes in the concentration of CDCA and 7-keto LCA according to the course were measured (Fig. 3a).

3A is a graph comparing the change in concentration of CDCA and 7-keto LCA over time of a reaction for generating 7-keto LCA from CDCA using 7αHSDH and LDH in phosphate buffer conditions.

As shown in Figure 3a, it was confirmed that the conversion rate of 7-keto LCA was about 87%.

Example 2-2: 7βHSDH (7 beta-hydroxy steroid dehydrogenase) enzyme reaction

^{7-keto LCA, NADP +} 1 mM, glucose 100mM, GDH 0.1 unit/mL, and 7βHSDH 2 unit/mL were added to a buffer solution (50 mM potassium phosphate, pH 8), and reacted for 4 hours. Changes in the concentration of 7-keto LCA and UDCA were measured (Fig. 3b).

3B is a graph comparing the change in concentration of 7-keto LCA and UDCA over time of the reaction for generating UDCA from 7-keto LCA using 7βHSDH and GDH in a phosphate buffer condition.

As shown in FIG. 3B, the reaction was terminated at the time point 30 minutes elapsed, and it was confirmed that the conversion rate of UDCA was about 48%.

However, it was confirmed that the reaction product was gelled.

Example 2-3: cascade enzyme reaction of 7αHSDH and 7βHSDH

^{CDCA 20 g/L, NAD +} 5 mM, sodium pyruvate 50 mM, LDH 0.1 unit/mL, and 7αHSDH 2 unit/mL were added to a buffer solution (50 mM potassium phosphate, pH 8), and reacted for 14 hours, and then the reaction product was heated. Thus, enzyme activity was removed. Then, NADP ⁺ 1 mM, glucose 100mM, GDH 0.1 unit/mL, and 7βHSDH 2 unit/mL were added to the reaction product, and after reacting for 4 hours, the concentration of CDCA, 7-keto LCA and UDCA according to the course of the reaction time The change was measured (Fig. 3c).

Figure 3c is a graph comparing the concentration change of CDCA, 7-keto LCA and UDCA over time of the reaction to generate UDCA from CDCA by a stepwise reaction using 7αHSDH, LDH, 7βHSDH and GDH in a phosphate buffer condition.

As shown in Figure 3c, it was confirmed that the conversion rate of UDCA was about 48%.

Example 2-4: One pot enzyme reaction of 7αHSDH and 7βHSDH

CDCA 20 g/L, NAD ⁺ 5 mM, NADP ⁺ 1 mM, sodium pyruvate 50 mM, glucose 100 mM, LDH 0.1 unit/mL, GDH 0.1 unit/mL, 7αHSDH 2 unit/ After adding mL and 2 unit/mL of 7βHSDH and reacting for 14 hours, changes in the concentration of CDCA, 7-keto LCA and UDCA were measured with the passage of the reaction time (FIG. 3D).

Figure 3d shows the change in concentration of CDCA, 7-keto LCA and UDCA over time of the reaction to generate UDCA from CDCA by a one-pot reaction using 7αHSDH, LDH, 7βHSDH and GDH in phosphate buffer conditions. This is a comparison graph.

As shown in FIG. 3D, UDCA was generated until 2 hours elapsed, but after that, UDCA was no longer generated, and only 7-keto LCA was generated, and it was confirmed that the conversion rate of UDCA was about 26%.

Example 2-5: Study on the gelation phenomenon that occurs during the 7βHSDH enzyme reaction

As shown in Example 2-2, it was confirmed that the reaction product gelled during the 7βHSDH enzyme reaction, and the cause of the gelation and the effect of the gelling reaction were to be investigated.

First, it was assumed that the cause of the gelation was gluconic acid converted from glucose, and this was to be confirmed.

Approximately, 20 g/L of 7-keto LCA or UDCA was added to a phosphate buffer (50 mM potassium phosphate, pH 8), followed by various concentrations (0, 10, 25, 50, 75 or 100 mM) of gluconic acid. Addition, it was confirmed whether a white precipitate was formed (Figs. 3e and 3f).

Figure 3e is a photograph showing the result of confirming whether a white precipitate is generated by adding gluconic acid to 7-keto LCA, and Figure 3f is a photograph showing the result of confirming whether a white precipitate is generated by adding gluconic acid to UDCA .

As shown in Figures 3e and 3f, when 10 mM of gluconic acid is added, it was confirmed that white precipitates were formed in 7-keto LCA and UDCA.

Since it was confirmed that the pH decreased in proportion to the treatment concentration of gluconic acid, it was analyzed that white precipitates were formed in 7-keto LCA and UDCA due to the decrease in pH due to the addition of gluconic acid.

In addition, it was attempted to confirm whether the 7βHSDH enzymatic reaction was affected by the gelation.

Roughly, three kinds of phosphate buffers were prepared, one 50 mM phosphate buffer (Experiment Group 1), the other 100 mM phosphate buffer (Experiment Group 2), and the other 50 mM phosphate buffer solution, but the 7βHSDH enzyme reaction process During the reaction time, a buffer solution (Experiment Group 3) maintaining pH 8.0 was prepared by performing a continuous titration.

To each of the three prepared phosphate buffers, 7-keto LCA 20 g/L, NADP ⁺ 1 mM, glucose 100 mM, GDH 0.1 unit/mL, and 7βHSDH 2 unit/mL were added, reacted for 9 hours, and then the reaction time The change in the concentration of UDCA according to the course of was measured, and the conversion rate of UDCA was calculated from this (Figs. 3g, 3h and 3i).

Figure 3g is a graph showing the result of measuring the pH change of the reaction solutions in Experimental Groups 1 and 2 with the passage of the reaction time, and Figure 3h is the UDCA measured in the reaction solutions of Experimental groups 1, 2 and 3 with the passage of the reaction time. It is a graph showing the level of change in the conversion rate, and FIG. 3i is a graph showing the result of comparing the UDCA conversion rate measured in the reaction solutions of Experimental Groups 1, 2 and 3 after 9 hours have elapsed.

As shown in Figs. 3g, 3h and 3i, the conversion reaction was terminated when 1 hour elapsed from the start of the reaction, and it was confirmed that the pH of the reactant decreased to 7.1 during the conversion reaction, and then gradually increased.

In addition, it was confirmed that the UDCA conversion rate was relatively high when the phosphate buffer was continuously appropriate for the reaction time, rather than the UDCA conversion rate when 50 mM phosphate buffer and 100 mM phosphate buffer were used.

Therefore, it was analyzed that the conversion rate of UDCA could be increased by suppressing the gelation phenomenon that occurs during the 7βHSDH enzyme reaction.

Example 3: Screening of deep eutectic solvent DES

As shown in the results of Example 2, in performing the method of producing UDCA (ursodeoxycholic acid) from CDCA (Chenodeoxycholic acid) in a one pot reaction using 7αHSDH and 7βHSDH, the phosphate buffer solution was inappropriate. As confirmed, it was attempted to select a eutectic solvent (DES) that could be used more appropriately.

Accordingly, choline chloride or betaine monohydrate is used as a candidate material for HBA (hydrogen bonding acceptor), and ethylene glycol (EG), glycerol ( Gly) or propylene glycol (PG) was used to select a eutectic solvent capable of dissolving CDCA.

First, in order to select a component suitable for HBA, the reaction conversion rate of choline chloride or betaine hydrate was compared.

As a result, it was confirmed that betaine hydrate showed a relatively high level of reaction conversion rate than choline chloride, and betaine hydrate was selected as HBA.

Next, in order to select HBD suitable for use with the selected betaine hydrate, each eutectic mixture of the betaine hydrate and the HBD candidate material at 1:1, 1:2 or 1:3 (molar ratio), respectively. A solvent was prepared. Then, CDCA, 7αHSDH and 7βHSDH were added to each of the prepared eutectic solvents to react, and then the concentration of CDCA, 7-keto-LCA or UDCA contained in the reaction product was measured and compared (FIG. 4 ).

4 is a photograph and graph showing the experimental process and results of selecting a eutectic solvent for producing UDCA from CDCA by a one-pot reaction.

As shown in FIG. 4, the case where the precipitate was generated by mixing the betaine hydrate and the HBD candidate by molar ratio was excluded because it did not proceed normally. It was confirmed that UDCA was produced only when a eutectic solvent in which betaine hydrate and propylene glycol (PG) were mixed in a ratio of 1:3 (molar ratio) was used among the reaction products in which the normal reaction proceeded.

Therefore, it was found that it is preferable to use a solvent in which betaine hydrate and propylene glycol (PG) are mixed at 1:3 (molar ratio) as a eutectic solvent for producing UDCA from CDCA by a one-pot reaction. Could.

Example 4: 7αHSDH enzyme reaction in eutectic solvent

7αHSDH enzymatic reaction was performed using the eutectic solvent selected in Example 3, and an appropriate concentration of the eutectic solvent was determined.

Approximately, by mixing the eutectic solvent and phosphate buffer (50mM potassium phosphate, pH 8), each reaction solvent having a concentration of the eutectic solvent of 0, 10, 20, 30, 40, 50 or 60% was prepared. CDCA 20 g/L, NAD ⁺ 5 mM, sodium pyruvate 50 mM, LDH 0.1 unit/mL, and 7αHSDH 2 unit/mL were added to each of the prepared reaction solvents, and reacted for 14 hours, followed by 7 The change in concentration of -keto LCA was measured, and the conversion rate of 7-keto LCA was calculated from this (FIG. 5 and Table 1).

5 is a graph showing the result of comparing the conversion rate of 7-keto LCA converted by the 7αHSDH enzymatic reaction using a eutectic solvent of various concentrations (20, 40 or 60%) over the course of the reaction time.

7-keto LCA conversion rate by eutectic solvent concentration Eutectic solvent concentration (%) Conversion rate (%) 0
10
20
30
40
50
60 87
90
93
90
71
24
8

As shown in Fig. 5 and Table 1, it was confirmed that the conversion rate of 7-keto LCA by the 7αHSDH enzyme reaction was changed according to the concentration of the eutectic solvent.

In particular, when the concentration of the eutectic solvent increased to 20%, the conversion rate of 7-keto LCA increased proportionally, showing the maximum conversion rate at 20%. However, when the concentration of the eutectic solvent increased further, the conversion rate of 7-keto LCA It can be seen that this decreases.

Therefore, it was found that the concentration of the eutectic solvent for optimizing the 7αHSDH enzymatic reaction was 20%.

Example 5: 7βHSDH enzyme reaction in eutectic solvent

7βHSDH enzymatic reaction was performed using the eutectic solvent selected in Example 3, and an appropriate concentration of the eutectic solvent was determined.

Approximately, by mixing the eutectic solvent and phosphate buffer (50mM potassium phosphate, pH 8), each reaction solvent having a concentration of the eutectic solvent of 0, 10, 20, 30, 40, 50 or 60% was prepared. To each of the prepared reaction solvents, 7-keto LCA 20 g/L, NADP ⁺ 1 mM, glucose 100 mM, GDH 0.1 unit/mL, and 7βHSDH 2 unit/mL were added, reacted for 6 hours, and then the reaction time elapsed. The change in the concentration of UDCA was measured, and the conversion rate of UDCA was calculated from this (FIG. 6A and Table 2).

6A is a graph showing the result of comparing the conversion rate of UDCA converted by the 7βHSDH enzymatic reaction using a eutectic solvent of various concentrations (20, 40 or 60%) over the course of the reaction time.

UDCA conversion rate by eutectic solvent concentration Eutectic solvent concentration (%) Conversion rate (%) 0
10
20
30
40
50
60 48
82
88
98
99
64
13

6A and Table 2, it was confirmed that the conversion rate of UDCA by the 7βHSDH enzyme reaction was changed according to the concentration of the eutectic solvent.

In particular, when the concentration of the eutectic solvent increased to 40%, the conversion rate of UDCA increased in proportion to this, showing the maximum conversion rate at 40%, but it was found that the conversion rate of UDCA decreased as the concentration of the eutectic solvent increased further. .

However, as shown in the reaction graph using 20% eutectic solvent and the reaction graph using 40% eutectic solvent, it was confirmed that the reaction termination time was delayed when 40% eutectic solvent was used.

Therefore, it was found that the concentration of the eutectic solvent for optimizing the 7βHSDH enzymatic reaction was 40% or less.

On the other hand, when the 7βHSDH enzymatic reaction was performed for 1.5 hours, it was confirmed that the reaction product was gelled (Fig. 6b).

6B is a photograph showing the result of photographing a reaction product obtained by performing a 7βHSDH enzymatic reaction using 0 to 20% eutectic solvent.

Example 6: One pot reaction using a eutectic solvent

Example 6-1: One-pot reaction using a eutectic solvent

According to the results of Examples 4 and 5, it was confirmed that the concentration of the eutectic solvent capable of satisfying both 7αHSDH and 7βHSDH enzymatic reaction was 20%, and a single reaction using the 20% eutectic solvent Was intended to convert CDCA to UDCA.

Approximately, 20 g/L of CDCA, NAD ⁺ 5 mM, NADP ⁺ 1 mM, sodium pyruvate 50 mM, glucose 100 mM, LDH 0.1 unit/mL, GDH 0.1 unit/mL, 7αHSDH 2 unit/mL and 7βHSDH 2 unit/mL was added, and after reacting for 14 hours, changes in the concentration of CDCA, 7-keto LCA, and UDCA were measured with the passage of the reaction time (FIG. 7A).

Figure 7a is the concentration of CDCA, 7-keto LCA, and UDCA over time of the reaction to generate UDCA from CDCA by a one-pot reaction using 7αHSDH, LDH, 7βHSDH and GDH under 20% eutectic solvent conditions. This is a graph comparing the changes.

As shown in FIG. 7A, the concentration of 7-keto LCA, an intermediate product in the reaction product, was maintained at 2.5% (molar ratio) or less, and it was confirmed that the final conversion rate of UDCA was about 89%.

Example 6-2: Optimal concentration of eutectic solvent for one-pot reaction

In order to compare the conversion rate of UDCA when performing a one-pot reaction using different concentrations of eutectic solvent, a one-pot reaction was performed using various concentrations of eutectic solvent.

^{Approximately, 20 g/L of CDCA, NAD +} 5 mM, NADP ⁺ 1 mM, sodium pyruvate 50 mM, glucose 100 mM, LDH in eutectic solvents of various concentrations (0, 10, 20, 30, 40, 50 or 60%) 0.1 unit/mL, GDH 0.1 unit/mL, 7αHSDH 2 unit/mL and 7βHSDH 2 unit/mL were added and reacted for 14 hours. It was measured (Figs. 7b, 7c and Table 3).

Figure 7b is a graph showing the result of comparing the final conversion (molar ratio) of CDCA, 7-keto LCA and UDCA when performing a one-pot reaction using a eutectic solvent of various concentrations, Figure 7c is When a one-pot reaction is performed using 0, 20, 40 or 60% eutectic solvent, the change in the conversion rate (molar ratio) of CDCA, 7-keto LCA and UDCA according to the elapse of the reaction time is compared. It is a graph showing one result.

UDCA conversion rate by concentration of eutectic solvent in one-pot reaction Eutectic solvent concentration (%) Conversion rate (%) 0
10
20
30
40
50
60 26
77
89
85
65
18
6

7b, 7c and Table 3, it was confirmed that the conversion rate of UDCA was changed by a one-pot reaction according to the concentration of the eutectic solvent.

In particular, when the concentration of the eutectic solvent increased to 20%, the conversion rate of UDCA increased in proportion to this, showing the maximum conversion rate at 20%, but it was found that the conversion rate of UDCA decreased as the concentration of the eutectic solvent increased further. .

Therefore, it was found that the concentration of the eutectic solvent for optimizing the one-pot reaction was 20%.

Example 6-3: One-pot reaction with changing 7βHSDH enzymatic reaction conditions

In order to analyze the contribution of the 7βHSDH enzymatic reaction to the one-pot reaction result, a one-pot reaction was performed under the condition of increasing the concentrations of 7βHSDH and GDH involved in the 7βHSDH enzymatic reaction.

^{Approximately, 20 g/L of CDCA, NAD +} 5 mM, NADP ⁺ 1 mM, sodium pyruvate 50 mM, glucose 100 mM, LDH in eutectic solvents of various concentrations (0, 10, 20, 30, 40, 50 or 60%) 0.1 unit/mL, GDH 1 unit/mL, 7αHSDH 2 unit/mL, and 7βHSDH 10 unit/mL were added and reacted for 14 hours, and then the change in the concentration of UDCA with the passage of the reaction time was measured (Figs. 7d and 7e. ).

7D is a graph showing the result of comparing the final conversion rate (molar ratio) of UDCA in the case of performing a one-pot reaction using a eutectic solvent of various concentrations under the condition of increasing the concentration of 7βHSDH and GDH. 7E is a case in which a one-pot reaction was performed using a eutectic solvent of 0, 20 or 40% under the condition of increasing the concentration of 7βHSDH and GDH, the conversion rate of UDCA according to the course of the reaction time ( This is a graph showing the result of comparing the change in molar ratio).

Comparing the results of FIG. 7b with the results of FIG. 7d, it was confirmed that the maximum conversion rate of UDCA by a one-pot reaction increased from 89% to 93% under the condition of increasing the concentration of 7βHSDH and GDH.

In addition, when comparing the results of FIG. 7c with the results of FIG. 7e, it was confirmed that the reaction time for ending the conversion of UDCA was shortened from 7 hours to 2 hours.

Therefore, it was analyzed that if the concentration of each component was changed during a one-pot reaction, the final conversion rate and reaction time of UDCA could be controlled.

Example 6-4: Increasing the conversion rate of UDCA according to the adjustment of the reaction conditions during a one-pot reaction

From the results of Example 6-3, it was analyzed that when the concentration of each component was changed when performing a one-pot reaction, the final conversion rate and reaction time of the final UDCA could be controlled, so the concentration of each component was A single-pot reaction was performed under the increased conditions (2.5 times).

Approximately, 50 g/L of CDCA, NAD ⁺ 5 mM, NADP ⁺ 1 mM, sodium pyruvate 125 mM, glucose 250 mM, LDH 0.25 unit/mL, GDH 0.25 unit/mL, 7αHSDH 5 unit/mL and 7βHSDH 5 unit/mL was added, and after reacting for 18 hours, changes in the concentration of CDCA, 7-keto LCA, and UDCA were measured with the passage of the reaction time (FIG. 7f).

Figure 7f is a single reaction (one-pot reaction) using a eutectic solvent of 20% under the condition of increasing the concentration of each component involved in the single reaction (one-pot reaction), the elapse of the reaction time It is a graph showing the result of comparing the change in the conversion rate (molar ratio) of CDCA, 7-keto LCA, and UDCA according to the above.

As shown in FIG. 7f, it was confirmed that the conversion of UDCA was terminated at about 12 hours after the start of the reaction, and the conversion rate of the terminated UDCA was about 70%.

When the conversion of UDCA was completed, 7αHSDH and 7βHSDH were added to the reaction product and the pH of the reactant was titrated to 8.0. As a result, a one-pot reaction further proceeded to increase the conversion rate of UDCA, and about 6 hours. It was confirmed that the conversion of UDCA was terminated at the time when this addition elapsed, and the conversion rate of UDCA that was terminated was about 93%.

Therefore, when a high concentration of CDCA (50g/L), which can be practically used in a 20% eutectic solvent condition, was used as a substrate, it exhibited a high conversion rate of 93%, which is expected to have high industrial production potential.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

Claims (19)

A eutectic solvent, 7αHSDH (7 alpha-hydroxy steroid dehydrogenase) and 7βHSDH (7 beta-hydroxy steroid dehydrogenase) containing, UDCA (ursodeoxycholic acid) production composition.
The method of claim 1,
The eutectic solvent is HBA (Hydrogen bonding acceptors) and HBD (Hydrogen Bonding Donor) containing the, UDCA production composition.
The method of claim 4,
The HBA is a betaine hydrate (Betaine monohydrate) that, UDCA production composition.
The method of claim 4,
The HBD is propylene glycol, the composition for producing UDCA.
The method of claim 1,
The composition is used to produce UDCA using CDCA (Chenodeoxycholic acid) as a substrate, a composition for producing UDCA.
The method of claim 1,
The 7αHSDH represents the enzymatic activity of converting CDCA into 7-keto LCA (7-Ketolithocholic acid), and the 7βHSDH represents the enzymatic activity of converting 7-keto LCA into UDCA, a composition for UDCA production.
A kit for producing UDCA (ursodeoxycholic acid) comprising the composition of any one of claims 1 to 6.
(a) obtaining 7-keto LCA (7-Ketolithocholic acid) by reacting CDCA (Chenodeoxycholic acid) and 7αHSDH (7 alpha-hydroxy steroid dehydrogenase) under a reaction solvent condition including a eutectic solvent; And,
(b) a two-step reaction comprising the step of reacting the obtained 7-keto LCA with 7βHSDH (7 beta-hydroxy steroid dehydrogenase) under the reaction solvent condition containing a eutectic solvent to obtain UDCA (ursodeoxycholic acid). UDCA production method by.
The method of claim 8,
The (a) step is ^{to be performed by using NAD +} , LDH and pyruvate (sodium pyruvate) additionally.
The method of claim 8,
The (b) step is ^{to be performed by using NADP +} , GDH and glucose additionally.
The method of claim 8,
The eutectic solvent is a method comprising betaine hydrate (HBA) and propylene glycol (HBD).
The method of claim 8,
The reaction solvent in step (a) is 1 to 40% (v/v) of the eutectic solvent.
The method of claim 11,
The reaction solvent of the step (b) is 1 to 50% (v/v) of the eutectic solvent.
The method of claim 8,
When performing the step (b), the method further comprising the step of titration (titration) so that the reactant maintains a pH of 8.0.
One reaction comprising the step of reacting by adding 7αHSDH (7 alpha-hydroxy steroid dehydrogenase) and 7βHSDH (7 beta-hydroxy steroid dehydrogenase) to CDCA (Chenodeoxycholic acid) under the conditions of a reaction solvent including a eutectic solvent. -pot reaction) production method of UDCA (ursodeoxycholic acid).
The method of claim 15,
The eutectic solvent is a method comprising betaine hydrate (HBA) and propylene glycol (HBD).
The method of claim 15,
The reaction solvent is to contain a eutectic solvent of 1 to 40% (v / v), the method.
The method of claim 15,
The reaction is NAD ⁺ , NADP ⁺ , LDH, GDH, pyruvate (sodium pyruvate) and glucose are further used to perform the method.
The method of claim 15,
The method of claim 1, further comprising titrating the reaction to maintain a pH of 8.0.

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