CN112210580A - Method for reducing inhibition of reaction components on enzyme activity - Google Patents
Method for reducing inhibition of reaction components on enzyme activity Download PDFInfo
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Abstract
In order to solve the problem that cholic acid and derivatives thereof inhibit enzyme activity, the method for reducing the enzyme activity inhibition of reaction components reduces the enzyme activity inhibition of the cholic acid and derivatives thereof by adding resin into a reaction system, and improves the biocatalytic conversion efficiency. The invention also provides a method for increasing the substrate conversion concentration by adding the resin. Specifically, the concentration of a substrate added into a biological catalytic reaction system is 20-200g/L, the mass concentration of resin added is 4-40g/L, and the reaction can be completed after the conversion is carried out for 8-12 h. The resin is added to adsorb a substrate in a reaction system, and the resin can adsorb a product after the reaction along with the reaction, so that the dynamic balance of the substrate and the product adsorbed by the resin is achieved, the concentration of reaction components in a solution is reduced, and the inhibition on enzyme activity is reduced. The addition of the resin does not reduce the reaction rate, and under the same experimental conditions, the reaction time is effectively shortened, and the reaction efficiency is improved. After the reaction is completed, the resin can be separated from the reaction system by filtration, and the reaction product on the resin is eluted by using an eluent.
Description
Technical Field
The invention relates to a method for reducing enzyme activity inhibition by using resin to adsorb reaction components and reducing the proportion of a substrate and a product in a solution, and application of the resin to the conversion process of cholic acid compounds, belonging to the field of bioengineering.
Background
Cholic acid and its derivatives have wide biological activity and are endogenous natural products. Due to its high degree of optical purity and structural characteristics of amphiphilic molecules, biological activity studies have been one of the research hotspots in the fields of pharmacology and drug discovery. Cholic acid drugs have been used clinically, for example, ursodeoxycholic acid and adeoxycholic acid of cattle have been used for treating liver diseases (the effect of ursodeoxycholic acid combination on treating fatty liver complicated with hyperlipidemia, Weihong, 2017, 30, 133). In 2015, two cholic acid drugs, namely a Cholham capsule (the main component is cholic acid) for treating rare diseases of bile acid synthesis disorder and a fat-dissolving injection Kybella (the main component is deoxycholic acid) for submental fat, are approved by FDA to be on the market.
The traditional method for synthesizing cholic acid and derivatives thereof is chemical conversion, and in recent years, the preparation of cholic acid and derivatives thereof such as ursodeoxycholic acid by using a biological method is also better in literature (NAD)+-Dependent Enzymatic Route for the infection of Hydroxysteroids, Fabio Toni, Linda G.Otten, Isabel W.C.E.Arends, 2018,11, 1-13; flavin Oxidored reductase-medial Regeneration of noncotinamide Adenide Diurea with Dioxygen and Catalytic amino of Flavin monoglucoside for One-Point Multi-enzyme Preparation of Ursoxyolic Acid, Xi Chen, Yunfeng Cui, Jinhui Feng, Yu Wang, Xiangtao Liu, Qiaqing Wu, Dunming Zhu, Yanhe Ma,2019, DOI: 10.1002/adsc.201900111). In the biological catalysis process of cholic acid and derivatives thereof, enzyme activity in the reaction process is obviously inhibited due to the amphiphilic structure characteristic of a reaction substrate, and the cholic acid and derivatives thereofThe concentration of the substance is often only 4-10 g/L. Ion resins are often used for post-treatment of separation and purification processes, but the use of ion resins to reduce the concentration of components in the reaction system and thus reduce its inhibition of biocatalysts, i.e., enzymes, has not been reported in the literature.
Disclosure of Invention
In order to solve the problem that cholic acid and derivatives thereof inhibit enzyme activity, the method reduces the inhibition of cholic acid and derivatives thereof on the enzyme activity by adding resin into the concentration of the cholic acid and derivatives thereof in a reaction system, and improves the biocatalytic conversion efficiency.
The invention also provides a method for increasing the substrate conversion concentration by adding the resin. Specifically, the concentration of a substrate added into a biological catalytic reaction system is 20-200g/L, the mass concentration of resin added is 4-40g/L, and the reaction can be completed after the conversion is carried out for 8-12 h.
The invention has the beneficial effects that: the resin is added to adsorb a substrate in a reaction system, and the resin can adsorb a product after the reaction along with the reaction, so that the dynamic balance of the substrate and the product adsorbed by the resin is achieved, the concentration of reaction components in a solution is reduced, and the inhibition on enzyme activity is reduced. The addition of the resin does not reduce the reaction rate, and under the same experimental conditions, the reaction time is effectively shortened, and the reaction efficiency is improved. After the reaction is completed, the resin can be separated from the reaction system by filtration, and the reaction product on the resin is eluted by using an eluent.
Drawings
FIG. 1 is a graph showing the detection of the adsorption capacity of different resins to cholic acid and derivatives thereof.
FIG. 2 shows the effect of resin addition on the conversion of chenodeoxycholic acid oxidation reaction.
FIG. 3 is a graph showing the effect of resin addition on the conversion of 7-Keto lithocholic acid reduction.
FIG. 4 is a graph showing the effect of resin addition on the conversion of cholic acid oxidation reactions.
FIG. 5 is a graph showing the effect of resin addition on the conversion of taurocholic acid oxidation.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention.
Example 1: adsorption of cholic acid and derivatives thereof by resin
Detection of adsorption capacity of different resins to cholic acid and derivatives thereof
50g/L of aqueous solution of cholic acid, taurocholic acid, glycocholic acid, 7-Keto-lithocholic acid, 7-Keto-cholic acid, chenodeoxycholic acid and ursodeoxycholic acid is prepared, and strong acidic ion resin (1), weak acidic ion resin (2), strong basic ion resin (3), weak basic ion resin (4), nonpolar adsorption resin (5) and polar adsorption resin (6) are respectively added, wherein the mass concentration is 5 g/L. After 1 hour of addition of the resin, the concentration of the remaining cholic acid and derivatives thereof in the aqueous solution was examined to examine the adsorption capacity of the resin.
Example 2: preparation of biocatalyst
The biocatalyst was obtained according to the literature (Flavin oxidized produced of noncoding amide adjacent dioxide with dioxide and Catalytic amino of Flavin monoglucoside for One-Point Multi-Enzymatic Preparation of Ursoxicolic Acid, Xi Chen, Yunfeng Cui, Jinhui Feng, Yu Wang, Xiangtao Liu, Qiaqing Wu, Dunming Zhu, Yanhe Ma,2019, DOI: 10.1002/adsc.201900111).
Example 3: influence of resin addition on the conversion of chenodeoxycholic acid oxidation reaction
The co-expression strain of 7 alpha-HSDH and flavan oxidant detect (MgFR) obtained according to the literature, and the reaction is constructed according to the literature method, and simultaneously resin is added to investigate the experimental transformation condition. The results show the same experimental conditions (0.15mM NAD)+0.44mM FMN), the rate of conversion of the substrate in the reaction system to which the resin was added was increased to 2-3 times the original rate for the same reaction time (16 hours) after the addition of the specific resin (10 g/L). Resin (0), strong acid ion resin (1), weak acid ion resin (2), strong alkaline ion resin (3), weak alkaline ion resin (4), nonpolar adsorption resin (5) and polar adsorption resin (6) are not added.
Example 4: effect of resin addition on the conversion of 7-Keto lithocholic acid reduction
The co-expression strain of 7 beta-HSDH and TbADH is obtained according to the literature, and the reaction is constructed according to the literature method, and meanwhile, the experimental transformation situation is investigated by adding resin. The results showed the same experimental conditions (0.15mM NADP)+1M isopropanol), the highest conversion rate of the substrate in the reaction system added with the resin can be increased to 5 times of the original conversion rate in the same reaction time (20 hours) after the specific resin (10g/L) is added. Resin (0), strong acid ion resin (1), weak acid ion resin (2), strong alkaline ion resin (3), weak alkaline ion resin (4), nonpolar adsorption resin (5) and polar adsorption resin (6) are not added.
Example 5: effect of resin addition on cholic acid Oxidation conversion
The co-expression strain of 7 alpha-HSDH and flavan oxidant detect (MgFR) obtained in literature is used for constructing reaction according to the method of example 3, and meanwhile, resin is added for investigating experimental transformation condition. The results show the same experimental conditions (0.15mM NAD)+0.44mM FMN), the rate of conversion of the substrate in the reaction system to which the resin was added was increased to 2-3 times the original rate for the same reaction time (16 hours) after the addition of the specific resin (10 g/L). Resin (0), strong acid ion resin (1), weak acid ion resin (2), strong alkaline ion resin (3), weak alkaline ion resin (4), nonpolar adsorption resin (5) and polar adsorption resin (6) are not added.
Example 6: effect of resin addition on the Oxidation conversion of taurocholic acid
The co-expression strain of 7 alpha-HSDH and flavan oxidant detect (MgFR) obtained in literature is used for constructing reaction according to the method of example 3, and meanwhile, resin is added for investigating experimental transformation condition. The results show the same experimental conditions (0.15mM NAD)+0.44mM FMN), the highest conversion rate of the substrate in the reaction system added with the resin can be increased to 6 times as high as the original conversion rate in the same reaction time (20 hours) after the addition of the specific resin (10 g/L). Resin (0) is not added, strong acid ion resin (1), weak acid ion resin (2), strong alkaline ion resin (3), weak alkaline ion resin (4), non-polar adsorption resin (5),And a polar adsorption resin (6).
Example 7: adding resin to perform oxidation reaction on chenodeoxycholic acid
Co-expression strains of 7 alpha-HSDH and flavin oxide detect (MgFR) obtained according to the literature are constructed according to the literature method, reaction (100mL) is established, 100g/L of substrate chenodeoxycholic acid is added, 20g/L of weak base ion resin is added at the same time, the reaction process is detected by thin layer chromatography and HPLC, after 20 hours, the substrate chenodeoxycholic acid is completely converted, the reaction is also adjusted to pH 9.0 after the resin is removed by filtration, thalli are removed by centrifugation, the pH is adjusted to 6.0 again, solid precipitate is obtained, and the solid precipitate is filtered and collected. And regenerating the ion resin obtained by filtering by using 0.4g/L NaOH solution, eluting the adsorbed chenodeoxycholic acid oxidation product 7-Keto lithocholic acid, adjusting the pH value of the obtained eluent to 6.0 to obtain solid precipitate, combining the precipitates, and drying to obtain a crude product of 9.8 g.
Example 8: adding resin to perform reduction reaction on 7-Keto lithocholic acid
The preparation method comprises the steps of obtaining a co-expression strain of 7 beta-HSDH and TbADH according to a literature, constructing a reaction (100mL) according to a literature method, adding 50g/L of a substrate 7-Keto lithocholic acid, simultaneously adding 4g/L of a weakly basic ion resin, detecting the reaction process by thin layer chromatography and HPLC, after 6 hours, completely converting the substrate 7-Keto lithocholic acid, filtering to remove the resin, adjusting the pH value of the reaction to 9.0, centrifuging to remove bacteria, adjusting the pH value to 6.0 again, obtaining a solid precipitate, filtering and collecting. The ion resin obtained by filtering is regenerated by using 0.4g/L NaOH solution, and simultaneously the adsorbed 7-Keto lithocholic acid reduction product ursodeoxycholic acid is eluted, the obtained eluent is adjusted to pH6.0 to obtain solid precipitate, and the solid precipitate is combined and dried to obtain 4.3g of crude product.
Example 9: adding resin to perform reduction reaction on 7-Keto lithocholic acid
The preparation method comprises the steps of obtaining a co-expression strain of 7 beta-HSDH and TbADH according to a literature, constructing a reaction (100mL) according to a literature method, adding 150g/L of a substrate 7-Keto lithocholic acid, adding 40g/L of a weakly basic ion resin, detecting the reaction process by thin layer chromatography and HPLC, after 30 hours, completely converting the substrate 7-Keto lithocholic acid, filtering to remove the resin, adjusting the pH value of the reaction to 9.0, centrifuging to remove bacteria, adjusting the pH value to 6.0 again, obtaining a solid precipitate, filtering and collecting. The ion resin obtained by filtering is regenerated by using 0.4g/L NaOH solution, and simultaneously the adsorbed 7-Keto lithocholic acid reduction product ursodeoxycholic acid is eluted, the obtained eluent is adjusted to pH6.0 to obtain solid precipitate, and the solid precipitate is combined and dried to obtain 13.9g of crude product.
Claims (6)
1. A method for reducing inhibition of reaction components on enzyme activity is characterized in that resin is added into a biotransformation system of cholic acid and derivatives thereof, after reaction, the resin is filtered and recycled, eluent is utilized to elute conversion products of the cholic acid and the derivatives thereof adsorbed on the resin, and residual products in reaction solution are obtained by filtering after the pH of the solution is adjusted to be less than 6.0.
2. The method of claim 1, wherein cholic acid and cholic acid derivatives thereof comprise cholic acid, taurocholic acid, glycocholic acid, 7-Keto-lithocholic acid, 7-Keto-cholic acid, and chenodeoxycholic acid.
3. The method of reducing the inhibition of enzyme activity by reactive species of claim 1, wherein the resin added is a weakly basic ionic resin.
4. The method for reducing inhibition of reaction components on enzyme activity according to claim 1, wherein the mass ratio of the added resin to the cholic acid and the derivative thereof is 1: 5-1: 20.
5. The method of claim 1, wherein the biotransformation system for cholic acid and its derivatives is an oxidation or reduction biotransformation system.
6. The method for reducing the inhibition of enzyme activity by reactive species of claim 1, wherein the substrate concentration of cholic acid and its derivatives is 20 to 200 g/L.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109929896A (en) * | 2019-04-23 | 2019-06-25 | 南京久安源环保科技有限公司 | A kind of production technology of ursodesoxycholic acid |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109929896A (en) * | 2019-04-23 | 2019-06-25 | 南京久安源环保科技有限公司 | A kind of production technology of ursodesoxycholic acid |
Non-Patent Citations (3)
| Title |
|---|
| XI CHEN等: "Flavin Oxidoreductase-Mediated Regeneration of Nicotinamide Adenine Dinucleotide with Dioxygen and Catalytic Amount of Flavin Mononucleotide for One-Pot Multi-Enzymatic Preparation of Ursodeoxycholic Acid", 《ADV.SYNTH.CATAL.》 * |
| 胡怀玉: "生物氧化4-甲基环己酮的研究", 《中国优秀博硕士学位论文全文数据库（硕士） 基础科学辑》 * |
| 马小雷: "熊去氧胆酸的分离与纯化", 《中国优秀博硕士学位论文全文数据库（硕士） 工程科技I辑》 * |
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Application publication date: 20210112 |