Hydrogel immobilized enzyme as well as preparation method and application thereof

Abstract

The invention provides a hydrogel immobilized enzyme and a preparation method and application thereof, the hydrogel immobilized enzyme comprises an active enzyme and a hydrogel carrier, the hydrogel carrier is formed by crosslinking polyacrylamide and sodium polyacrylate, and the active enzyme is ethanol dehydrogenase and / or acetaldehyde dehydrogenase. Alcohol dehydrogenase (ADH) or acetaldehyde dehydrogenase (ALDH) is embedded in a hydrogel carrier to obtain the hydrogel immobilized enzyme which is relatively strong in acid-base and temperature stability and relatively excellent in mechanical property, and the hydrogel immobilized enzyme can directly reach the stomach through oral administration to achieve the purpose of dispelling the effects of alcohol; alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) are embedded in a hydrogel carrier to form an immobilized enzyme with double-enzyme coupling catalysis capability, ethanol in the stomach can be quickly converted into acetic acid, so that the effect of dispelling the effects of alcohol is achieved, accumulation of an intermediate product acetaldehyde is avoided through double-enzyme coupling, and the effect of dispelling the effects of alcohol is achieved. And the stability and bioavailability of the enzyme are improved by using a hydrogel embedding technology.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis and drug development, and specifically relates to a hydrogel immobilized enzyme and a preparation method and application thereof. Background Art

[0002] Enzymes are a type of biological catalyst with high efficiency and specificity, and usually play a catalytic role under mild conditions such as normal temperature and pressure. However, after the enzyme leaves its specific environment, its stability is poor and it is difficult to repeat. These problems in applications can be overcome by enzyme immobilization technology. Professor Hassan Mohamed El-Said Azzazy [Maghraby YR, El-Shabasy RM, Ibrahim AH, Azzazy HME. Enzyme Immobilization Technologies and Industrial Applications. ACS Omega. 2023 Jan 31; 8(6): 5184-5196.] introduced that enzyme immobilization technology is an indispensable component in large-scale industries such as medicine, food, detergents, textiles and pharmaceuticals.

[0003] Alcohol dehydrogenase and acetaldehyde dehydrogenase are important enzymes for sobering up, but they cannot be taken orally directly. They are generally coated with carriers. Existing technologies use Fe3O4@SiO2 nanoparticles or silica as carriers to coat alcohol dehydrogenase or acetaldehyde dehydrogenase. The preparation process of Fe3O4@SiO2 nanoparticles is relatively complicated and the preparation conditions are harsh, which will lead to increased costs. In addition, alcohol dehydrogenase or acetaldehyde dehydrogenase coated with Fe3O4@SiO2 nanoparticles cannot be taken orally and cannot achieve the purpose of sobering up.

[0004] When using silica as a carrier, the pore structure and pore size of silica are crucial to the immobilization of the enzyme. If the pore size is too small, it may limit the diffusion of enzyme molecules and the transport of substrates, thereby affecting the catalytic efficiency. On the other hand, if the pore size is too large, it may cause the enzyme molecules to easily fall off the carrier, reducing the immobilization efficiency. However, silica-coated alcohol dehydrogenase or acetaldehyde dehydrogenase cannot be taken orally and cannot achieve the purpose of sobering up.

[0005] The number of drinkers in China has exceeded 600 million, of which 84.1% are male and 15.9% are female. The liver is an important organ of the human body, responsible for more than 500 metabolic reactions. About 80% of alcohol is converted into acetaldehyde by alcohol dehydrogenase in the liver, and then converted into acetic acid by acetaldehyde dehydrogenase, and finally decomposed into water and carbon dioxide and excreted from the body. Drinking a lot of alcohol in a short period of time can lead to acute alcohol poisoning, causing movement disorders, impaired consciousness, and even life-threatening. Long-term heavy drinking can lead to alcoholic liver damage, including fatty degeneration, hepatitis, cirrhosis and liver cancer. Among heavy drinkers, more than 80% have a certain degree of fatty liver, of which 10% to 35% may develop alcoholic hepatitis, and 10% to 20% may further develop cirrhosis. The market is in urgent need of alcohol detoxification drugs that can efficiently decompose ethanol.

[0006] Existing alcohol relieving drugs mainly include the following technologies:

[0007] 1. Chinese medicine preparations: The Chinese invention patent document with publication number CN103610803A discloses a Chinese medicine composition for protecting the liver and relieving alcohol, which uses puerarin and other substances in Pueraria root to activate the activity of superoxide dismutase in the body and remove oxidative free radicals produced during ethanol metabolism.

[0008] 2. Chemical preparations: The Chinese invention patent document with publication number CN113069527A discloses a composition with alcohol sobering and liver protecting functions and its preparation method, which uses metadoxine to inhibit the inactivation of alcohol dehydrogenase, alleviate alcohol poisoning, and protect the liver. However, metadoxine is a prescription drug, and inhibits the inactivation of alcohol dehydrogenase alone without considering the activity of acetaldehyde dehydrogenase, which will cause the accumulation of acetaldehyde, an intermediate product of the ethanol metabolic pathway, and is harmful to human health.

[0009] 3. Probiotic preparations: The Chinese invention patent document with publication number CN111471660A discloses a recombinant acetaldehyde dehydrogenase gene and its lactic acid bacteria vector and application. The acetaldehyde dehydrogenase expressed by the lactic acid bacteria vector is used to decompose acetaldehyde, an intermediate product in the ethanol metabolic pathway. Such probiotic products have similar problems: the colonization rate of probiotics in the body is generally not high, and the main colonization site is the small intestine. When drinking, ethanol absorption mainly occurs in the stomach. Even if the probiotics are successfully colonized and quickly express a large amount of enzymes that decompose ethanol, they cannot be transmitted to the stomach to decompose ethanol. Summary of the invention

[0010] The technical problem to be solved by the present invention is how to coat alcohol dehydrogenase or acetaldehyde dehydrogenase through a carrier so as to achieve the purpose of sobering up.

[0011] The present invention solves the above technical problems through the following technical means:

[0012] The first aspect of the present invention provides a hydrogel-immobilized enzyme, comprising an active enzyme and a hydrogel carrier, wherein the hydrogel carrier is formed by cross-linking polyacrylamide and sodium polyacrylate, and the active enzyme is alcohol dehydrogenase and / or acetaldehyde dehydrogenase.

[0013] Beneficial effects: The present invention embeds alcohol dehydrogenase (ADH) or acetaldehyde dehydrogenase (ALDH) in a hydrogel carrier to obtain a hydrogel-immobilized enzyme with strong acid-base and temperature stability and excellent mechanical properties. The hydrogel-immobilized enzyme can directly reach the stomach through oral administration to achieve the purpose of sobering up; alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) are embedded in a hydrogel carrier to form an immobilized enzyme with dual enzyme coupling catalysis ability, which can quickly convert ethanol in the stomach into acetic acid, thereby achieving the effect of sobering up. The present invention avoids the accumulation of the intermediate product acetaldehyde through dual enzyme coupling, and uses hydrogel encapsulation technology to improve the stability and bioavailability of the enzyme.

[0014] Preferably, the mass ratio of polyacrylamide to sodium polyacrylate is 3:2-2:3.

[0015] Preferably, the volume ratio of the alcohol dehydrogenase to the acetaldehyde dehydrogenase is 1:2.

[0016] Preferably, the ratio of active enzyme to hydrogel carrier is 30-120ul:100mg.

[0017] The second aspect of the present invention provides a method for preparing the above hydrogel-immobilized enzyme, comprising the following steps:

[0018] S1 dissolves sodium polyacrylate and sodium polyacrylate in water in a mass ratio of 3:2-2:3 to obtain a mixed solution;

[0019] S2: stirring the mixed solution at 30-50° C. for 0.5-4 h, and centrifuging to remove bubbles to obtain a hydrogel carrier solution;

[0020] S3: Mix ethanol dehydrogenase and acetaldehyde dehydrogenase in a volume ratio of 1:2 to obtain active enzyme, mix 30-120ul of active enzyme with 100mg of hydrogel carrier solution at 30-40°C, then centrifuge to remove bubbles, inject all of it into a syringe, and drip it into a calcium chloride solution through a syringe. The fixation time is 0-25min, and the hydrogel-immobilized enzyme is obtained after washing.

[0021] Preferably, the centrifugal degassing in S2 is performed by stirring the reacted solution in a water bath at 20-30° C. until the bubbles are removed.

[0022] Preferably, the mass volume concentration of the calcium chloride solution in S3 is 10%.

[0023] Preferably, the fixation time in S3 is 13-23 min.

[0024] Preferably, the ratio of active enzyme to hydrogel carrier solution in S2 is 52ul:100mg.

[0025] The third aspect of the present invention provides the use of the hydrogel-immobilized enzyme, which is the use of the hydrogel-immobilized enzyme in an oral alcohol-resolving drug, wherein the active enzymes of the hydrogel-immobilized enzyme are alcohol dehydrogenase and acetaldehyde dehydrogenase.

[0026] The advantages of the present invention are:

[0027] The present invention simultaneously fixes alcohol dehydrogenase and acetaldehyde dehydrogenase through a hydrogel carrier to form a dual enzyme system that can directly decompose ethanol in the stomach, thereby achieving a hangover-relieving effect from the root and avoiding the accumulation of ethanol in the body and the generation of acetaldehyde, a toxic intermediate product in the metabolic process.

[0028] The hydrogel carrier encapsulates and protects the activity of the dual enzymes in the gastric acid environment, improves the stability and bioavailability of the enzymes, and ensures that the enzymes work effectively in the stomach. The hydrogel-immobilized enzymes maintain high activity over a wide range of pH values ​​and temperatures. Compared with free enzymes, they exhibit stronger acid and alkali resistance and heat resistance, are more adaptable, and have better reusability. They can still maintain high activity after multiple uses, reducing the cost of use and improving economy and feasibility.

[0029] The preparation method of the hydrogel-immobilized enzyme is simple, easy to operate, suitable for large-scale production, and reduces the manufacturing cost. The dual enzyme system of the hydrogel-immobilized enzyme of the present invention not only improves the efficiency of sobering up, reduces the damage of ethanol and acetaldehyde to the liver, but also achieves an efficient, economical and reliable sobering up effect by optimizing the enzymatic properties and improving the stability of the enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a pH-enzyme activity curve diagram of the immobilized alcohol dehydrogenase and the free alcohol dehydrogenase of Example 1;

[0031] Figure 2 is a pH-enzyme activity curve diagram of the immobilized acetaldehyde dehydrogenase and the free acetaldehyde dehydrogenase of Example 2;

[0032] Figure 3 is a temperature-enzyme activity curve diagram of the immobilized alcohol dehydrogenase and the free alcohol dehydrogenase in Example 1;

[0033] Figure 4 is a temperature-enzyme activity curve diagram of the immobilized acetaldehyde dehydrogenase and the free acetaldehyde dehydrogenase of Example 2;

[0034] Figure 5 is an ethanol concentration-enzyme activity curve diagram of the immobilized alcohol dehydrogenase and the free alcohol dehydrogenase of Example 1;

[0035] Figure 6 is an acetaldehyde concentration-enzyme activity curve of the immobilized acetaldehyde dehydrogenase and the free acetaldehyde dehydrogenase of Example 2;

[0036] Figure 7 It is the enzyme loading-relative enzyme activity curve of the hydrogel-immobilized enzyme of Example 1 and Examples 1-1 to 1-4;

[0037] Figure 8 is a graph showing the immobilization time-enzyme activity curve of the hydrogel-immobilized enzyme of Example 1 and Comparative Example 1;

[0038] Fig. 9 is a graph of the number of cycles-residual activity of the hydrogel-immobilized enzyme in Example 1;

[0039] Fig.10 is a graph of the number of cycles-residual activity of the hydrogel-immobilized enzyme in Example 2;

[0040] Fig.11 This is a graph showing the HPLC results of the hydrogel-immobilized enzyme in Example 3. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0043] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0044] Example 1

[0045] This embodiment provides a hydrogel-immobilized enzyme and a preparation method, which specifically includes the following contents:

[0046] A hydrogel-immobilized enzyme comprises an active enzyme and a hydrogel carrier, wherein the hydrogel carrier is formed by cross-linking polyacrylamide and sodium polyacrylate, and the active enzyme is alcohol dehydrogenase; the mass ratio of the polyacrylamide to the sodium polyacrylate is 2:3, and the ratio of the alcohol dehydrogenase to the hydrogel carrier is 52ul:100mg.

[0047] The method for preparing the hydrogel-immobilized enzyme of this embodiment comprises the following steps:

[0048] 0.2 g of polyacrylamide and 0.3 g of sodium polyacrylate were dissolved in 10 ml of deionized water to obtain a mixed solution; the mixed solution was stirred on a magnetic stirrer at 500 rpm at 40°C for 3 h, and then stirred at 150 rpm in a water bath at 30°C to remove bubbles to obtain a hydrogel carrier solution.

[0049] 52ul of alcohol dehydrogenase and 100mg of hydrogel carrier solution were mixed at 30°C, then centrifuged to remove bubbles, and then all of them were injected into a syringe, and then dropped into a calcium chloride solution through the syringe for 15 minutes to form gel balls. Distilled water was used to wash away excess calcium ions on the surface of the gel balls to obtain a hydrogel-immobilized enzyme, which was named immobilized alcohol dehydrogenase.

[0050] Examples 1-1 to 1-4 provide a hydrogel-immobilized enzyme and a preparation method, which are different from Example 1 in that the ratio of alcohol dehydrogenase to hydrogel carrier is different, and the specific situation is as follows:

[0051]

[0052]

[0053] Comparative Example 1

[0054] This comparative example provides a hydrogel-immobilized enzyme and a preparation method. The difference between this comparative example and Example 1 is that the preparation method is different from Example 1 in that the immobilization time is set to 0, 5 min, 20 min and 25 min.

[0055] Example 2

[0056] This embodiment provides a hydrogel-immobilized enzyme and a preparation method, which specifically includes the following contents:

[0057] A hydrogel-immobilized enzyme comprises an active enzyme and a hydrogel carrier, wherein the hydrogel carrier is formed by cross-linking polyacrylamide and sodium polyacrylate, and the active enzyme is acetaldehyde dehydrogenase; the mass ratio of the polyacrylamide to the sodium polyacrylate is 2:3, and the ratio of the acetaldehyde dehydrogenase to the hydrogel carrier is 52ul:100mg.

[0058] The method for preparing the hydrogel-immobilized enzyme of this embodiment comprises the following steps:

[0059] 0.2 g of polyacrylamide and 0.3 g of sodium polyacrylate were dissolved in 10 ml of deionized water to obtain a mixed solution; the mixed solution was stirred on a magnetic stirrer at 500 rpm at 40°C for 3 h, and then stirred at 150 rpm in a water bath at 30°C to remove bubbles to obtain a hydrogel carrier solution.

[0060] 52ul of acetaldehyde dehydrogenase and 100mg of hydrogel carrier solution were mixed at 30°C, then centrifuged to remove bubbles, and then all of them were injected into a syringe, which was then dripped into a calcium chloride solution through a syringe for 15 minutes to form gel balls. Distilled water was used to wash away excess calcium ions on the surface of the gel balls to obtain a hydrogel-immobilized enzyme, which was named immobilized acetaldehyde dehydrogenase.

[0061] Example 3

[0062] This embodiment provides a hydrogel-immobilized enzyme and a preparation method, which specifically includes the following contents:

[0063] A hydrogel-immobilized enzyme comprises an active enzyme and a hydrogel carrier, wherein the hydrogel carrier is formed by cross-linking polyacrylamide and sodium polyacrylate, the active enzymes are alcohol dehydrogenase and acetaldehyde dehydrogenase, the volume ratio of the alcohol dehydrogenase to the acetaldehyde dehydrogenase is 1:2, the mass ratio of the polyacrylamide to the sodium polyacrylate is 2:3, and the ratio of the active enzyme to the hydrogel carrier is 52ul:100mg.

[0064] The method for preparing the hydrogel-immobilized enzyme of this embodiment comprises the following steps:

[0065] 0.2 g of polyacrylamide and 0.3 g of sodium polyacrylate were dissolved in 10 ml of deionized water to obtain a mixed solution; the mixed solution was stirred on a magnetic stirrer at 500 rpm at 40°C for 3 h, and then stirred at 150 rpm in a water bath at 30°C to remove bubbles to obtain a hydrogel carrier solution.

[0066] 52ul of active enzyme and 100mg of hydrogel carrier solution were mixed at 30°C, then centrifuged to remove bubbles, and all of them were injected into a syringe. Calcium chloride solution was dripped into the syringe and fixed for 15min to form gel balls. Distilled water was used to wash away excess calcium ions on the surface of the gel balls to obtain hydrogel-immobilized enzyme, which was named fixed dual enzyme.

[0067] Experimental example

[0068] 1. Effect of pH on the enzyme activity of free enzyme and hydrogel-immobilized enzyme

[0069] First, buffer solutions with different pH values ​​of pH 4.0-10.0 are prepared respectively, and the buffer solutions are: sodium hydrogen phosphate-sodium citrate buffer solution (pH 4.0-6.0), phosphate buffer solution (pH 6.0-7.0), Tris-HCl buffer solution (pH 8.0-10.0), and the optimal pH of the free enzyme and the hydrogel-immobilized enzyme is found according to the enzyme activity determination method of the free enzyme and the hydrogel-immobilized enzyme. The enzyme refers to alcohol dehydrogenase or acetaldehyde dehydrogenase.

[0070] The specific operations are as follows:

[0071] (1) Determination of free alcohol dehydrogenase activity: 0.05 ml of alcohol dehydrogenase was added to 3 mL of 0.1 mol / L buffer solutions of different pH values, and the mixture was incubated at 37°C for 5 min. Then, 0.4 mL of 0.5 mol / L ethanol and 0.1 mL of 10 mmol / L NAD were added. + , and the absorbance was measured at a wavelength of 340 nm. No NAD was added to the blank control. + , and replace it with an equal volume of distilled water.

[0072] (2) Determination of the enzyme activity of immobilized alcohol dehydrogenase: The prepared immobilized alcohol dehydrogenase was added to 3 mL of 0.1 mol / L buffer solutions of different pH values, incubated at 37°C for 5 min, and then 0.4 mL of 0.5 mol / L ethanol and 0.1 mL of 10 mmol / L NAD were added. + , and the absorbance was measured at a wavelength of 340 nm. No NAD was added to the blank control. + , and replace it with an equal volume of distilled water.

[0073] The experimental results are as follows Figure 1 As shown, it is clearly revealed that the optimal pH value of alcohol dehydrogenase is 8. Specifically, free alcohol dehydrogenase exhibits the highest enzyme activity at pH 8. Similarly, immobilized alcohol dehydrogenase also reaches its peak enzyme activity at pH 8. This shows that alcohol dehydrogenase can exert its best catalytic efficiency in a pH environment close to neutral, whether in a free state or after immobilization.

[0074] However, during the entire measurement process, the immobilized alcohol dehydrogenase showed stronger acid and alkali resistance. The experimental results showed that the immobilized alcohol dehydrogenase can maintain a relatively high level of enzyme activity in a wide range of pH values ​​between 4 and 10. This specific performance significantly shows that compared with free alcohol dehydrogenase, immobilized alcohol dehydrogenase shows higher stability in different acid and alkali environments. The immobilization process enhances the resistance of alcohol dehydrogenase molecules to external pH changes, allowing them to continue to effectively play a catalytic role under a wider range of pH conditions. Therefore, immobilized alcohol dehydrogenase has more significant advantages in practical applications, especially in situations where complex and changeable environmental conditions need to be dealt with. Therefore, compared with free alcohol dehydrogenase, immobilized alcohol dehydrogenase shows better adaptability and stability under a wide range of pH conditions.

[0075] (3) Determination of free aldehyde dehydrogenase activity: 0.005 mL of aldehyde dehydrogenase was added to 0.3 mL of 0.1 mol / L buffer solutions of different pH values, and the mixture was incubated at 37°C for 5 min. Then, 0.025 mL of 0.02 mol / L acetaldehyde and 0.02 mL of 10 mmol / L NAD were added. + , and the absorbance was measured at a wavelength of 340 nm. No NAD was added to the blank control. + , and replace it with an equal volume of distilled water.

[0076] (4) Determination of the activity of immobilized acetaldehyde dehydrogenase: The prepared immobilized acetaldehyde dehydrogenase was added to 0.3 mL of 0.1 mol / L buffer solutions of different pH values, incubated at 37°C for 5 min, and then 0.025 mL of 0.02 mol / L acetaldehyde and 0.02 mL of 10 mmol / L NAD were added. + , and the absorbance was measured at a wavelength of 340 nm. No NAD was added to the blank control. + , and replace it with an equal volume of distilled water.

[0077] The experimental results are as follows Figure 2 As shown, the optimal pH value of acetaldehyde dehydrogenase is clearly revealed. Specifically, free acetaldehyde dehydrogenase exhibits the highest enzyme activity at pH 8, and similarly, immobilized acetaldehyde dehydrogenase reaches its peak enzyme activity at pH 9. This shows that acetaldehyde dehydrogenase can exert its best catalytic efficiency in a pH environment close to alkaline, whether in a free state or after immobilization.

[0078] However, during the entire measurement process, the immobilized acetaldehyde dehydrogenase showed stronger acid and alkali resistance. The experimental results showed that the immobilized enzyme can maintain a relatively high level of enzyme activity in a wide range of pH values ​​between 4 and 10. This specific performance significantly shows that the immobilized acetaldehyde dehydrogenase shows higher stability in different acid and alkali environments compared with the free acetaldehyde dehydrogenase. The immobilization process seems to enhance the resistance of the acetaldehyde dehydrogenase molecules to external pH changes, allowing it to continue to effectively play a catalytic role under a wider range of pH conditions. Therefore, immobilized acetaldehyde dehydrogenase has more significant advantages in practical applications, especially in situations where complex and changeable environmental conditions need to be dealt with. Therefore, compared with free acetaldehyde dehydrogenase, immobilized acetaldehyde dehydrogenase shows better adaptability and stability under a wide range of pH conditions.

[0079] 2. Effect of temperature on the enzyme activity of free enzyme and hydrogel-immobilized enzyme

[0080] The free enzyme and the hydrogel-immobilized enzyme were tested for enzyme activity at 27°C, 37°C, 47°C, 57°C, and 67°C to find out the optimal temperature for the free enzyme and the hydrogel-immobilized enzyme. The enzyme refers to alcohol dehydrogenase or acetaldehyde dehydrogenase.

[0081] The specific operations are as follows:

[0082] (1) Determination of free alcohol dehydrogenase activity: 0.05 mL of alcohol dehydrogenase was added to 3 mL of 0.1 mol / L phosphate buffer solution (pH 8), and the mixture was incubated for 5 min at 27°C, 37°C, 47°C, 57°C, and 67°C, respectively. Then, 0.4 mL of 0.5 mol / L ethanol and 0.1 mL of 10 mmol / L NAD were added. + , and the absorbance was measured at a wavelength of 340 nm. No NAD was added to the blank control. + , and replace it with an equal volume of distilled water.

[0083] (2) Determination of the enzyme activity of immobilized alcohol dehydrogenase: The prepared immobilized alcohol dehydrogenase was added to 3 mL of 0.1 mol / L phosphate buffer solution (pH 8), and the reaction was incubated at 27°C, 37°C, 47°C, 57°C, and 67°C for 5 min, followed by the addition of 0.4 mL of 0.5 mol / L ethanol and 0.1 mL of 10 mmol / L NAD. + , and the absorbance was measured at a wavelength of 340 nm. No NAD was added to the blank control. + , and replace it with an equal volume of distilled water.

[0084] according to Figure 3 It can be seen that: in the temperature range of 27℃ to 67℃, the activity of free alcohol dehydrogenase and immobilized alcohol dehydrogenase usually increases with the increase of temperature, because more molecules become activated molecules. However, when the temperature reaches a certain level, the enzyme protein will denature, resulting in enzyme inactivation, and thus the enzyme activity will decrease. Therefore, when conducting experiments, it is necessary to find the optimal temperature of the enzyme, such as Figure 2 As shown in the figure, the immobilized alcohol dehydrogenase reaches the best enzyme activity at 37°C. Compared with the free alcohol dehydrogenase, the immobilized alcohol dehydrogenase shows better heat resistance and maintains a higher enzyme activity in a wider temperature range. This shows that the immobilized alcohol dehydrogenase has better stability and applicability under different temperature conditions.

[0085] (3) Determination of free acetaldehyde dehydrogenase activity: 0.005 mL of acetaldehyde dehydrogenase was added to 0.3 mL of 0.1 mol / L phosphate buffer solution (pH 8), and the mixture was incubated for 5 min at 27°C, 37°C, 47°C, 57°C, and 67°C, respectively. Then, 0.025 mL of 0.02 mol / L acetaldehyde and 0.02 mL of 10 mmol / L NAD were added. + , and the absorbance was measured at a wavelength of 340 nm. No NAD was added to the blank control. + , and replace it with an equal volume of distilled water.

[0086] (4) Determination of the activity of immobilized acetaldehyde dehydrogenase: The prepared immobilized acetaldehyde dehydrogenase was added to 0.3 mL of 0.1 mol / L phosphate buffer solution (pH 8), and the mixture was incubated for 5 min at 27°C, 37°C, 47°C, 57°C, and 67°C, respectively. Then, 0.025 mL of 0.02 mol / L acetaldehyde and 0.02 mL of 10 mmol / L NAD were added. + , and the absorbance was measured at a wavelength of 340 nm. No NAD was added to the blank control. + , and replace it with an equal volume of distilled water.

[0087] according to Figure 4 It can be seen that in the temperature range of 27°C to 67°C, the immobilized acetaldehyde dehydrogenase reaches the best enzyme activity at 37°C. Compared with the free acetaldehyde dehydrogenase, the immobilized acetaldehyde dehydrogenase exhibits better heat resistance and maintains a higher enzyme activity in a wider temperature range. This shows that the immobilized acetaldehyde dehydrogenase has better stability and applicability under different temperature conditions.

[0088] 3. Effects of different concentrations of ethanol on the enzyme activity of free and immobilized alcohol dehydrogenase

[0089] Using ethanol as the enzyme reagent, different concentrations of ethanol were set, and the enzyme activities of free alcohol dehydrogenase and immobilized alcohol dehydrogenase were measured at different ethanol concentrations. The ethanol concentrations were 0.01, 0.02, 0.04, 0.1, 0.5, 1.0, and 2.0 M, respectively, to find out the optimal ethanol concentrations of free and immobilized alcohol dehydrogenase.

[0090] The specific operations are as follows:

[0091] (1) Determination of free alcohol dehydrogenase activity: 0.05 ml of alcohol dehydrogenase was added to 3 mL of 0.1 mol / L phosphate buffer solution (pH 8), and the mixture was incubated at 37°C for 5 min. Then, 0.4 mL of 0.01, 0.02, 0.04, 0.1, 0.5, 1.0, and 2.0 M ethanol and 0.1 mL of 10 mmol / L NAD were added.+ , and the absorbance was measured at a wavelength of 340 nm. No NAD was added to the blank control. + , and replace it with an equal volume of distilled water.

[0092] (2) Determination of the activity of immobilized alcohol dehydrogenase: The prepared immobilized alcohol dehydrogenase was added to 3 mL of 0.1 mol / L phosphate buffer solution (pH 8), incubated at 37°C for 5 min, and then 0.4 mL of 0.01, 0.02, 0.04, 0.1, 0.5, 1.0, and 2.0 M ethanol and 0.1 mL of 10 mmol / L NAD were added. + , and the absorbance was measured at a wavelength of 340 nm. No NAD was added to the blank control. + , and replace it with an equal volume of distilled water.

[0093] according to Figure 5 It can be seen that the enzyme activity of immobilized alcohol dehydrogenase increases with the increase of ethanol concentration, but decreases after it is higher than 1.0M. This may be due to the inactivation of the active center of alcohol dehydrogenase caused by excessively high ethanol concentration. Therefore, the optimal ethanol concentration is 0.5M.

[0094] 4. Effects of different concentrations of acetaldehyde on the enzyme activity of free acetaldehyde dehydrogenase and immobilized acetaldehyde dehydrogenase

[0095] Using acetaldehyde as the enzyme reagent, different concentrations of acetaldehyde were set, and the enzyme activities of free acetaldehyde dehydrogenase and immobilized acetaldehyde dehydrogenase were measured at different acetaldehyde concentrations. The acetaldehyde concentrations were 0.004, 0.02, 0.1, 0.5 or 2.5 M, respectively, to find out the optimal acetaldehyde concentrations of free acetaldehyde dehydrogenase and immobilized acetaldehyde dehydrogenase.

[0096] The specific operations are as follows:

[0097] (1) Determination of free acetaldehyde dehydrogenase activity: 0.05 mL of acetaldehyde dehydrogenase was added to 3 mL of 0.1 mol / L phosphate buffer solution (pH 8), and the mixture was incubated at 37°C for 5 min. Then, 0.4 mL of 0.004, 0.02, 0.1, 0.5 or 2.5 M acetaldehyde and 0.1 mL of 10 mmol / L NAD were added. + , and the absorbance was measured at a wavelength of 340 nm. No NAD was added to the blank control. + , and replace it with an equal volume of distilled water.

[0098] (2) Determination of the activity of immobilized acetaldehyde dehydrogenase: The prepared immobilized acetaldehyde dehydrogenase was added to 3 mL of 0.1 mol / L phosphate buffer solution (pH 8), incubated at 37°C for 5 min, and then 0.4 mL of 0.004, 0.02, 0.1, 0.5 or 2.5 M acetaldehyde and 0.1 mL of 10 mmol / L NAD were added. + , and the absorbance was measured at a wavelength of 340 nm. No NAD was added to the blank control. + , and replace it with an equal volume of distilled water.

[0099] according to Figure 6 It can be seen that the activity of immobilized acetaldehyde dehydrogenase increases with the increase of acetaldehyde concentration, but decreases after exceeding a certain concentration. This may be due to the inactivation of the active center of alcohol dehydrogenase due to excessive acetaldehyde concentration. Therefore, the optimal acetaldehyde concentration is 0.02M.

[0100] 5. Effect of enzyme loading on the activity of hydrogel-immobilized enzymes

[0101] The hydrogel-immobilized enzymes of Example 1 and Examples 1-1 to 1-4 were subjected to enzyme activity assay, and the optimal enzyme loading amount was obtained by relative enzyme activity. The relative enzyme activity (%) = (enzyme activity under certain conditions / maximum enzyme activity under the conditions) × 100%

[0102] The specific operations are as follows:

[0103] Determination of the activity of the hydrogel-immobilized enzyme: The hydrogel-immobilized enzymes of Examples 1-1 to 1-4 of Example 1 were added to 0.3 mL of 0.1 mol / L phosphate buffer solution (pH 8), incubated at 37 ° C for 5 min, and then 0.025 mL of 0.5 mol / L ethanol and 0.1 mL of 10 mmol / L NAD were added. + , and the absorbance was measured at a wavelength of 340 nm. No NAD was added to the blank control. + , and replace it with an equal volume of distilled water.

[0104] according to Figure 7 It can be seen that the hydrogel-immobilized enzyme is formed by cross-linking sodium polyacrylate and polyacrylamide to form a hydrogel carrier, and the alcohol dehydrogenase is fixed in the hydrogel carrier by physical embedding adsorption. The effect of enzyme loading on relative enzyme activity is studied. The results are as follows Figure 7 As shown in the figure, with the increase of enzyme loading, the relative enzyme activity first increased and then decreased. This is because at a higher enzyme loading, the interaction force between the hydrogel carrier and alcohol dehydrogenase decreased, resulting in a decrease in relative enzyme activity. The optimal enzyme loading was 3.5 mg / g.

[0105] 6. Effect of calcium chloride fixation time on the enzyme activity of hydrogel-immobilized enzyme

[0106] The enzyme activity of the hydrogel-immobilized enzymes in Example 1 and Comparative Example 1 was measured, and the optimal immobilization time was obtained according to the enzyme activity.

[0107] according to Figure 8 It can be seen that the present invention adopts a chemical precipitation method, immerses the hydrogel carrier in a calcium chloride solution, and quickly rinses the calcium chloride solution on the surface with water after immersion for a certain period of time. The toughness of the hydrogel itself changes, and the activity of the immobilized enzyme increases with the extension of the cross-linking time. However, if the cross-linking time is too long, the condensation reaction on the hydrogel carrier is too much, or the hydrogel carrier over-modifies the enzyme, resulting in a gradual decrease in the activity of the enzyme. It can be seen that the optimal fixation time of the hydrogel immobilized enzyme is 15 minutes.

[0108] 7. Determination of the number of times the hydrogel-immobilized enzyme can be reused

[0109] The enzyme activity was calculated according to the enzyme activity determination method. After each determination, the immobilized enzyme was taken out from the reaction system and washed with buffer for 3 times, then put into new buffer for enzyme activity determination. The highest enzyme activity value was used as the control (100%), and the residual enzyme activity of the immobilized enzyme was recorded after each repeated use (parallel determination 3-5 times).

[0110] The hydrogel-immobilized enzymes of Example 1 and Example 2 were used in a continuous cycle for 6 times, and the enzyme activity was recorded each time. Figure 9-10 It can be seen that the hydrogel-immobilized enzyme of the present invention showed high activity in the first use, and the enzyme activity decreased after the second use. When used for the fifth time, the enzyme activity decreased by about 50% compared with the first time, which means that the hydrogel-immobilized enzyme can be reused many times, making it more economical and feasible in application.

[0111] 8. Investigation of the enzymatic properties of the hydrogel-immobilized enzyme in Example 3

[0112] Example 3: Alcohol dehydrogenase and acetaldehyde dehydrogenase were immobilized simultaneously on a hydrogel carrier to obtain an immobilized dual enzyme. The immobilized dual enzyme of Example 3 was placed in 1.5 mL of 0.1 mol / L phosphate buffer solution (pH 8), incubated at 37°C for 5 min, and then 0.2 mL of 0.5 mol / L ethanol and 0.025 mL of 10 mmol / L NAD were added. + The reaction was continued for 24 hours, and the supernatant was taken and acidified before separation on a Foley FL2000 high performance liquid chromatograph with a C18 column, and the mobile phase was a 5% methanol solution containing 0.1 M NaH2PO4. Fig.11 As shown, acetic acid (2.1 minutes) was generated after the reaction, with only a small amount of acetaldehyde intermediate (3.5 minutes), and the peak at 4.9 minutes was NAD + / NADH combined peak. It can be seen that the immobilized dual enzyme of the present invention not only improves the efficiency of sobering up and reduces the damage of ethanol and acetaldehyde to the liver, but also achieves efficient, economical and reliable sobering up effect by optimizing enzymatic properties and improving enzyme stability.

[0113] The present invention embeds alcohol dehydrogenase (ADH) or acetaldehyde dehydrogenase (ALDH) in a hydrogel carrier to obtain a hydrogel-immobilized enzyme with strong acid-base and temperature stability and excellent mechanical properties. The hydrogel-immobilized enzyme can directly reach the stomach through oral administration to achieve the purpose of sobering up. Alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) are embedded in the hydrogel carrier to form an immobilized enzyme with dual enzyme coupling catalysis ability, which can quickly convert ethanol in the stomach into acetic acid, thereby achieving the effect of sobering up. The present invention avoids the accumulation of the intermediate product acetaldehyde through dual enzyme coupling, and utilizes hydrogel embedding technology to improve the stability and bioavailability of the enzyme.

[0114] The present invention simultaneously fixes alcohol dehydrogenase and acetaldehyde dehydrogenase through a hydrogel carrier to form a dual enzyme system that can directly decompose ethanol in the stomach, thereby achieving a hangover-relieving effect from the root and avoiding the accumulation of ethanol in the body and the generation of acetaldehyde, a toxic intermediate product in the metabolic process.

[0115] The hydrogel carrier encapsulates and protects the activity of the dual enzymes in the gastric acid environment, improves the stability and bioavailability of the enzymes, and ensures that the enzymes work effectively in the stomach. The hydrogel-immobilized enzymes maintain high activity over a wide range of pH values ​​and temperatures. Compared with free enzymes, they exhibit stronger acid and alkali resistance and heat resistance, are more adaptable, and have better reusability. They can still maintain high activity after multiple uses, reducing the cost of use and improving economy and feasibility.

[0116] The preparation method of the hydrogel-immobilized enzyme is simple, easy to operate, suitable for large-scale production, and reduces the manufacturing cost. The dual enzyme system of the hydrogel-immobilized enzyme of the present invention not only improves the efficiency of sobering up, reduces the damage of ethanol and acetaldehyde to the liver, but also achieves an efficient, economical and reliable sobering up effect by optimizing the enzymatic properties and improving the stability of the enzyme.

[0117] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A hydrogel-immobilized enzyme, characterized in that: The invention comprises an active enzyme and a hydrogel carrier, wherein the hydrogel carrier is formed by cross-linking polyacrylamide and sodium polyacrylate, and the active enzyme is alcohol dehydrogenase and / or acetaldehyde dehydrogenase.

2. The hydrogel-immobilized enzyme according to claim 1, characterized in that: The mass ratio of the polyacrylamide to sodium polyacrylate is 3:2-2:

3.

3. The hydrogel-immobilized enzyme according to claim 1, characterized in that: The volume ratio of the alcohol dehydrogenase to the acetaldehyde dehydrogenase is 1:

2.

4. The hydrogel-immobilized enzyme according to claim 1, characterized in that: The ratio of active enzyme to hydrogel carrier is 30-120ul:100mg.

5. The method for preparing a hydrogel-immobilized enzyme according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1 dissolves sodium polyacrylate and sodium polyacrylate in water in a mass ratio of 3:2-2:3 to obtain a mixed solution; S2: stirring the mixed solution at 30-50° C. for 0.5-4 h, and centrifuging to remove bubbles to obtain a hydrogel carrier solution; S3: Mix 30-120ul of active enzyme with 100mg of hydrogel carrier solution at 30-40℃, centrifuge to remove bubbles, inject all of it into a syringe, and drip it into calcium chloride solution through the syringe. The fixation time is 0-25min, and the hydrogel-immobilized enzyme is obtained after washing.

6. The method for preparing a hydrogel-immobilized enzyme according to claim 5, characterized in that: The centrifugal degassing in S2 is performed by stirring the reacted solution in a water bath at 20-30° C. until the bubbles are removed.

7. The method for preparing a hydrogel-immobilized enzyme according to claim 5, characterized in that: The mass volume concentration of the calcium chloride solution in S3 is 10%.

8. The method for preparing a hydrogel-immobilized enzyme according to claim 5, characterized in that: The fixation time in S3 was 13-23 min.

9. The method for preparing a hydrogel-immobilized enzyme according to claim 5, characterized in that: The ratio of active enzyme to hydrogel carrier solution in S2 was 52ul:100mg.

10. The use of the hydrogel-immobilized enzyme according to any one of claims 1 to 4, characterized in that: Application of hydrogel-immobilized enzyme in oral alcohol-relief medicine, wherein the active enzymes of the hydrogel-immobilized enzyme are alcohol dehydrogenase and acetaldehyde dehydrogenase.

Images