Enzyme immobilization carrier and preparation method therefor, immobilized enzyme and preparation method therefor

Abstract

To provide an enzyme immobilization carrier, an immobilized enzyme, and a preparation method therefor.SOLUTION: The enzyme immobilization carrier is one in which super-crosslinked polyvinyl alcohol is modified with an amino group or cyanuric chloride. Use of the enzyme immobilization carrier of the present invention may effectively improve stability and reusability of immobilized enzyme. Since a form of enzyme covalent linkage is used, compared with an embedding method, it is not necessary to perform immersion with a chemical reagent, which is advantageous for maintaining own activity of the enzyme, and allows the immobilized enzyme to have better activity while being stable and reusable.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the technical field of biocatalysis, and more particularly to an enzyme-immobilized carrier and a method for producing the same, and an immobilized enzyme and a method for producing the same. [Background technology]

[0002] Compared with chemical catalysts, enzymes, which are biological catalysts, are widely used in industrial production due to their high activity, selectivity, and substrate specificity. Biocatalysts are becoming an important component in the manufacturing of chemicals, intermediates, fine chemicals, and final drug molecules. Enzymes have high regioselectivity, high catalytic efficiency, and mild reaction conditions. However, free enzymes are easily inactivated because their catalytic activity is affected by temperature, pH, solvent, etc. As process demands continue to expand, the demands for enzyme utilization efficiency and economy are also increasing. Therefore, it is necessary to not only improve enzyme activity, specificity, and productivity, but also to improve the recoverability and reusability of enzymes.

[0003] Immobilizing enzymes by immobilizing them on solid supports to obtain heterogeneous immobilized enzyme systems provides an excellent solution to the above problems. Immobilized enzymes maintain their inherent catalytic activity while offering a number of advantages, including high stability, easy separation and recovery, reusability, and simple processing. The performance of immobilized enzymes depends primarily on the immobilization method and the support material used. Currently, enzyme immobilization methods are classified into adsorption, covalent bonding, crosslinking, and embedding, depending on the method of binding between the enzyme and the support. Among these, adsorption, which utilizes physical adsorption, van der Waals forces, and hydrophobic interactions between the enzyme and the support, is the most commonly used method for enzyme immobilization due to its advantages of simple and convenient operation, large adsorption capacity, and low industrial costs. However, these methods have the drawback of weak binding strength between the support and enzyme, which can easily detach from the support during repeated use, resulting in reduced conversion. Covalent bonding methods involve covalently linking the enzyme to the support. Therefore, the carrier must have a corresponding functional group that can react with the enzyme to form a covalent bond.Compared to the adsorption method, the bond between the enzyme and the carrier is stronger, there is no problem of the enzyme falling off during use, and stability and recyclability are better.

[0004] Polyvinyl alcohol (PVA) is a polymeric organic compound with no toxicity or side effects to the human body and good biocompatibility. It is widely used in the medical field, including the manufacture of hydrogels used in soft contact lenses, oral patches applied to affected areas to treat bacterial infections, and medical sponges used to absorb fluids and blood during surgery. Polyvinyl alcohol can be produced using established methods, such as the petroleum ethylene method, the natural gas acetylene method, and the carbide acetylene method, making it easy to produce, readily available, and inexpensive. Furthermore, unlike other vinyl polymers (e.g., polystyrene), polyvinyl alcohol can be used as a carbon source by bacteria, is degradable, and is an environmentally friendly material.

[0005] In recent years, extensive research has been conducted both in China and abroad on the immobilization of bioactive substances using PVA as a carrier. The main methods for producing PVA are the PVA-boric acid method and the freeze-thaw method. Bioactive substances embedded using the boric acid method have high mechanical strength, long service life, and good elasticity. However, the drawbacks are that boric acid is toxic to the embedded organisms (enzymes), low bioretention, and the immobilized particles are prone to adhesion. The freeze-thaw method uses physical crosslinking to form gels. While the gels produced by this method have a high water content, their stability is inferior to that of gels obtained by chemical crosslinking, making them difficult to meet the requirements of some bioreactors, especially fluidized-bed reactors. Furthermore, in addition to non-toxicity and stability, mass transport and loading capacity are also important for immobilization carriers. However, current embedding methods in immobilized microorganism research require immobilized microorganisms to be immersed or frozen in chemical reagents, which can affect microbial activity and application efficiency. Furthermore, the low porosity of the embedding carrier limits mass transport and loading capacity.

[0006] Therefore, how to efficiently immobilize enzymes with PVA so that the immobilized enzymes have high activity, better stability, and reusability is an urgent issue to be solved in this field. Summary of the Invention [Problem to be solved by the invention]

[0007] The main object of the present invention is to provide an enzyme immobilization carrier and a method for producing the same, and an immobilized enzyme and a method for producing the same, in order to solve the problem of the prior art that polyvinyl alcohol-immobilized enzymes cannot have high activity, good stability, and reusability. [Means for solving the problem]

[0008] In order to achieve the above object, according to one aspect of the present invention, there is provided an enzyme-immobilized support which is made by modifying hypercrosslinked polyvinyl alcohol with an amino group or cyanuric chloride.

[0009] Furthermore, hypercrosslinked polyvinyl alcohol is obtained by sequentially subjecting polyvinyl alcohol to oxidation, self-crosslinking, and crosslinking with a crosslinking agent.

[0010] Furthermore, for amino group modification, 3-aminopropyltriethoxysilane is used as an amino group modification reagent.

[0011] According to another aspect of the present invention, there is provided an immobilized enzyme in which the enzyme-immobilization support and the enzyme are covalently bonded together.

[0012] Furthermore, the enzyme is any one or more selected from transaminase, monooxygenase, ketoreductase, enereductase, and amino acid dehydrogenase. Preferably, the transaminase is a transaminase derived from Chromobacterium violaceum DSM30191, a transaminase derived from Arthrobacter citreus, or a transaminase derived from B. thuringiensis. Preferably, the monooxygenase is a cyclohexanone monooxygenase derived from Brachymonas petroleovorans or a cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1. Preferably, the ketoreductase is a ketoreductase derived from Acetobacter sp. CCTCC M209061 or a ketoreductase derived from Candida macedoniensis AKU4588. Preferably, the enereductase is a ketoreductase derived from Saccharomyces Preferably, the amino acid dehydrogenase is leucine dehydrogenase from Bacillus cereus and phenylalanine dehydrogenase from Bacillus sphaericus.

[0013] According to another aspect of the present invention, there is further provided a method for producing the above-mentioned enzyme-immobilized support, comprising the steps of providing hyper-crosslinked polyvinyl alcohol and modifying the hyper-crosslinked polyvinyl alcohol with an amino group or cyanuric chloride to obtain an enzyme-immobilized support.

[0014] Furthermore, hypercrosslinked polyvinyl alcohol can be produced by a method including: an oxidation step in which polyvinyl alcohol is dissolved in water, and an oxidizing agent is added to carry out an oxidation reaction to obtain an oxidation product system, preferably at a temperature of 20-30°C; a self-crosslinking step in which the hydrogen chloride concentration of the oxidation product system is adjusted to 0.1-1 mol / L with hydrochloric acid, and then a self-crosslinking reaction is carried out at a temperature of 70-90°C to obtain polyvinyl alcohol gel particles; and a crosslinking step in which the polyvinyl alcohol gel particles are dispersed in water, and a crosslinking agent is added to carry out a reaction to obtain hypercrosslinked polyvinyl alcohol, preferably at a temperature of 40-60°C, and hydrochloric acid is added to the system during the reaction to adjust the hydrogen chloride concentration of the system to 0.1-1 mol / L.

[0015] Furthermore, the step of modifying the hyper-crosslinked polyvinyl alcohol with amino groups includes a step of mixing and reacting the hyper-crosslinked polyvinyl alcohol, a first solvent, and an amino group modifying reagent to obtain an enzyme-immobilized support, wherein the amino group modifying reagent is preferably 3-aminopropyltriethoxysilane, and the amount of the amino group modifying reagent added is preferably 0.3 to 1 mL per 1 g of the hyper-crosslinked polyvinyl alcohol in the amino group modification reaction, and the pH value of the reaction is preferably 2 to 3 and the reaction temperature is preferably 60 to 90°C, and the first solvent is preferably water.

[0016] Furthermore, the step of modifying the hyper-crosslinked polyvinyl alcohol with cyanuric chloride includes a step of dispersing the hyper-crosslinked polyvinyl alcohol in a second solvent, adding cyanuric chloride to react, and obtaining an enzyme-immobilized support, wherein the amount of cyanuric chloride added is preferably 0.25 to 1 g per 1 g of hyper-crosslinked polyvinyl alcohol, the reaction temperature in the cyanuric chloride modification reaction is preferably 0 to 10°C, and the second solvent is preferably acetone.

[0017] According to another aspect of the present invention, there is further provided a method for producing an immobilized enzyme, which comprises the step of covalently linking the enzyme-immobilization support and an enzyme to obtain an immobilized enzyme.

[0018] Furthermore, when the enzyme-immobilized support is hypercrosslinked polyvinyl alcohol modified with amino groups, the method for producing an immobilized enzyme includes the steps of: dispersing the enzyme-immobilized support in a glutaraldehyde solution to activate it, thereby obtaining an activated support; and reacting the activated support with an enzyme solution containing an enzyme, thereby covalently linking the enzyme and the enzyme-immobilized support to obtain an immobilized enzyme. Preferably, the glutaraldehyde solution has a concentration of 1 to 2 mass %, and more preferably, the glutaraldehyde solution is a mixed solution of glutaraldehyde and a phosphate buffer solution. Preferably, in the activation step, the activation temperature is 20 to 30°C, and the activation time is 1 to 3 hours. Preferably, in the process of reacting the activated support with the enzyme solution, the reaction temperature is 20 to 30°C. Preferably, 1 g of the activated support corresponds to 2 to 6 mL of enzyme solution, and the protein content of the enzyme solution is 30 to 40 mg / mL.

[0019] Furthermore, when the enzyme-immobilized support is a hypercrosslinked polyvinyl alcohol modified with cyanuric chloride, the method for producing an immobilized enzyme includes the steps of wetting the enzyme-immobilized support with a phosphate buffer solution and reacting the wetted enzyme-immobilized support with an enzyme solution containing the enzyme to covalently link the enzyme and the enzyme-immobilized support to obtain an immobilized enzyme. Preferably, in the process of reacting the wetted enzyme-immobilized support with the enzyme solution, the reaction temperature is 20 to 30°C, and preferably, 1 g of the enzyme-immobilized support corresponds to 2 to 6 mL of enzyme solution, and the protein content in the enzyme solution is 30 to 40 mg / mL.

[0020] The present invention further provides an enzyme immobilization support which is a hypercrosslinked polyvinyl alcohol modified with an amino group or cyanuric chloride. By modifying the hypercrosslinked polyvinyl alcohol with an amino group or cyanuric chloride, the polyvinyl alcohol molecular chain is modified with an amino group or [ka] These modifying groups react with the enzyme, resulting in the effect of linking the two together via a covalent bond. Therefore, when the enzyme-immobilized support of the present invention is used, the stability and reusability of the immobilized enzyme are effectively improved. Furthermore, since the enzyme is linked by a covalent bond, there is no need for immersion in a chemical reagent, as compared with the embedding method, which is advantageous in maintaining the activity of the enzyme itself, making the immobilized enzyme stable and reusable while maintaining better activity. DETAILED DESCRIPTION OF THE INVENTION

[0021] It should be noted that, unless there is a contradiction, the embodiments and features of the embodiments of the present application may be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0022] As described in the Background Art section, in the prior art, it is difficult to combine high activity, good stability, and reusability in polyvinyl alcohol-immobilized enzymes.

[0023] In order to solve the above problems, the present invention provides an enzyme immobilization carrier which is a hypercrosslinked polyvinyl alcohol modified with an amino group or cyanuric chloride. By modifying the hypercrosslinked polyvinyl alcohol with an amino group or cyanuric chloride, the polyvinyl alcohol molecular chain is modified with an amino group or [ka] These modifying groups react with the enzyme, resulting in the effect of linking the two together via a covalent bond. Therefore, when the enzyme-immobilized support of the present invention is used, the stability and reusability of the immobilized enzyme are effectively improved. Furthermore, since the enzyme is linked by a covalent bond, there is no need for immersion in a chemical reagent, as compared with the embedding method, which is advantageous in maintaining the activity of the enzyme itself, making the immobilized enzyme stable and reusable while maintaining better activity.

[0024] In the present invention, the term "hyper-crosslinked" in the context of hyper-crosslinked polyvinyl alcohol means that the degree of crosslinking is so high that the polymer becomes insoluble in good solvents for polyvinyl alcohol, such as water and organic solvents.

[0025] In a preferred embodiment, hypercrosslinked polyvinyl alcohol is obtained by sequentially oxidizing polyvinyl alcohol, self-crosslinking, and crosslinking with a crosslinking agent. During oxidation, the carbon-carbon bond of the end-to-end o-diol structure in the PVA segment is broken to form two aldehyde groups. During self-crosslinking, the aldehyde group obtained by oxidation reacts with the hydroxyl group on the PVA chain to form the corresponding acetal structure (the reactions during the oxidation and self-crosslinking processes are shown below). When glutaraldehyde crosslinking agent is added, it further reacts with the hydroxyl group on the PVA chain to form a hypercrosslinked PVA resin. [ka]

[0026] In order to improve the efficiency of amino group modification, in a preferred embodiment, 3-aminopropyltriethoxysilane is used as an amino group modification reagent for amino group modification. This 3-aminopropyltriethoxysilane reacts with the hydroxyl groups on the molecular chain of the hypercrosslinked polyvinyl alcohol to form hydroxyl groups on the molecular chain. [ka] More importantly, the amino group modification using this group has a more appropriate size, and the group gradually changes from rigid to flexible in the direction away from the molecular backbone, which is advantageous for the subsequent immobilization reaction with the enzyme, and can better immobilize the enzyme, further improving the stability and reusability of the immobilized enzyme, and imparting higher activity to the immobilized enzyme.

[0027] According to another aspect of the present invention, there is further provided an immobilized enzyme in which the enzyme-immobilized support and the enzyme are covalently linked. As described above, the enzyme-immobilized support of the present invention is a hypercrosslinked polyvinyl alcohol modified with an amino group or cyanuric chloride, whereby the molecular chain of the polyvinyl alcohol is bonded to the amino group or [ka] These modifying groups react with the enzyme, resulting in a covalent bond between the two. Therefore, the use of this enzyme immobilization carrier effectively improves the stability and reusability of the immobilized enzyme. Furthermore, because the enzyme is covalently linked, there is no need for immersion in chemical reagents, as compared with the embedding method, which is advantageous for maintaining the activity of the enzyme itself, making the immobilized enzyme stable and reusable while also providing better activity.

[0028] The immobilized enzyme of the present invention has broad applicability to enzymes, including, but not limited to, any one or more of transaminases, monooxygenases, ketoreductases, enereductases, and amino acid dehydrogenases, but is more particularly applicable to the following enzymes: The transaminase is a transaminase derived from Chromobacterium violaceum DSM30191, a transaminase derived from Arthrobacter citreus, or a transaminase derived from B. thuringiensis; the monooxygenase is a cyclohexanone monooxygenase derived from Brachymonas petroleovorans or a cyclohexanone monooxygenase derived from Rhodococcus ruber-SD1; the ketoreductase is a ketoreductase derived from Acetobacter sp. CCTCC M209061 or a ketoreductase derived from Candida macedoniensis AKU4588; and the amino acid dehydrogenase is a leucine dehydrogenase derived from Bacillus cereus and a phenylalanine dehydrogenase derived from Bacillus sphaericus.

[0029] According to another aspect of the present invention, there is provided a method for producing the above-mentioned enzyme-immobilized carrier, which comprises the steps of providing a hyper-crosslinked polyvinyl alcohol and modifying the hyper-crosslinked polyvinyl alcohol with an amino group or cyanuric chloride to obtain an enzyme-immobilized carrier. The enzyme-immobilized carrier produced by this method has a hyper-crosslinked polyvinyl alcohol as the carrier body and has an amino group or a cyanuric chloride on its molecular chain. [ka] These modifying groups react with enzymes to immobilize them, forming immobilized enzymes, effectively improving the stability and reusability of the immobilized enzymes. Furthermore, because the enzyme is covalently linked, this method does not require immersion in chemical reagents, as compared with the embedding method, and is therefore advantageous in maintaining the activity of the enzyme itself, making the immobilized enzyme stable and reusable while also providing better activity.

[0030] In a preferred embodiment, hypercrosslinked polyvinyl alcohol is produced by a process that includes the following steps: an oxidation step in which polyvinyl alcohol is dissolved in water and an oxidizing agent is added to conduct an oxidation reaction to obtain an oxidation product system; a self-crosslinking step in which the hydrogen chloride concentration of the oxidation product system is adjusted to 0.1-1 mol / L with hydrochloric acid and a self-crosslinking reaction is conducted at a temperature of 70-90°C to obtain polyvinyl alcohol gel particles; and a crosslinking step in which the polyvinyl alcohol gel particles are dispersed in water and a crosslinking agent is added to react with the polyvinyl alcohol gel particles to obtain hypercrosslinked polyvinyl alcohol. During oxidation, the carbon-carbon bond of the end-to-end o-diol structure in the PVA segment is broken to form two aldehyde groups. During self-crosslinking, the aldehyde group obtained by oxidation reacts with the hydroxyl groups on the PVA chain to form the corresponding acetal structure. The addition of glutaraldehyde crosslinking agent further reacts with the hydroxyl groups on the PVA chain to obtain a hypercrosslinked PVA resin. Preferably, the oxidizing agent is sodium periodate. Sodium periodate facilitates the oxidation reaction. Preferably, the oxidation reaction temperature is 20 to 30°C. Preferably, the crosslinking agent is glutaraldehyde, and the reaction temperature in the crosslinking step using the crosslinking agent is 40 to 60°C. Hydrochloric acid is added during the reaction to adjust the hydrogen chloride concentration in the system to 0.1 to 1 mol / L. The above crosslinking agent and crosslinking conditions allow the reaction with the hydroxy groups on the PVA chains to proceed more thoroughly, resulting in a hypercrosslinked polyvinyl alcohol with a more stable structure.

[0031] To promote dissolution of polyvinyl alcohol, in actual operation, polyvinyl alcohol may be added to the first solvent and then dissolved at approximately 90°C until dissolved. The oxidation may be carried out at room temperature, and the reaction time is preferably 1 to 3 hours. In the self-crosslinking reaction step, the reaction time is preferably 3 to 6 hours. In the crosslinking step using a crosslinking agent, the reaction time is preferably 5 to 8 hours.

[0032] In a preferred embodiment, the step of modifying the hyper-crosslinked polyvinyl alcohol with amino groups includes the step of mixing and reacting the hyper-crosslinked polyvinyl alcohol, the first solvent, and an amino group modifying reagent to obtain an enzyme immobilized support, and preferably, the amino group modifying reagent is 3-aminopropyltriethoxysilane. This 3-aminopropyltriethoxysilane reacts with the hydroxyl groups on the molecular chain of the hyper-crosslinked polyvinyl alcohol to form an amino group on the molecular chain. [ka] More importantly, when the amino group is modified with this group, the size of the group is more appropriate, and the group gradually changes from rigid to flexible in the direction away from the molecular backbone, which is advantageous for the subsequent immobilization reaction with the enzyme, and the enzyme can be better immobilized, further improving the stability and reusability of the immobilized enzyme and having better activity.

[0033] Preferably, in the amino group modification reaction step, the amount of amino group modification reagent added is 1 to 3 ml per 1 g of hypercrosslinked polyvinyl alcohol. By controlling the amount of amino group modification reagent added within the above range, the reaction with the hydroxyl groups on the molecular chains of hypercrosslinked polyvinyl alcohol can be more thoroughly carried out, thereby improving the amount and stability of the enzyme supported in the subsequent enzyme immobilization step.

[0034] To further increase the reaction efficiency, in a preferred embodiment, in the amino group modification reaction step, the pH value of the reaction is 2 to 3, and the reaction temperature is 60 to 90°C. Specifically, the pH value of the reaction system can be adjusted with a pH adjuster, such as concentrated hydrochloric acid. Preferably, the first solvent is water. Preferably, the reaction time is 5 to 24 hours.

[0035] In a preferred embodiment, the step of modifying the hypercrosslinked polyvinyl alcohol with cyanuric chloride includes dispersing the hypercrosslinked polyvinyl alcohol in a second solvent, followed by adding cyanuric chloride to react with the resulting enzyme-immobilized support. Compared to using amino groups as the modifying group, modification with cyanuric chloride eliminates the need for an additional crosslinking agent such as glutaraldehyde in the subsequent enzyme immobilization step; instead, the enzyme-immobilized support can be directly reacted with the enzyme solution. Preferably, the amount of cyanuric chloride added is 0.25 to 1 g per 1 g of hypercrosslinked polyvinyl alcohol. Controlling the amount of cyanuric chloride used within the above range allows for a more thorough reaction between the hydroxyl groups on the molecular chain and the cyanuric chloride, thereby increasing the amount of enzyme supported on the support and further enhancing the stability and activity of the immobilized enzyme.

[0036] To further enhance the stability and efficiency of the reaction, in a preferred embodiment, the reaction temperature in the cyanuric chloride modification reaction step is 0 to 10°C. Preferably, the second solvent is acetone. Preferably, the reaction time is 2 to 6 hours.

[0037] In yet another aspect of the present invention, there is provided a method for producing an immobilized enzyme, comprising the step of covalently linking the enzyme to the enzyme-immobilization support to obtain an immobilized enzyme. In the above support, the amino group and the cyanuric chloride modifying group are reacted with the enzyme to form a covalent bond between them. Compared with physical adsorption immobilization, the immobilized enzyme formed by covalent linkage has better stability and reusability. Compared with methods such as embedding, the covalent linkage promotes the immobilized enzyme to maintain high enzymatic activity, providing a significant improvement in maintaining stable catalytic activity over a long period of time.

[0038] In a preferred embodiment, when the enzyme-immobilized support is a hypercrosslinked polyvinyl alcohol modified with amino groups, the method for producing an immobilized enzyme includes the steps of dispersing the enzyme-immobilized support in a glutaraldehyde solution to activate it, thereby obtaining an activated support, and reacting the activated support with an enzyme solution containing an enzyme to covalently bond the enzyme to the enzyme-immobilized support, thereby obtaining an immobilized enzyme. Activating the activated support with glutaraldehyde and reacting it with the enzyme solution containing the enzyme promotes the completion of a chemical reaction between the modified amino groups and the enzyme, thereby immobilizing the enzyme. One aldehyde group of glutaraldehyde reacts with an amino group on the support to form an imine bond, and the other aldehyde group reacts with a free amino group on the enzyme, thereby linking the enzyme and the support together.

[0039] To ensure sufficient activation, the glutaraldehyde solution preferably has a concentration of 1 to 2% by mass, and more preferably is a mixed solution of glutaraldehyde and a phosphate buffer. The phosphate buffer is preferably a phosphate buffer with a pH of 7.0 (which may be disodium hydrogen phosphate buffer or sodium dihydrogen phosphate buffer). Preferably, in the activation step, the activation temperature is 20 to 30°C and the activation time is 1 to 3 hours. Activation with glutaraldehyde at these temperatures provides better results and more stable reactions. Preferably, the reaction temperature during the reaction between the activating carrier and the enzyme solution is 20 to 30°C. Performing the enzyme immobilization reaction at these temperatures provides better reaction efficiency and stability. Preferably, 1 g of the activating carrier corresponds to 2 to 6 mL of enzyme solution, and the protein content in the enzyme solution is 30 to 40 mg / mL. This method is advantageous in that the enzyme is more thoroughly immobilized on the carrier, increasing the enzyme loading and promoting improved activity of the immobilized enzyme. Preferably, the reaction time between the activating carrier and the enzyme solution is 15 to 25 hours.

[0040] In a preferred embodiment, when the enzyme-immobilization support is a hypercrosslinked polyvinyl alcohol modified with cyanuric chloride, the method for producing an immobilized enzyme includes the steps of wetting the enzyme-immobilization support with a phosphate buffer solution and reacting the wetted enzyme-immobilization support with an enzyme solution to covalently bond the enzyme to the enzyme-immobilization support, thereby obtaining an immobilized enzyme. The phosphate buffer solution is preferably a phosphate buffer solution with a pH of 7.0. By wetting the support, the cyanuric chloride-modified groups in the support undergo an immobilization reaction with the enzyme in the enzyme solution, forming a covalent bond between them.

[0041] In order to enhance stability during the reaction process and increase reaction efficiency, in a preferred embodiment, the reaction temperature during the reaction between the wetted enzyme-immobilized carrier and the enzyme solution is 20 to 30°C. More preferably, 1 g of the enzyme-immobilized carrier corresponds to 2 to 6 mL of enzyme solution, and the protein content in the enzyme solution is 30 to 40 mg / mL. This is advantageous in that the enzyme is more thoroughly immobilized, the amount supported is improved, and the immobilized enzyme is promoted to have higher catalytic activity. Preferably, the reaction time between the enzyme-immobilized carrier and the enzyme solution is 15 to 25 hours.

[0042] The present application will now be described in more detail with reference to specific examples, which should not be construed as limiting the scope of the patent claims of the present application.

[0043] The origins of the enzymes used in the following examples are shown in Table 1 below. [Table 1]

[0044] Example 1 Example of manufacturing immobilization carrier PVA oxidation: PVA (10 g) was added to 100 mL of pure water and stirred at 90°C until completely dissolved. After that, it was cooled to room temperature, and sodium periodate (0.9 g) was added thereto and stirred for 1 hour to react.

[0045] PVA self-crosslinking: The PVA solution oxidized in the previous step was adjusted with 36% concentrated hydrochloric acid until the hydrogen chloride concentration reached 0.1 mol / L, and then heated to 70°C and left to react for 3 hours to form a PVA gel. The resulting self-crosslinked PVA gel was sieved through a 20-mesh sieve to obtain gel particles (SCL-PVA), which were then washed three times with pure water.

[0046] Further crosslinking of PVA colloidal particles: The SCL-PVA colloidal particles obtained above were dispersed in 100 mL of pure water, to which 50 wt% glutaraldehyde (20 mL) and 36% concentrated hydrochloric acid (25 mL) were added. After uniform stirring, the mixture was heated to 50°C and reacted for 6 hours. The resulting colloidal particles were then washed with pure water until neutral and dried to obtain macroporous hypercrosslinked PVA (MP-PVA).

[0047] Example 2 Example of producing an immobilization carrier by amino group modification 3 g of MP-PVA was added to 100 mL of deionized water and 2 mL of APTES, and the mixture was stirred at room temperature for 30 min. The pH was then adjusted to 3.5 with concentrated hydrochloric acid. After stirring for 5 min, the mixture was heated to 80°C and reacted for 20 h under N2 protection (reaction scheme is shown below). After the reaction was complete, the mixture was washed with ethanol and then with deionized water to obtain the amino group-modified immobilized support MP-PVA-NH2. [ka]

[0048] Example 3 Example of cyanuric chloride-modified immobilization support 1 g of the baked MP-PVA was added to 40 mL of acetone and stirred in an ice-water bath (0 °C) for 30 min, after which 0.5 g of cyanuric chloride was added and the reaction was continued for another 4 h in an ice-water bath. After the reaction was completed, the mixture was washed with acetone and suction filtered to dryness to obtain the cyanuric chloride-modified enzyme immobilized support MP-PVA-CC. [ka]

[0049] Example 4 MP-PVA-NH2 enzyme immobilization A 1 wt% glutaraldehyde solution was prepared using 20 mM sodium dihydrogen phosphate buffer (pH 7.0), and 1 g of MP-PVA-NH2 was weighed and dispersed in the solution. After incubation at 30 °C for 1 h, the mixture was washed three times with deionized water. A predetermined amount of the prepared enzyme solution (protein content: 30-40 mg / mL) was added to the glutaraldehyde-activated MP-PVA-NH2 (4 mL of enzyme solution corresponds to 1 g of carrier), and the mixture was immobilized at 20 °C for 20 h to obtain the MP-PVA-NH2-immobilized enzyme.

[0050] Example 5 MP-PVA-CC enzyme immobilization The MP-PVA-CC was wetted with 20 mM sodium dihydrogen phosphate buffer (pH 7.0) and washed three times. A predetermined amount of the prepared enzyme solution (protein content: 30-40 mg / mL) was added to the MP-PVA-CC (4 mL of enzyme solution corresponds to 1 g of carrier), and the immobilization was carried out at 20°C for 20 hours to obtain the immobilized enzyme on MP-PVA-CC.

[0051] Application Example 1 Testing the activity and reusability of immobilized transaminase (TA) of MP-PVA-NH2 and MP-PVA-CC The enzyme-catalyzed reaction scheme is as follows: [ka]

[0052] 0.5 mL of methanol was added to a 20 mL reaction flask, 0.1 g of carbonyl substrate was dissolved, 15 eq of isopropylamine hydrochloride and 25.0 mg of PLP (pyridoxal 5'-phosphate) were added, and 0.1 M phosphate buffer (PB8.0) was added to the reaction solution until the final volume reached 5 mL to form a reaction system.

[0053] 0.1 g of transaminase enzyme powder or an immobilized enzyme prepared with an enzyme solution corresponding to 0.1 g of transaminase enzyme powder was added to the reaction system, which was then stirred at 47°C for 20 hours. The conversion rate of the system was measured by HPLC, and after each reaction, the immobilized enzyme was separated and reused in the next reaction, and the number of times it was reused was determined. The reaction data are as follows: [Table 2]

[0054] The transaminase is derived from a transaminase derived from Aspergillus fumigatus and has the following sequence: SEQ ID NO:1: MQKQRTCSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDCEGNKIIDGMAGLWCVNVGYGRKDFAEAAARRQMEELPFYNTFFGTTHPPVVELSSLLAEVTPAGFDRV FYTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWNGYHGSTIGGASLGGMKYMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQ GAGGVIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPLGAVFVGDRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGI VQRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMMLAFTLVKNKAKRELFPDFGEIGTLCEDIFFRNNLIMTAQGDHIVSAPPLVMTRAEVDEMLAVAERCLEEFEQTLKARGLA

[0055] Application Example 2 Testing the activity and reusability of MP-PVA-NH2 and MP-PVA-CC immobilized amino acid dehydrogenase (AADH) The scheme of the enzyme-catalyzed reaction is as follows: [ka]

[0056] In a 10 mL reaction flask, add 5 mL of 0.1 M Tris-Cl (pH 8.0), then add 100 mg of the main material and 108 mg of ammonium chloride, adjust the pH to 7.5, and then add nicotinamide adenine dinucleotide (NAD + ) and 50 mg of glucose dehydrogenase (GDH: glucose-1-dehydrogenase derived from Lysinibacillus sphaericus G10, coenzyme, for NADP cycle) were added to form a reaction system.

[0057] 100 mg of AADH enzyme (or immobilized AADH prepared with an enzyme solution corresponding to 100 mg of free enzyme) was added to each reaction system. After 20 hours of reaction at 30°C, the conversion rate was tested. The test results are shown in the table below. [Table 3]

[0058] The amino acid dehydrogenase is derived from leucine dehydrogenase from Bacillus cereus and has the following sequence: SEQ ID NO:2: MRDVFEMMMDRYGHEQVIFCRHPQTGLKAIIALHNTTAGPALGGCRMIPYASTDEALEDVLRLSKGMTYKCSLADVDFGGGKMVIIGDPKKDKSPELFRVIGRFVGGLNGRFYTGTDMGTNPEDFVHAARESKSFAGLPKSYGGKGDTSIPTALGVFHGMRATARFLWGTDQLKGRVVAIQGVG KVGERLLQLLVEVGAYCKIADIDSVRCEQLKEKYGDKVQLVDVNRIHKESCDIFSPCAKGGVVNDDTIDEFRCLAIVGSANNQLVEDRHGALLQKRSICYAPDYLVNAGGLIQVADELEGFHEERVLAKTEAIYDMVLDIFHRAKNENITTCEAADRIVMERLKKLTDIRRILLEDPRNSARR

[0059] Application Example 3 MP-PVA-NH2 and MP-PVA-CC immobilized ketoreductase (KRED) activity and reusability tests: The scheme of the enzyme-catalyzed reaction is as follows: [ka]

[0060] Add 0.5 mL of isopropanol (IPA) to a 10 mL reaction flask, dissolve 0.1 g of the main ingredient, and add 0.5 mL of 0.1 M PB 7.0 and nicotinamide adenine dinucleotide (NAD + ) 10 mg was added to form a reaction waiting system.

[0061] To the reaction system, 0.05 g of ketoreductase enzyme powder or an immobilized enzyme prepared with an enzyme solution corresponding to 0.05 g of ketoreductase enzyme powder was added separately, and the mixture was stirred at 30°C for 20 hours. The conversion rate of the system was measured by HPLC, and after each reaction, the immobilized enzyme was separated and reused in the next reaction, and the number of times it was reused was determined. The reaction data are as follows: [Table 4]

[0062] The ketoreductase is derived from the ketoreductase of Candida macedoniensis AKU4588 and has the following sequence: SEQ ID NO:3: MKAIQYTRIGAEPELTEIPKPEPGPGEVLLEVTAAGVCHSDDFIMSLPEEQYTYGLPLTLGHEGAGKVAAVGEGVEGLDIGTNVVVYGPWGCGNCWHCSQGLENYCSRAQELGINPPGLGAPGALAEFMIVDSPRHLVPIGDLDPVKTVPLTDAGLTPYHAIKRSLPKLRGGSY AVVIGTGGLGHVAIQLLRHLSAATVIALDVSADKLELATKVGAHEVVLSDKDAAENVRKITGSQGAALVLDFVGYQPTIDTAMAVAGVGSDVTIVGIGDGQAHAKVGFFQSPYEASVTVPYWGARNELIELIDLAHAGIFDIAVETFSLDNGAEAYRRLAAGTLSGRAVVVPGL

[0063] Application Example 4 Testing the activity and reusability of immobilized monooxygenase (CHMO) of MP-PVA-NH2 and MP-PVA-CC The scheme of the enzyme-catalyzed reaction is as follows: [ka]

[0064] A 10 mL reaction flask was charged with 0.3 mL of isopropanol, followed by 3 mL of 0.1 M PB (pH 8.0) containing 500 mg of the main ingredient and 5 mg of NADP+. This was followed by 50 mg of ADH-Tb (alcohol dehydrogenase derived from Thermoanaerobium brockii, a coenzyme for the NADP cycle) and 100 mg of monooxygenase enzyme powder (or immobilized monooxygenase prepared with an enzyme solution equivalent to 100 mg of free enzyme). The reaction was run for 20 hours at 30°C, and the conversion rate was tested. After each reaction, the immobilized enzyme was separated and reused in the next reaction. The number of cycles was then determined. The results are shown in the table below. [Table 5]

[0065] The monooxygenase is derived from cyclohexanone monooxygenase from Brachymonas petroleovorans and has the following sequence: SEQ ID NO:4: MTTSIDREALRRKYAEERDKRIRPDGNDQYIRLDHVDGWSHDPYMPITPREPKLDHVTFAFIGGGFSGLVTAARLRESGVESVRIIDKAGDFGGVWYWNRYPGAMCDTAAMVYMPLLEETGYMPTEKYAHGPEILEHCQRIGKHYDLYDD ALFHTEVTDLVWQEHDQRWRISTNRGDHFTAQFVGMGTGPLHVAQLPGIPGIESFRGKSFHTSRWDYDYTGGDALGAPMDKLADKRVAVIGTGATAVQCVPELAKYCRELYVVQRTPSAVDERGNHPIDEKWFAQIATPGWQKRWLDSFTA IWDGVLTDPSELAIEHEDLVQDGWTALGQRMRAAVGSVPIEQYSPENVQRALEEADDEQMERIRARVDEIVTDPATAAQLKAWFRQMCKRPCFHDDYLPAFNRPNTHLVDTGGKGVERITENGVVVAGVEYEVDCIVYASGFEFLGTGYTD RAGFDPTGRDGVKLSEHWAQGTRTLHGMHTYGFPNLFVLQLMQGAALGSNIPHNFVEAARVVAAIVDHVLSTGTSSVETTKEAEQAWVQLLLDHGRPLGNPECTPGYYNNEGKPAELKDRLNVGYPAGSAAFFRMMDHWLAAGSFDGLTFR

[0066] Application Example 5 Testing the activity and reusability of immobilized ene reductase (ERED) from MP-PVA-NH2 and MP-PVA-CC The scheme of the enzyme-catalyzed reaction is as follows: [ka]

[0067] A 10 mL reaction flask was charged with 3 mL of 0.1 M sodium dihydrogen phosphate buffer solution (pH 7.0), and then 100 mg of substrate was added, followed by 10 mg of nicotinamide adenine dinucleotide phosphate (NADP+), 80 mg of ammonium formate, and 20 mg of formate dehydrogenase (FDH: formate dehydrogenase derived from Candida boidinii, coenzyme, for the NADP cycle) to form a reaction waiting system.

[0068] 100 mg of ERED enzyme (or immobilized ERED enzyme prepared with an enzyme solution equivalent to 100 mg of free enzyme) was added to the reaction system. The reaction was carried out at 30°C for 20 hours, and the conversion rate was tested. After each reaction, the immobilized enzyme was separated and reused in the next reaction, and the number of times it was reused was determined. The results are shown in the table below. [Table 6]

[0069] The enereductase is derived from the enereductase from Saccharomyces cerevisiae and has the following sequence: SEQ ID NO:5: MNTMLFSPYTIRGLTLKNRIVMSPMCMYSCDTKDGAVRTWHKIHYPARAVGQVGLIIVEATGVTPQGRISERDLGIWSDDHIAGLRELVGLVKEHGAAIGIQLAHAGRKSQVPGEIIAPSAVPFDDSSPTPKEMTKADIEETVQAFQNGARRAKEAGFDVIEIHAAHGYL INEFLSPLSNRRQDEYGGSPENRYRFLGEVIDAVREVWDGPLFVRISASDYHPDGLTAKDYVPYAKRMKEQGVDLVDVSSGAIVPARMNVYPGYQVPFAELIRREADIPTGAVGLITSGWQAEEILQNGRADLVFLGRELLRNPYWPYAAARELGAKISAPVQYERGWRF

[0070] The above is merely a preferred embodiment of the present invention, and is not intended to limit the present invention. Those skilled in the art can make various modifications and changes to the present invention. All modifications, equivalent replacements, improvements, etc. made without departing from the spirit and principles of the present invention shall be included in the patent scope of the present invention.

Claims

1. An enzyme immobilization carrier characterized in that it is a hypercrosslinked polyvinyl alcohol modified with an amino group or cyanuric chloride.

2. 2. The enzyme immobilization carrier according to claim 1, wherein the hyper-crosslinked polyvinyl alcohol is obtained by sequentially subjecting polyvinyl alcohol to oxidation, self-crosslinking, and crosslinking with a crosslinking agent.

3. 3. The enzyme-immobilized carrier according to claim 1, wherein 3-aminopropyltriethoxysilane is used as an amino group modifying reagent for the modification with amino groups.

4. 4. An immobilized enzyme, characterized in that the enzyme-immobilized support according to claim 1 and an enzyme are covalently bonded to each other.

5. the enzyme is any one or more selected from the group consisting of transaminase, monooxygenase, ketoreductase, enereductase, and amino acid dehydrogenase; the transaminase is a transaminase derived from Chromobacterium violaceum DSM 30191, a transaminase derived from Arthrobacter citreus, or a transaminase derived from B. thuringiensis; the monooxygenase is cyclohexanone monooxygenase derived from Brachymonas petroleovorans or cyclohexanone monooxygenase derived from Rhodococcus rubber-SD1; the ketoreductase is a ketoreductase derived from Acetobacter sp. CCTCC M209061 or a ketoreductase derived from Candida macedoniensis AKU4588; the ene-reductase is derived from Saccharomyces cerevisiae and derived from Chryseobacterium sp. CA49; 5. The immobilized enzyme according to claim 4, wherein the amino acid dehydrogenase is leucine dehydrogenase derived from Bacillus cereus and phenylalanine dehydrogenase derived from Bacillus sphaericus.

6. A method for producing an enzyme-immobilized support according to any one of claims 1 to 3, comprising: providing a hypercrosslinked polyvinyl alcohol; and modifying the hyper-crosslinked polyvinyl alcohol with an amino group or cyanuric chloride to obtain the enzyme-immobilized support.

7. The hypercrosslinked polyvinyl alcohol is an oxidation step of dissolving polyvinyl alcohol in water and adding an oxidizing agent to carry out an oxidation reaction to obtain an oxidation product system; a self-crosslinking step of adjusting the concentration of hydrogen chloride in the oxidation product system to 0.1 mol / L to 1 mol / L with hydrochloric acid, and then carrying out a self-crosslinking reaction at a temperature of 70°C to 90°C to obtain polyvinyl alcohol gel particles; 7. The method for producing an enzyme immobilized carrier according to claim 6, further comprising a cross-linking step of dispersing the polyvinyl alcohol gel particles in water, adding a cross-linking agent to cause a reaction, and obtaining the hyper-cross-linked polyvinyl alcohol.

8. 8. The method for producing an enzyme-immobilized carrier according to claim 7, wherein the oxidizing agent is sodium periodate, and the oxidation reaction temperature is 20°C to 30°C.

9. 8. The method for producing an enzyme-immobilized carrier according to claim 7, wherein the crosslinking agent is a glutaraldehyde solution having a concentration of 2 wt% to 10 wt%, the reaction temperature of the crosslinking step using the crosslinking agent is 40°C to 60°C, and hydrochloric acid is added to the system during the reaction to adjust the hydrogen chloride concentration in the system to 0.1 mol / L to 1 mol / L.

10. The step of modifying the hyper-crosslinked polyvinyl alcohol with the amino group comprises: The method for producing an enzyme-immobilized support according to any one of claims 6 to 9, further comprising the step of mixing and reacting the hyper-crosslinked polyvinyl alcohol, a first solvent, and an amino group modifying reagent to obtain the enzyme-immobilized support.

11. the amino group modifying reagent is 3-aminopropyltriethoxysilane; 11. The method for producing an enzyme-immobilized support according to claim 10, wherein in the amino group modification reaction, the amount of the amino group modification reagent added is 0.3 mL to 1 mL per 1 g of the hyper-crosslinked polyvinyl alcohol.

12. 12. The method for producing an enzyme-immobilized carrier according to claim 11, wherein the amino group modification reaction is carried out at a pH value of 2 to 3 and a reaction temperature of 60 to 90°C.

13. modifying the hypercrosslinked polyvinyl alcohol with the cyanuric chloride, The method for producing an enzyme-immobilized support according to any one of claims 6 to 9, further comprising the step of dispersing the hyper-crosslinked polyvinyl alcohol in a second solvent, and then adding cyanuric chloride to cause a reaction, thereby obtaining the enzyme-immobilized support.

14. 14. The method for producing an enzyme-immobilized support according to claim 13, wherein the amount of cyanuric chloride added is 0.25 g to 1 g per 1 g of the hypercrosslinked polyvinyl alcohol.

15. 14. The method for producing an enzyme-immobilized support according to claim 13, wherein the reaction temperature in the cyanuric chloride modification reaction is 0°C to 10°C.

16. 6. A method for producing the immobilized enzyme according to claim 4 or 5, comprising a step of covalently linking the enzyme to the enzyme-immobilization support according to any one of claims 1 to 3 to obtain the immobilized enzyme.

17. When the enzyme-immobilized support is a hypercrosslinked polyvinyl alcohol modified with an amino group, the method for producing the immobilized enzyme includes the steps of: a step of activating the enzyme-immobilized carrier by dispersing it in a glutaraldehyde solution to obtain an activated carrier; and reacting the activated carrier with an enzyme solution containing an enzyme to covalently bond the enzyme to the enzyme-immobilized carrier, thereby obtaining the immobilized enzyme.

18. 18. The method for producing an immobilized enzyme according to claim 17, wherein the glutaraldehyde solution has a concentration of 1% by mass to 2% by mass.

19. The method for producing an immobilized enzyme according to claim 18, wherein in the activation step, the activation temperature is 20°C to 30°C, the activation time is 1 hour to 3 hours, the reaction temperature in the process of reacting the activating carrier with the enzyme solution is 20°C to 30°C, 1 g of the activating carrier corresponds to 2 mL to 6 mL of the enzyme solution, and the protein content in the enzyme solution is 30 mg / mL to 40 mg / mL.

20. When the enzyme-immobilized support is a hypercrosslinked polyvinyl alcohol modified with cyanuric chloride, the method for producing the immobilized enzyme includes the steps of: Wetting the enzyme-immobilized support with a phosphate buffer solution; and reacting the wetted enzyme-immobilization carrier with an enzyme solution containing the enzyme to covalently bond the enzyme to the enzyme-immobilization carrier, thereby obtaining the immobilized enzyme.

21. 21. The method for producing an immobilized enzyme according to claim 20, wherein in the process of reacting the wetted enzyme-immobilized carrier with the enzyme solution, the reaction temperature is 20°C to 30°C, 2 mL to 6 mL of the enzyme solution per 1 g of the enzyme-immobilized carrier corresponds to 2 mL to 6 mL of the enzyme solution, and the protein content in the enzyme solution is 30 mg / mL to 40 mg / mL.

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