Quasi-immobilized enzyme catalysis system and application method thereof

Abstract

According to the quasi-immobilized enzyme catalysis system and the application method thereof provided by the invention, the adsorption carrier is directly added in the free enzyme method reaction to construct the quasi-immobilized enzyme catalysis system, so that the production efficiency can be effectively improved, and the defects of complex preparation process, high cost and difficult maintenance of the immobilized enzyme are avoided; meanwhile, the advantages and characteristics of the immobilized enzyme can be exerted. And when the adsorption carrier is separated and recycled after the reaction is completed, the reaction activity cannot be reduced, and the catalytic efficiency is further enhanced along with the increase of the recycling times. The preparation method is simple and convenient to operate, is environment-friendly, does not need to add equipment and additional chemical reagents, has a wide application range, and can be applied to various occasions needing to improve the catalytic efficiency and stability of the enzyme.

Description

Technical Field

[0001] The present invention relates to the technical field of biocatalysis, and particularly to a quasi-immobilized enzyme catalytic system and an application method thereof. Background Art

[0002] As a biocatalyst, biocatalyst is prone to inactivation under actual operating environments or extreme conditions (such as high temperature, strong acid or strong base, or organic solvents, etc.), and is not easy to recover and reuse, which greatly limits the industrial application of enzymes. To solve the problem of industrial application of enzymes, the technology of immobilized enzymes emerged. Immobilization of enzymes refers to a class of technologies that use solid carriers to bind or confine enzymes within a certain area, still maintaining the unique catalytic activity of the enzymes, and enabling them to be recovered and reused. Immobilized enzymes have the following advantages: through the protection of the carrier, they still maintain activity in a relatively wide range of pH, high temperature, or organic solvents, so as to improve the stability of the enzymes; filtration, centrifugation and other methods can be used to simply separate the enzymes from the reaction solution and recycle them, improving the separation efficiency and reusability of the enzymes; by the hydrophilic and hydrophobic properties of the carrier itself, the microenvironment of the enzyme and its reaction environment with the substrate are adjusted, thereby promoting the enzyme-catalyzed reaction and improving the catalytic activity of the enzyme; through the size screening effects provided by the steric hindrance or pore size of the carrier, substrates of a certain size can be selected to enter the carrier for catalysis, improving the selectivity of the enzyme. The development of enzyme immobilization strategies has led to the production of customized enzymes, and the stability (such as thermal stability, acid-base stability, chemical stability, and storage stability) of these enzymes has been significantly improved.

[0003] According to the binding mode between the enzyme and the carrier, the enzyme immobilization methods are mainly divided into adsorption method, embedding method, covalent binding method, crosslinking method, and in-situ synthesis method developed in recent years. However, the immobilized enzymes prepared by the above enzyme immobilization methods also have some deficiencies: for example, the immobilization process affects the structure of the enzyme, resulting in a decrease in activity, the immobilization process is complex, the yield of the immobilized enzyme is low, the price of new carriers is expensive, it is not easy to industrialize, the activity of the immobilized enzyme continuously decreases during long-term use, and it needs to be replaced regularly, resulting in high industrial costs and inconvenient operation, etc.

[0004] Chinese Patent CN119286841A discloses a nano-flower-shaped immobilized enzyme, its preparation method and application. It uses zinc phosphate as an inorganic carrier, and through the coordination between metal ions and enzyme protein molecules, glucose dehydrogenase and carbonyl reductase are co-immobilized to form an immobilized enzyme, and it is applied to the production of (S)-4-chloro-3-hydroxybutyric acid ethyl ester. However, the activity of this immobilized enzyme can only be maintained at 80% after being reused 6 times, and only 60% after being reused 8 times, which requires continuous supplementation of the immobilized enzyme in actual production, resulting in an increase in production costs and inconvenient operation. Summary of the Invention

[0005] Aiming at the above deficiencies, the purpose of the present invention is to develop a quasi-immobilized enzyme catalytic system and its application method in view of the disadvantages existing in the use of existing immobilized enzymes, such as the continuous reduction of the activity of immobilized enzymes with cyclic use, the need for continuous supplementation, resulting in an increase in production costs, etc. The system can directly improve the reaction conversion rate, effectively improve production efficiency, can be recycled repeatedly, does not require subsequent addition of adsorption carriers, does not change the existing production mode, does not require large-scale equipment transformation of enterprises, and can effectively improve the reaction rate on the basis of the original equipment and process, with broad application prospects.

[0006] The specific content of the present invention is as follows:

[0007] A quasi-immobilized enzyme catalytic system is constructed by adding an adsorption carrier to the reaction using free bioenzymes. By directly adding the adsorption carrier to the reactor using free bioenzymes, the enzyme reaches an equilibrium between the solution and the surface of the adsorption carrier and simultaneously participates in the catalysis of the reaction, thus constructing the quasi-immobilized enzyme catalytic reaction system.

[0008] Furthermore, in the quasi-immobilized enzyme catalytic system, the bioenzyme reaches a dynamic adsorption equilibrium on the surface of the adsorption carrier, and the catalytic action of the enzyme is directly realized through two ways: free solution and dynamic immobilization in the reaction system.

[0009] Furthermore, the bioenzymes applicable to the quasi-immobilized enzyme catalytic reaction system include reductase and transaminase. The reductase is carbonyl reductase and is applicable to catalyze the asymmetric reduction of carbonyl to hydroxyl. A free bioenzyme catalytic reaction system is constructed by a carbonyl raw material, a buffer solution, carbonyl reductase, and a coenzyme. Isopropanol or glucose is used as the hydrogen donor substance, and then an adsorption carrier is added to dynamically immobilize part of the free bioenzymes to construct a quasi-immobilized enzyme catalytic reaction system, and then the catalytic reaction is carried out; the coenzyme is NADP+ or a mixed system of NADP+ and NADPH; the buffer solution is citric acid / sodium citrate buffer solution or phosphate buffer solution. When glucose is used as the hydrogen donor substance, the corresponding glucose reductase needs to be added. The reaction general formula of the carbonyl reductase catalytic reaction is:

[0010]

[0011] The dosages of the above various substances are adjusted according to actual needs;

[0012] The transaminase catalyzes the asymmetric conversion of carbonyl groups into amino groups. Using carbonyl compounds as raw materials, a free biocatalytic reaction system is constructed by combining a buffer solution, a solvent, a transaminase, and a coenzyme. Isopropylamine is used as the amine source, and an adsorption carrier is added to dynamically immobilize some of the free biocatalysts to construct a quasi-immobilized enzyme catalytic reaction system, and then the catalytic reaction is carried out; the coenzyme is pyridoxal phosphate; the buffer solution is a phosphate buffer solution; the reaction general formula of the transaminase-catalyzed reaction is as follows:

[0013]

[0014] The dosages of the above various substances are adjusted according to actual needs;

[0015] After the above two quasi-immobilized enzyme catalytic reaction systems are completed, the adsorption carrier is separated from the quasi-immobilized enzyme system, and the obtained adsorption carrier is directly applied to the construction of the next batch of enzyme-catalyzed reaction quasi-immobilized enzyme catalytic reaction systems.

[0016] Furthermore, the adsorption carrier is one or more of mesoporous molecular sieve materials, calcium alginate, macroporous resins, magnetic nanoparticles, or covalent organic framework materials.

[0017] Furthermore, the dosage of the adsorption carrier is 5% - 35% of the weight of the reaction system raw materials. A reasonable dosage reduces unnecessary costs, ensures the smooth progress of the reaction process, avoids uneven reactions, and prolongs the service life of the carrier and the enzyme.

[0018] Furthermore, the pH of the citric acid / sodium citrate buffer solution is 6, which is convenient for maintaining the reaction activity of the carbonyl reductase and providing an appropriate reaction environment.

[0019] Furthermore, the pH of the phosphate buffer solution is 7, which is convenient for maintaining the reaction activity of the transaminase and providing an appropriate reaction environment.

[0020] Furthermore, the molar ratio of the NADP+ / NADPH mixed system is 1:1, which helps the efficient progress of the catalytic reaction and also ensures the stability and continuity of the reaction system.

[0021] Furthermore, the biocatalysts applicable to the quasi-immobilized enzyme catalytic reaction system also include oxidase and lipase.

[0022] A quasi-immobilized enzyme catalytic system, and its application method includes the following steps:

[0023] In the first step, in the reaction using free biocatalysts, an adsorption carrier is directly added, and the dosage of the adsorption carrier is 5% - 35% of the weight of the reaction system raw materials to construct a quasi-immobilized enzyme catalytic system for enzymatic reaction;

[0024] In the second step, after the reaction is completed, the adsorption carrier is separated and recycled to the next batch of enzymatic reactions, and the remaining reaction solution is processed in a normal manner to obtain the product.

[0025] The beneficial effects of the present invention are as follows: 1. The quasi-immobilized catalytic system of the present invention can effectively improve production efficiency, avoid the disadvantages of the complex preparation process, high cost and difficult maintenance of immobilized enzymes, and at the same time can exert the advantages and characteristics that immobilized enzymes should have; 2. When the adsorption carrier of the present invention is separated and recycled after the reaction is completed, it will not only not cause a decrease in reaction activity, but on the contrary, with the increase in the number of recycling times, the catalytic efficiency will be further enhanced. This preparation method is not only simple to operate, but also environmentally friendly, without the need to add equipment and additional chemical reagents, and has a wide range of applications, and can be used in various occasions where it is necessary to improve the catalytic efficiency and stability of enzymes. Specific embodiments

[0026] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0027] Example 1: Application of the quasi-immobilized enzyme catalytic system to the reaction for preparing (S)-ethyl 4-chloro-3-hydroxybutyrate by carbonyl reductase

[0028] Experiment 1 (control reaction): In a reaction flask, add 3.3 g mM ethyl 4-chloroacetoacetate, 3.6 g glucose, 50 ml of citric acid / sodium citrate buffer solution with pH = 6, 0.5 g carbonyl reductase, 0.5 g glucose dehydrogenase, 0.2 g NADPH, 0.2 g NADP+, stir and heat to 25 °C for reaction, and keep the reaction until the content of ethyl 4-chloroacetoacetate < 1%. The reaction is qualified after about 10 h. After the reaction is completed, the reaction solution is post-treated to obtain 3.15 g of (S)-ethyl 4-chloro-3-hydroxybutyrate with a purity of 99.1% and a yield of 94.2%.

[0029] Experiment 2 (Reaction 1): In a reaction flask, 3.3 g of mM ethyl 4-chloroacetoacetate, 3.6 g of glucose, 50 ml of citric acid / sodium citrate buffer with pH = 6, 0.5 g of carbonyl reductase, 0.5 g of glucose dehydrogenase, 0.2 g of NADPH, 0.2 g of NADP+, and 0.5 g of macroporous resin (HZ818, Shanghai Huazhen Technology Co., Ltd.) were added. The mixture was stirred and heated to 25 °C for reaction, and the reaction was kept at this temperature until the content of ethyl 4-chloroacetoacetate was < 1%. The reaction was qualified after about 9 h. After the reaction, the macroporous resin was separated by centrifugation and reused in the next batch. The remaining reaction solution was post-treated to obtain 3.15 g of (S)-ethyl 4-chloro-3-hydroxybutyrate with a purity of 99.1% and a yield of 94.3%.

[0030] Experiment 3 (Recycling Reaction): In a reaction flask, 3.3 g of mM ethyl 4-chloroacetoacetate, 3.6 g of glucose, 50 ml of citric acid / sodium citrate buffer with pH = 6, 0.5 g of carbonyl reductase, 0.5 g of glucose dehydrogenase, 0.2 g of NADPH, 0.2 g of NADP+, and the macroporous resin separated in Experiment 2 were added. The mixture was stirred and heated to 25 °C for reaction, and the reaction was kept at this temperature until the content of ethyl 4-chloroacetoacetate was < 1%. The reaction was qualified after about 8.5 h. After the reaction, the macroporous resin was separated by centrifugation and reused in the next batch. The remaining reaction solution was post-treated to obtain 3.14 g of (S)-ethyl 4-chloro-3-hydroxybutyrate with a purity of 99.2% and a yield of 94.0%.

[0031] Example 2: Application of the quasi-immobilized enzyme catalytic system to the reaction for preparing S-(-)-phenylethyl alcohol by carbonyl reductase

[0032] Experiment 1 (Control Reaction): In a reaction flask, 50 ml of isopropanol, 30 ml of 0.1 M phosphate buffer (pH = 7.0), 10 g of acetophenone, 4.0 g of carbonyl reductase, and 0.5 g of NADP+ were added. The mixture was stirred and heated to 30 °C for reaction, and the reaction was kept at this temperature until the content of acetophenone was < 3%. The reaction was qualified after about 15 h. After the reaction, the reaction solution was post-treated to obtain 9.57 g of S-(-)-phenylethyl alcohol with a purity of 99.1% and a yield of 94.2%.

[0033] Experiment 2 (Reaction 1): In a reaction flask, 50 ml of isopropanol, 30 ml of 0.1 M phosphate buffer (pH = 7.0), 10 g of acetophenone, 4.0 g of carbonyl reductase, 0.5 g of NADP+, and 1.5 g of calcium alginate were added. The mixture was stirred and heated to 30 °C for reaction, and the reaction was kept at this temperature until the content of acetophenone was < 3%. The reaction was qualified after about 13 h. After the reaction, the calcium alginate was separated by centrifugation and reused in the next batch. The remaining reaction solution was post-treated to obtain 9.60 g of S-(-)-phenylethyl alcohol with a purity of 99.3% and a yield of 94.5%.

[0034] Experiment 3 (Recycling Reaction): In a reaction flask, add 50 ml of isopropanol, 30 ml of 0.1 M phosphate buffer (pH = 7.0), 10 g of acetophenone, 4.0 g of carbonyl reductase, 0.5 g of NADP+, and the calcium alginate obtained from Experiment 2. Stir and heat to 30 °C for reaction, and keep the reaction at a constant temperature until the content of acetophenone is < 3%. The reaction is qualified after about 13 h. After the reaction is completed, centrifuge to separate the calcium alginate and recycle it to the next batch. The remaining reaction solution is post-treated to obtain 9.56 g of S-(-)-phenylethyl alcohol with a purity of 99.3% and a yield of 94.1%.

[0035] Example 3: Application of the quasi-immobilized enzyme catalytic system in the reaction for preparing (R)-1-tert-butoxycarbonyl-3-aminopiperidine

[0036] Experiment 1 (Control Reaction): In a reaction flask, add 100 ml of 0.2 M phosphate buffer (pH = 7.0), 4.0 g of isopropylamine, 20 ml of DMSO, 4.0 g of N-tert-butoxycarbonyl-3-piperidone, 4.0 g of transaminase, and 0.4 g of pyridoxal phosphate. Stir and heat to 35 °C for reaction, and keep the pH of the reaction system between 7.0 and 7.5. React until the content of N-tert-butoxycarbonyl-3-piperidone is < 2%. The reaction is qualified after about 24 h. After the reaction is completed, the reaction solution is post-treated to obtain 3.62 g of (R)-1-tert-butoxycarbonyl-3-aminopiperidine with a purity of 98.5% and a yield of 90.2%.

[0037] Experiment 2 (Reaction 1): In a reaction flask, add 100 ml of 0.2 M phosphate buffer (pH = 7.0), 4.0 g of isopropylamine, 20 ml of DMSO, 4.0 g of N-tert-butoxycarbonyl-3-piperidone, 4.0 g of transaminase, 0.4 g of pyridoxal phosphate, and 1.4 g of AB-8 resin (Tianjin Nankai Hecheng Technology Co., Ltd.). Stir and heat to 35 °C for reaction, and keep the pH of the reaction system between 7.0 and 7.5. React until the content of N-tert-butoxycarbonyl-3-piperidone is < 2%. The reaction is qualified after about 20 h. After the reaction is completed, centrifuge to separate the AB-8 resin and recycle it to the next batch. The remaining reaction solution is post-treated to obtain 3.64 g of (R)-1-tert-butoxycarbonyl-3-aminopiperidine with a purity of 98.7% and a yield of 90.5%.

[0038] Experiment 3 (Recycling reaction): In a reaction flask, add 100 ml of 0.2 M phosphate buffer (pH = 7.0), 4.0 g of isopropylamine, 20 ml of DMSO, 4.0 g of N-Boc-3-piperidone, 4.0 g of transaminase, 0.4 g of pyridoxal phosphate, and the AB-8 resin separated in Experiment 2. Stir and heat to 35 °C for reaction, maintaining the pH of the reaction system between 7.0 and 7.5. React until the content of N-Boc-3-piperidone is <2%. The reaction is qualified after about 19 h. After the reaction is completed, centrifuge to separate the AB-8 resin and recycle it to the next batch. The remaining reaction solution is post-treated to obtain 3.62 g of (R)-1-Boc-3-aminopiperidine with a purity of 98.9% and a yield of 90.2%.

[0039] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be encompassed within the present invention.

[0040] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A quasi-immobilized enzyme catalytic system, characterized in that: In a reactor using free biocatalysts, an adsorption carrier is directly added. The biocatalysts reach an equilibrium between the solution and the surface of the adsorption carrier, and simultaneously participate in the catalysis of the reaction, thus constructing a quasi-immobilized biocatalyst reaction system.

2. The quasi-immobilized enzyme catalysis system according to claim 1, wherein: In the described quasi-immobilized biocatalyst system, the dynamic adsorption equilibrium of the biocatalysts on the surface of the adsorption carrier is utilized, and the catalytic action of the biocatalysts is directly achieved through two pathways in the reaction system: free solution and dynamic immobilization.

3. The quasi-immobilized enzyme catalytic system according to claim 1, wherein: The biocatalysts applicable to the described quasi-immobilized biocatalyst reaction system include reductases and transaminases. The reductase is a carbonyl reductase and is applicable to catalyze the asymmetric reduction of carbonyl to hydroxyl. A free biocatalyst reaction system is constructed by using a carbonyl raw material, a buffer solution, a carbonyl reductase, and a coenzyme. Isopropanol or glucose is used as the hydrogen donor. Then, an adsorption carrier is added to dynamically immobilize a part of the free biocatalysts to construct a quasi-immobilized biocatalyst reaction system, and then the catalytic reaction is carried out. The coenzyme is NADP+ or a mixed system of NADP+ and NADPH. The buffer solution is a citric acid / sodium citrate buffer solution or a phosphate buffer solution. When glucose is used as the hydrogen donor, the corresponding glucose reductase needs to be added. The reaction general formula for the catalytic reaction of the carbonyl reductase is: The transaminase is used to catalyze the asymmetric conversion of carbonyl to amino. Using a carbonyl compound as the raw material, a free biocatalyst reaction system is constructed by combining a buffer solution, a solvent, a transaminase, and a coenzyme. Isopropylamine is used as the amine source. Then, an adsorption carrier is added to dynamically immobilize a part of the free biocatalysts to construct a quasi-immobilized biocatalyst reaction system, and then the catalytic reaction is carried out. The coenzyme is pyridoxal phosphate. The buffer solution is a phosphate buffer solution. The reaction general formula for the catalytic reaction of the transaminase is: After the reactions of the above two quasi-immobilized biocatalyst reaction systems are completed, the adsorption carrier is separated from the quasi-immobilized biocatalyst system, and the obtained adsorption carrier is directly applied to the construction of the quasi-immobilized biocatalyst reaction system for the next batch of enzymatic reactions.

4. A quasi-immobilized enzyme catalytic system according to claim 1, characterized in that: The described adsorption carrier is one or several of mesoporous molecular sieve materials, calcium alginate, macroporous resins, magnetic nanoparticles, or covalent organic framework materials.

5. A quasi-immobilized enzyme catalytic system according to claim 1, characterized in that: The feeding amount of the adsorption carrier is 5% - 35% of the weight of the raw materials in the reaction system.

6. The quasi-immobilized enzyme catalytic system according to claim 1, wherein: The pH of the citric acid / sodium citrate buffer solution is 6.

7. A quasi-immobilized enzyme catalytic system according to claim 1, wherein: The pH of the phosphate buffer solution is 7.

8. A quasi-immobilized enzyme catalysis system according to claim 1, characterized in that: The molar ratio of the NADP+ and NADPH mixed system is 1:

1.

9. A quasi-immobilized enzyme catalytic system according to claim 1, characterized in that: The biocatalysts applicable to the described quasi-immobilized biocatalyst reaction system also include oxidases and lipases.

10. A quasi-immobilized enzyme catalytic system according to claim 1, characterized in that Its application method includes the following steps: First step, in the reaction using free biocatalysts, an adsorption carrier is directly added to construct a quasi-immobilized biocatalyst system, and an enzymatic reaction is carried out. Second step, after the reaction is completed, the adsorption carrier is separated and applied to the next batch of enzymatic reactions, and the remaining reaction solution is processed in the normal way to obtain the product.

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