Immobilized DCH enzyme as well as preparation method and application thereof

Abstract

The invention relates to the technical field of catalyst preparation, in particular to an immobilized DCH enzyme and a preparation method and application thereof, and the preparation method comprises the following steps: balancing an epoxy resin carrier with a phosphate buffer solution I with the pH value of 7.0-8.0; mixing the DCH enzyme liquid with the pretreated epoxy resin carrier, and reacting at 20-35 DEG C under the condition of slow oscillation for 8-16 hours to obtain a reaction mixture; separating the reaction mixture, washing with a phosphate buffer solution II with the pH value of 7.0-8.0 until no protein is detected in the washing solution to obtain the immobilized DCH enzyme, and then storing the immobilized DCH enzyme in a phosphate buffer solution III with the pH value of 7.0-8.0 at 4 DEG C; the problems that the free DCH enzyme is poor in stability, difficult to recycle and incapable of being repeatedly used in an existing biological catalysis resolution method, and an existing immobilization method is low in immobilization rate, poor in enzyme activity recycling and insufficient in operation stability and reusability are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of catalyst preparation, in particular to a kind of immobilized DCH enzyme and its preparation method and application. BACKGROUND

[0002] D-3-acetyl mercapto-2-methyl propionic acid (D-AT) is a key chiral intermediate for synthesizing captopril antihypertensive drugs, and its optical purity directly affects the efficacy and safety of the final drug. Currently, the main methods for preparing D-AT include chemical resolution and biological catalytic resolution. Chemical resolution has the disadvantages of harsh reaction conditions, complicated steps, high cost of chiral inducers, serious environmental pollution, and low optical purity of products, and is gradually replaced by green and environmentally friendly, efficient and specific biological catalytic resolution.

[0003] Biological catalytic resolution mainly utilizes enzymes with stereoselectivity to catalyze the asymmetric hydrolysis of DL-3-acetyl thio-2-methyl propionate methyl ester (DL-MAT), wherein DCH enzyme is the key catalyst in the reaction, which can specifically hydrolyze the D enantiomer in DL-MAT to generate the target product D-AT, while the L enantiomer (L-MAT) remains in the reaction system, which can be separated by chemical resolution and then re-resolved, thereby improving the utilization rate of raw materials.

[0004] However, free DCH enzyme has many limitations in practical application: on the one hand, free enzyme has poor stability and is easily inactivated by factors such as temperature, pH value and organic solvent during the reaction, resulting in decreased reaction efficiency; on the other hand, free enzyme is difficult to separate and recover from the reaction system, which cannot be reused, not only increasing the production cost, but also possibly causing enzyme protein residues in the product, affecting the separation and purification of the product and its quality. Therefore, it is crucial to immobilize DCH enzyme to improve its stability and reusability for its industrial application.

[0005] However, the existing DCH enzyme immobilization method has the problems of low immobilization rate, poor enzyme activity recovery rate, insufficient stability, poor reusability and large mass transfer resistance, which cannot meet the needs of industrial production. Therefore, it is of great practical significance to develop an efficient, stable and easy-to-recover DCH enzyme immobilization method. SUMMARY

[0006] In order to overcome the problems of poor stability, difficult recovery and non-reusability of free DCH enzyme in the existing biological catalytic resolution method, and the problems of low immobilization rate, poor enzyme activity recovery, insufficient operational stability and reusability of the existing immobilization method, the present application provides a kind of immobilized DCH enzyme and its preparation method and application.

[0007] The present application is realized by adopting the following technical solutions: A method for preparing immobilized DCH enzyme includes the following steps: S1: Carrier pretreatment: Equilibrate the epoxy resin carrier with phosphate buffer I with a pH of 7.0-8.0; S2: Enzyme immobilization: The DCH enzyme solution is mixed with the epoxy resin carrier pretreated in step S1, and reacted at 20-35℃ with slow shaking for 8-16 hours to obtain the reaction mixture. S3: Washing and storage: The reaction mixture obtained in step S2 is separated and washed with phosphate buffer II at pH 7.0-8.0 until no protein is detected in the washing solution to obtain immobilized DCH enzyme. Then, the immobilized DCH enzyme is stored at 4°C in phosphate buffer III at pH 7.0-8.0.

[0008] Further, in step S1, the epoxy resin carrier is a polyacrylate resin with epoxy groups, having a particle size of 150-350 μm, a pore size of 40-60 nm, and a specific surface area ≥120 m². 2 / g.

[0009] Furthermore, in step S2, the amount of enzyme protein added to the DCH enzyme solution is 10-50 mg / g carrier, and the pH value of the enzyme immobilization reaction is 7.5.

[0010] An immobilized DCH enzyme is prepared using the method for preparing an immobilized DCH enzyme as described in this invention, wherein the immobilized DCH enzyme is formed by covalently binding a DCH enzyme with an epoxy resin carrier.

[0011] An application of an immobilized DCH enzyme for catalyzing the hydrolytic resolution of methyl DL-3-acetylthio-2-methylpropionate to prepare D-3-acetylthio-2-methylpropionate.

[0012] Furthermore, the hydrolysis and resolution reaction is carried out in a phosphate buffer solution with a pH of 7.0-8.0, at a reaction temperature of 30-40°C, and with a concentration of 50-200 mM of the substrate DL-3-acetylthio-2-methylpropionate.

[0013] Furthermore, after the hydrolysis and resolution reaction is completed, the immobilized DCH enzyme is recovered by filtration or centrifugation and used in the next hydrolysis and resolution reaction.

[0014] This invention provides an immobilized DCH enzyme, its preparation method, and its application, which have the following advantages compared with the prior art: 1. High efficiency of immobilization method: The preparation process of the method is mild and simple, and the immobilized DCH enzyme has the characteristics of high immobilization rate and high enzyme activity recovery rate.

[0015] 2. Significantly improved enzyme performance: The obtained immobilized DCH enzyme has good stability and excellent reusability. It can be recovered multiple times and maintains high catalytic activity, which significantly improves the efficiency and economy of enzyme use.

[0016] 3. Excellent catalytic effect: In the reaction of catalyzing the hydrolysis and resolution of DL-MAT to synthesize D-AT, the immobilized DCH enzyme exhibits high stereoselectivity, and the product D-AT has high optical purity (ee value > 98%), high conversion rate (close to the theoretical maximum value of 50%), and the reaction efficiency is comparable to that of the free enzyme.

[0017] In summary, this invention not only effectively solves the problems of poor stability, difficulty in recovery, and inability to be reused of free DCH enzymes, but also overcomes the shortcomings of existing immobilization methods, such as low immobilization rate, poor enzyme activity recovery, and insufficient operational stability and reusability, providing a high-performance biocatalyst for the green and efficient industrial production of D-AT. Attached Figure Description

[0018] Figure 1 This is the reaction equation for the preparation of D-AT by hydrolysis and resolution of DL-MAT catalyzed by immobilized DCH enzyme in this invention. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Example 1

[0020] A method for preparing immobilized DCH enzyme includes the following steps: S1: Carrier Pretreatment: Weigh 1.0 g of epoxy resin carrier (model: LX-1000EP, particle size 150-350μm, pore size 40-60nm, specific surface area ≥120 m²). 2 (g), equilibrate with 50 mL of 0.1 M phosphate buffer I at pH 7.5 at 25°C for 30 minutes, then filter to remove the equilibration solution.

[0021] S2: Enzyme immobilization: The DCH enzyme solution is mixed with the epoxy resin carrier pretreated in step S1 and reacted in a constant temperature shaker at 30°C and 150 rpm for 15 hours to obtain a reaction mixture; the DCH enzyme solution is 50 mL of phosphate buffer containing 40 mg of DCH enzyme protein, i.e., the loading is 40 mg / g carrier.

[0022] S3: Washing and storage: The reaction mixture obtained in step S2 was filtered and separated through a Buchner funnel to collect the immobilized enzyme particles. The particles were repeatedly washed with phosphate buffer II at pH 7.5 and detected by the Bradford method until no protein was detected in the washing solution to obtain the immobilized DCH enzyme. The immobilized DCH enzyme was then stored at 4°C in phosphate buffer III at pH 7.5.

[0023] This invention uses epoxy resin as a carrier to legally immobilize DCH enzymes via covalent bonding. The epoxy groups can undergo alkylation reactions with nucleophilic groups such as amino, thiol, and hydroxyl groups on the enzyme molecule surface, forming strong covalent bonds.

[0024] The immobilized DCH enzyme prepared in this embodiment was analyzed and measured: Immobilization rate: calculated by measuring the protein content in the DCH enzyme solution before and after immobilization.

[0025] Immobilization rate (%) = (Initial protein content - Protein content in washing solution) / Initial protein content × 100%.

[0026] Enzyme activity assay: The activities of free and immobilized enzymes were measured separately. Under standard reaction conditions (30℃, pH=7.5), using DL-MAT as a substrate, the amount of D-AT generated per unit time was determined by gas chromatography (GC) to define enzyme activity (U).

[0027] Activity recovery rate: (total activity of immobilized enzyme / total activity of free enzyme used for immobilization) × 100%.

[0028] GC detection conditions included: Agilent CP Chirasil-Dex CB chiral column (25 m × 0.32 mm × 0.25 μm), vaporization temperature 270 ℃, FID detector temperature 275 ℃, carrier gas nitrogen, column inlet pressure 0.4 MPa, air column inlet pressure 0.2 MPa, hydrogen column inlet pressure 0.2 MPa, split ratio 30:1, and a temperature program of 80 ℃ held for 40 min, then ramped to 150 ℃ at a rate of 5 ℃ / min and held for 30 min.

[0029] The results of the preparation of immobilized DCH enzyme obtained through experiments are shown in Table 1. As can be seen from the data in the table, the present invention successfully prepared a high-performance immobilized DCH enzyme with an immobilization rate and activity recovery rate of 80% or more, indicating that the immobilization process is highly efficient and has little impact on enzyme activity.

[0030] Table 1 Example 2

[0031] The immobilized DCH enzyme prepared in Example 1 was used in the reaction that catalyzes the hydrolysis and resolution of DL-MAT to prepare D-AT. The reaction equation is shown in the attached figure. Figure 1 As shown. The total volume of the hydrolysis and resolution reaction system was 50 mL, containing 100 mM of the substrate DL-MAT, and using 0.1 M phosphate buffer at pH 7.5. The reaction temperature was 35 °C.

[0032] To verify the advantages of the immobilized DCH enzyme of this invention compared to the free enzyme, a control experiment was set up as follows: A free DCH enzyme with the same initial enzyme activity was used as a control in the same reaction system (50 mL, 0.1 M, pH 7.5 phosphate buffer containing 100 mM DL-MAT) and under the same reaction conditions (35°C). Samples were taken at different time points during the reaction, and the substrate conversion rate and the enantiomeric excess value (ee value) of the product D-AT were determined by GC analysis.

[0033] Table 2 shows the comparison of the catalytic performance of immobilized DCH enzyme and free enzyme. The data in the table indicate that, within the same reaction time, the conversion rate of the immobilized DCH enzyme (48.0%) is comparable to that of the free enzyme (48.5%), and the ee values ​​of the D-AT products of both are higher than 98%, indicating that the immobilized enzyme maintains catalytic efficiency similar to that of the free enzyme and extremely high stereoselectivity. The conversion rate of the immobilized enzyme is close to the theoretical maximum of 50%, fully demonstrating its high efficiency.

[0034] Table 2

[0035] The reusability of the immobilized DCH enzyme after the hydrolysis and resolution reaction described in this embodiment was investigated, and the specific process is as follows: The immobilized DCH enzyme was recovered by filtration after the hydrolysis and resolution reaction, washed thoroughly with phosphate buffer (pH 7.5), and then added to a new reaction system with identical conditions. This process was repeated, and the remaining relative enzyme activity (with the enzyme activity of the first reaction as 100%) was measured after each reaction. The experimental results are shown in Table 3. The data in the table show that the immobilized DCH enzyme exhibits excellent reusability; after eight batches of reuse, the relative enzyme activity remained at 64%, greatly improving the utilization efficiency of the DCH enzyme and demonstrating promising prospects for industrial application.

[0036] Table 3 Example 3

[0037] The difference between this embodiment and Example 1 is that: the pH value of phosphate buffer I, phosphate buffer II, and phosphate buffer III is 7.0; the reaction time of the DCH enzyme solution mixed with the pretreated epoxy resin carrier in a constant temperature shaker is 8 hours, and the temperature of the constant temperature shaker is 20°C; the amount of enzyme protein added to the DCH enzyme solution is 10 mg / g carrier; the remaining raw material ratios, preparation steps, and process parameters are the same as in Example 1, and immobilized DCH enzyme is prepared. Example 4

[0038] The difference between this embodiment and Example 1 is that: the pH value of phosphate buffer I, phosphate buffer II, and phosphate buffer III is 8.0; the reaction time of the DCH enzyme solution mixed with the pretreated epoxy resin carrier in a constant temperature shaker is 16 hours, and the temperature of the constant temperature shaker is 35°C; the amount of enzyme protein added to the DCH enzyme solution is 50 mg / g carrier; the remaining raw material ratios, preparation steps, and process parameters are the same as in Example 1, and immobilized DCH enzyme is prepared. Example 5

[0039] The immobilized DCH enzyme prepared in Example 1 was used in the reaction catalyzing the hydrolysis and resolution of DL-MAT to prepare D-AT. The total volume of the hydrolysis and resolution reaction system was 50 mL, containing 50 mM of the substrate DL-MAT, and 0.1 M phosphate buffer with a pH of 7.0 was used. The reaction temperature was 30 °C. Example 6

[0040] The immobilized DCH enzyme prepared in Example 1 was used in the reaction catalyzing the hydrolysis and resolution of DL-MAT to prepare D-AT. The total volume of the hydrolysis and resolution reaction system was 50 mL, containing 200 mM of the substrate DL-MAT, and 0.1 M phosphate buffer with a pH of 8.0 was used. The reaction temperature was 40 °C.

[0041] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing immobilized DCH enzyme, characterized in that: Includes the following steps: S1: Carrier pretreatment: Equilibrate the epoxy resin carrier with phosphate buffer I with a pH of 7.0-8.0; S2: Enzyme immobilization: The DCH enzyme solution is mixed with the epoxy resin carrier pretreated in step S1, and reacted at 20-35℃ with slow shaking for 8-16 hours to obtain the reaction mixture. S3: Washing and storage: The reaction mixture obtained in step S2 is separated and washed with phosphate buffer II at pH 7.0-8.0 until no protein is detected in the washing solution to obtain immobilized DCH enzyme. Then, the immobilized DCH enzyme is stored at 4°C in phosphate buffer III at pH 7.0-8.

0.

2. The method for preparing an immobilized DCH enzyme according to claim 1, characterized in that: In step S1, the epoxy resin carrier is a polyacrylate resin with epoxy groups, having a particle size of 150-350 μm, a pore size of 40-60 nm, and a specific surface area ≥120 m². 2 / g.

3. The method for preparing an immobilized DCH enzyme according to claim 1, characterized in that: In step S2, the amount of enzyme protein added to the DCH enzyme solution is 10-50 mg / g carrier, and the pH value of the enzyme immobilization reaction is 7.

5.

4. An immobilized DCH enzyme, prepared using the method for preparing an immobilized DCH enzyme as described in any one of claims 1-3, characterized in that: The immobilized DCH enzyme is formed by covalently binding DCH enzyme and epoxy resin carrier.

5. The application of the immobilized DCH enzyme according to claim 4, characterized in that: It was used to catalyze the hydrolytic resolution of methyl DL-3-acetylthio-2-methylpropionate to prepare D-3-acetylthio-2-methylpropionate.

6. The application of the immobilized DCH enzyme according to claim 5, characterized in that: The hydrolysis and resolution reaction was carried out in a phosphate buffer solution with a pH of 7.0-8.0 at a temperature of 30-40°C, and the concentration of the substrate DL-3-acetylthio-2-methylpropionate was 50-200 mM.

7. The application of the immobilized DCH enzyme according to claim 6, characterized in that: After the hydrolysis and resolution reaction is completed, the immobilized DCH enzyme is recovered by filtration or centrifugation and used in the next hydrolysis and resolution reaction.

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