Immobilization and application of ketoreductase
By mutating the amino acid sequence of ketone reductase and immobilizing it on protein adsorption magnetic beads, the problem of low ketone reductase immobilization rate was solved, achieving efficient preparation of ezetimibe intermediate and reducing the amount of ketone reductase used and the preparation cost.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHANGZHOU PHARMA FACTORY
- Filing Date
- 2024-11-20
- Publication Date
- 2026-05-22
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biocatalytic synthesis technology, specifically relating to the immobilization of a ketone reductase for preparing the intermediate (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidinyl-2-one from ezetimibe. This invention also relates to the research, preparation and application of the immobilization method of this ketone reductase. Background Technology
[0002] Ezetimibe's mechanism of action is to inhibit the absorption of cholesterol from food in the intestines, reduce the transport of cholesterol from the small intestine to the liver, and lower plasma cholesterol levels. It can be used for various types of hyperlipidemia, especially for patients who do not respond well to or cannot tolerate statins, as well as some patients with hereditary and drug-induced hyperlipidemia. Merck's market sales reached $2.52 billion in 2015, and it has become a blockbuster drug in the lipid-lowering drug market with excellent sales performance. Its structural formula is...
[0003]
[0004] The compound (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazacyclopentan-2-one (CAS: 189028-95-3) is a key chiral intermediate in the synthesis of ezetimibe active pharmaceutical ingredient. Its structural formula is...
[0005]
[0006] The synthesis of this compound using traditional chemical synthesis methods typically involves asymmetric reduction using catalysts and ligands to reduce the carbonyl group to a chiral alcohol hydroxyl group. This method is difficult to achieve, has high costs, and is difficult to industrialize.
[0007] The most advantageous of the reported preparation processes is the asymmetric reduction of carbonyl groups catalyzed by ketoreductase (KRED). Enzyme immobilization technology refers to the process of binding free enzymes to a specific space or an insoluble support, thereby restricting the free flow of the enzyme and enabling it to exert its catalytic activity for a long time and be recyclable. After the enzyme is immobilized, the interaction between it and the immobilization matrix may change the spatial structure outside the enzyme's catalytic active site, thereby improving the enzyme's stability in strong pH, high temperature, or organic solvents. Simultaneously, the target product can be separated from the enzyme reaction system. Thus, the immobilized enzyme can be removed by simple mechanical separation or centrifugation, avoiding the disadvantages of free enzymes such as difficulty in filtration and easy emulsification during post-processing. While enzyme immobilization for asymmetric reduction of carbonyl groups can achieve good optical purity and yield, the current method has a low immobilization rate of ketoreductase, requires a large amount of ketoreductase, and is difficult to reuse repeatedly.
[0008] Therefore, in the industrial-scale preparation of ezetimibe intermediates, given the shortcomings of existing technologies, improving the immobilization rate of ketone reductase, reducing the amount of ketone reductase used, and increasing the number of applications are urgent challenges that need to be addressed. Summary of the Invention
[0009] The technical problem to be solved by this invention is how to improve the immobilization rate of ketone reductase, reduce the amount of ketone reductase used, and increase the number of times it is applied during the preparation of ezetimibe intermediate.
[0010] The technical solution of this invention to solve the above-mentioned technical problems is as follows: This invention mutates the amino acid sequence of ketone reductase to add a targeting tag, improves the protein immobilization rate, and then uses protein adsorption magnetic beads as a carrier to obtain an immobilized ketone reductase with high activity and multiple reusability. This enzyme is then used in the preparation of ezetimibe intermediate ((4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazacyclopentan-2-one).
[0011] The technical solution of the present invention to solve the above-mentioned technology is as follows: a method for immobilizing ketone reductase expressed by Escherichia coli fermentation onto amino acid-targeting tag protein adsorption magnetic beads by covalent binding, characterized by comprising the following steps:
[0012] Step 1): The ketone reductase sequence was mutated by PCR, and an amino acid linker was added to the end of the amino acid sequence to obtain a wet bacterial solution;
[0013] In step 1), the ketone reductase is selected from Escherichia coli, and the amino acid sequence is shown in SEQ ID NO:1. Adding an amino acid linker tag, such as histidine, lysine, or cysteine, to the end of the amino acid sequence can improve the protein immobilization rate and enhance the activity of the immobilized enzyme. Histidine and lysine are preferred.
[0014] Where the immobilization rate (%) = {(original enzyme liquid protein content - supernatant protein content after adsorption) / original enzyme liquid protein content} * 100%.
[0015] In step 1), the recombinant plasmid with added histidine, lysine, and cysteine linkage tags was transformed into BL21(DE3) host cells. The ketone reductase-engineered bacteria were inoculated into LB liquid medium containing kanamycin resistance, and then transferred to fermenter medium and cultured until the bacterial OD600 reached 15-20. IPTG (isopropyl-β-D-thiogalactoside) was added, and the culture was induced at 25-35℃ for 12-14 hours.
[0016] In step 1), the LB liquid medium for kanamycin resistance can be controlled at 50ug / mL-100ug / mL;
[0017] In step 1), the conditions of the culture medium are selected from a culture temperature of 20-40℃, a reaction speed of 200rpm-1000rpm, and a culture time of 8h-12h.
[0018] The conditions for the fermentation broth in step 1) are selected from 20-40℃ and 200rpm-1000rpm;
[0019] In step 1), the final concentration of IPTG (isopropyl-β-D-thiogalactopyranoside) is selected from 0.5 mM to 1 mM.
[0020] Step 2): Add 3-5 times the amount of 0.1M pH 7.0 phosphate buffer to the wet bacterial cell solution, stir well, and then homogenize using a homogenizer;
[0021] In step 2), the buffer solution can also be selected from 0.1M pH 7.0-7.5 Tris-HCl buffer.
[0022] In step 2), the reaction temperature is controlled at 10-15℃;
[0023] Step 3): Centrifuge the broken wet bacterial cell solution at 8000-10000 rpm for 5-10 min to remove cell debris and obtain the centrifuged enzyme solution;
[0024] After the intermediate fermentation process in step 3) is completed, centrifugation, filtration and other methods can be used to separate the cells. For example, the supernatant can be discarded to obtain the ketone reductase wet cells after adding the label, i.e. the enzyme solution after centrifugation. This process can be repeated multiple times depending on the actual operation.
[0025] Step 4): After centrifugation, add protein adsorption magnetic bead carrier to the enzyme solution, react at 10-20℃, stir for 20-24h to complete the incubation, and obtain immobilized ketone reductase magnetic beads.
[0026] Step 4) is a physical adsorption process in which the centrifuged enzyme solution is adsorbed onto the protein magnetic bead carrier to obtain immobilized enzyme.
[0027] In step 4), enzyme immobilization involves binding enzyme molecules to a specific support using physical or chemical methods, rendering them insoluble in water while retaining their native activity. This method not only facilitates separation from the reaction solution but also allows for enzyme reuse, thereby improving enzyme utilization efficiency and reducing costs.
[0028] Step 4) involves methods for preparing immobilized enzymes, including adsorption, encapsulation, covalent bonding, and cross-linking. Each method has its advantages and disadvantages, and the appropriate method depends on the specific application requirements and the properties of the enzyme. For example, adsorption is simple and easy to implement, but the enzyme is prone to detachment and is unstable; covalent bonding is more robust and can be reused multiple times, but the operation is complex.
[0029] In step 4), protein adsorption magnetic beads are added to the enzyme solution after centrifugation. The protein adsorption magnetic beads used are produced by Xiamen Primai Biotechnology Co., Ltd., and are used for protein adsorption, purification, immobilization, etc.
[0030] In step 4), the type or model of the protein adsorption magnetic beads can be selected from agarose protein L magnetic beads, agarose protein G magnetic beads, agarose protein A magnetic beads, Strep-tag II protein purification magnetic beads, GST fusion protein purification agarose magnetic beads, His-tag protein purification magnetic beads, with His-tag protein purification magnetic beads being preferred.
[0031] In step 4), the mass ratio of wet bacterial cells to protein-adsorbing magnetic beads is 1g:4-5g.
[0032] Step 5): After the fermentation broth has been incubated, filter it and wash the filter cake 2-3 times with 0.1M pH7.0 phosphate buffer to obtain immobilized ketone reductase magnetic beads.
[0033] In step 5), the buffer solution can also be selected from Tris-HCl buffer solution or triethanolamine buffer solution.
[0034] In addition, the present invention also provides a method for preparing the ezetimibe intermediate (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidinyl-2-one, characterized by comprising the following steps:
[0035] In this process, under the action of glucose dehydrogenase and immobilized enzyme magnetic beads, buffer solution is added, and (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone is used as the substrate, NAD or NADP as the coenzyme, and glucose as the hydrogen donor. Asymmetric reduction is then carried out to obtain (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidinyl-2-one.
[0036] (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone was used as the reaction substrate, and the substrate concentration was controlled at 50mg-100mg / ml.
[0037] NAD refers to nicotinamide adenine dinucleotide (coenzyme I), and NADP refers to nicotinamide adenine dinucleotide.
[0038] GDH refers to glucose dehydrogenase.
[0039] The substrate used was (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone, and the amount of NAD or NADP used was 0.05%-0.5% of the substrate mass.
[0040] The substrate used was (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone, and the amount of glucose used was 1.1-2 molar equivalents of the substrate.
[0041] The substrate is (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone, and the amount of glucose dehydrogenase is 0.1%-1% of the substrate mass.
[0042] The immobilized ketone reductase magnetic beads, using (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone as the substrate, account for 2%-10% of the substrate mass.
[0043] The asymmetric reduction reaction is carried out at a temperature of 30-40℃, a pH of 6.5-8.0, and a reaction time of 18-24h.
[0044] To accelerate or promote the rapid progress of the reaction, one or more of the following can be added as a co-solvent: dimethyl sulfoxide, methyl tert-butyl ether, isopropyl acetate, N,N-dimethylformamide, ethanol, acetonitrile, methanol, isopropanol, or tetrahydrofuran.
[0045] The amount of co-solvent used is 20%-50% of the volume of the added buffer solution.
[0046] The buffer solutions were selected from 0.1M pH 7.0-pH 7.5 phosphate buffer and 0.1M pH 7.0-7.5 Tris-HCl buffer.
[0047] Furthermore, this invention also provides a method for detecting the conversion rate of the ezetimibe intermediate (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidinyl-2-one:
[0048] The liquid chromatography (LC) detection conditions were as follows: LC column: ZORBAX Eclipse XDB-C18 (4.6*150mm, 5-Micron); mobile phase: 0.5% trifluoroacetic acid:acetonitrile (50:50); flow rate: 1.0 mL / min; column temperature: 35℃; retention time: product: 5.8 min; substrate: 7.5 min; detection wavelength: 210 nm.
[0049] The ee value detection conditions were selected as follows: chromatographic column: CHIPALPAKO-IC 4.6mm*250mm, 5um, mobile phase: n-hexane:isopropanol 90:10, flow rate: 0.6mL / min, wavelength: 210nm, column temperature: 35C, injection volume: 5uL, elution: 30min, blank solvent: isopropanol, and test solution: 1.0mg / mL.
[0050] The formula for calculating product eep is: eep=(CR-CS) / (CR+CS)×100%.
[0051] The protein concentration was detected using Beyotime's Bradford protein concentration assay kit, which was used to determine the protein concentration in the original enzyme solution and the supernatant after resin adsorption.
[0052] Where the immobilization rate (%) = {(original enzyme liquid protein content - supernatant protein content after adsorption) / original enzyme liquid protein content} * 100%.
[0053] The present invention achieves beneficial effects
[0054] 1. This invention discloses for the first time that immobilized enzymes can be achieved using a protein adsorption magnetic bead method. By modifying the amino acid sequence of existing ketoreductases, adding amino acid tags, and then immobilizing them onto His-Tag protein adsorption magnetic beads, the protein immobilization rate can be increased from 42% to 81%. Even after 20 consecutive applications, the conversion rate can still reach over 98%, effectively and significantly reducing costs, making it suitable for industrial production.
[0055] 2. This invention is the first to use immobilized amino acid-targeting tag protein adsorption magnetic beads in the synthesis of ezetimibe intermediates, which can solve the shortcomings of traditional chemical methods such as difficulty in filtration and easy emulsification. Attached Figure Description
[0056] Figure 1 : Reaction structure of ezetimibe intermediate
[0057] Figure 2 Protein concentration standard curve Detailed Implementation
[0058] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:
[0059] Example 1: Mutation of the amino acid sequence of the original enzyme protein. Histidine (His), lysine (Lys), and cysteine (Cys) linker tags were added to the ends of the amino acid sequences. PCR system: primers, template plasmid, PCR enzyme, and pure water. Specific parameters are shown in Table 1 below.
[0060] Table 1: Primer Design
[0061]
[0062]
[0063] Table 2: PCR reaction system (50 μL)
[0064]
[0065] The PrimeSTAR Max Premix (2x) was purchased from Takara Bio Reagents Ltd.
[0066] Table 3: PCR amplification program
[0067]
[0068] After PCR, the products were detected by agarose gel electrophoresis:
[0069] (1) Weigh 1% (w / v) of agarose into an Erlenmeyer flask, add an appropriate amount of 1×TAE Buffer, heat in a microwave oven until the agarose is completely dissolved, add 1 / 50000 of Gel Red dye while it is hot, mix well and pour into a gel casting plate and insert a toothed comb.
[0070] (2) Wait until the agarose gel solidifies, then pull out the comb vertically. After opening the gel, tilt the side with the well towards the cathode (black) and place it in the electrophoresis tank. Add 1×TAE Buffer until the tank solution covers the well.
[0071] (3) The sample to be tested is injected steadily and vertically into the gel well with a pipette. The lane is then adjusted to be completely parallel to the current direction. The voltage is adjusted to 120V. When the sample front reaches 2 / 3 of the total gel length from the well, the electrophoresis apparatus is turned off. The gel block is then transferred and the electrophoresis results are observed under a UV lamp.
[0072] The recombinant plasmid with histidine (His), lysine (Lys), and cysteine (Cys) linkage tags after mutation was transformed into BL21(DE3) competent cells, plated on LB plates containing 50 μg / ml kanamycin resistance, and cultured at 35°C for 8-12 h. Clones were randomly selected, plasmids were extracted and sequenced for identification, and recombinant plasmids containing the expression mutant plasmids were obtained through screening.
[0073] The mutant ketone reductase bacteria were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance and cultured at 37°C and 200 rpm for 8 h. The culture was then transferred to fermenter medium and cultured at 35°C and 800 rpm until the bacterial OD600 reached 15–20. IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.5 mM and the culture was induced at 25°C for 12–14 h. After fermentation, the bacteria were centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected to obtain wet ketone reductase cells with histidine (His), lysine (Lys), and cysteine (Cys) linkage tags.
[0074] Add 200g of wet bacterial cells to 800ml of 0.1M pH 7.0 phosphate buffer solution, stir well, homogenize in a homogenizer at 10-20℃, and centrifuge to obtain enzyme solution for later use.
[0075] Example 2
[0076] Pretreatment of His-Tag protein adsorption magnetic beads: 20g of His-Tag protein adsorption magnetic beads purchased from Xiamen Primai Biotechnology Co., Ltd. were soaked in 0.1M pH 7.0 phosphate buffer solution and filtered.
[0077] Step 1: Take 100ml of the broken enzyme solution, add 20g of pretreated magnetic beads, and stir at 10-20℃ for 20-24h to complete the incubation.
[0078] Step 2: After adsorption is complete, filter the solution, retain the filtrate, and rinse the filter cake with deionized water.
[0079] Step 3: Detect protein concentration and calculate immobilization rate. Beyotime's Bradford Protein Concentration Assay Kit is used to detect the protein concentration in the original enzyme solution and the supernatant after resin adsorption.
[0080] The method for plotting the protein concentration standard curve is as follows:
[0081] Dilute the protein standards (5 mg / ml BSA) provided in the kit with phosphate buffer. Prepare protein standards of 0, 0.125, 0.25, 0.5, 0.75, 1, and 1.5 mg / ml according to Table 4 below. Ensure thorough mixing for each dilution.
[0082] Table 4
[0083]
[0084]
[0085] Add 5 μl of protein standards at different concentrations and the test sample to each well of a 96-well plate. Then add 250 μl of G250 staining solution to each well. Set the detection wavelength of the microplate reader to 595 nm and measure the absorbance over two hours. Plot the protein concentration standard curve y = 0.5035x + 0.4485. Calculate the protein concentration x based on the value y measured by the microplate reader. Figure 2 .
[0086] The samples to be tested include the original enzyme solution after disruption and the supernatant enzyme solution after fixation.
[0087] Where the immobilization rate (%) = {(original enzyme liquid protein content - supernatant protein content after adsorption) / original enzyme liquid protein content} * 100%.
[0088] The calculation results are as follows:
[0089]
[0090] According to the test results, HA was immobilized with amino resin and EA was immobilized with epoxy resin. After improvement, the immobilization rate of protein magnetic beads in this application can be increased from the initial 42% to 81%, which fully demonstrates that more enzymes are adsorbed on the unit carrier and the activity of the carrier after adsorption is relatively higher, laying a practical foundation for subsequent multiple applications.
[0091] Example 3
[0092] Take the immobilized ketone reductase magnetic beads from Example 2, 2.0 g of reaction substrate ZT-4, 30 ml of 0.1 M pH 7.0 phosphate buffer, 0.5 g of the immobilized ketone reductase magnetic beads, 10 ml of co-solvent (selected from one or more of dimethyl sulfoxide, methyl tert-butyl ether, isopropyl acetate, N,N-dimethylformamide, ethanol, acetonitrile, methanol, isopropanol, or tetrahydrofuran), 2.5 g of glucose, 20 mg of GDH, and 20 mg of NADP. React at 37°C and 220 RPM for 24 h. Samples were taken to measure the conversion rate, finding it to be 100%.
[0093] ZT-4 is (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone.
[0094] Example 4
[0095] Under the condition of substrate ZT-4 concentration of 100 g / L, the ratio of immobilized ketone reductase magnetic beads to substrate was screened. The following solutions were used: 10.0 g substrate, 0.1 M pH 7.0 buffer, 70 ml PBS, 30 ml DMSO, 12.0 g glucose, 100 mg GDH, and 50 mg NADP. Immobilized ketone reductase magnetic beads from Example 2 were used at concentrations of 0.1 g, 0.5 g, 1.0 g, 1.5 g, and 2.0 g, respectively. The reaction was carried out at 37°C and 220 RPM for 24 h. The conversion rate after 24 h showed that 5% of the substrate amount of enzyme was sufficient for complete conversion. Therefore, the preferred amount of immobilized ketone reductase magnetic beads is 2%-10% of the substrate weight.
[0096] Example 5
[0097] The reaction temperature was controlled at 30-40℃. 0.5g of immobilized ketone reductase magnetic beads from Example 2, 10.0g of substrate, 70ml of 0.1M pH 7.5 phosphate buffer solution, 30ml of DMSO, 12.0g of glucose, 100mg of GDH, and 50mg of NADP were taken and reacted at 220RPM for 24h. The conversion rate was measured to be 100%.
[0098] Example 6
[0099] The reaction was controlled at pH 6.5-8.0. 0.5 g of immobilized ketone reductase magnetic beads (from Example 2), 10.0 g of substrate, 70 ml of 0.1 M phosphate buffer, 30 ml of DMSO, 12.0 g of glucose, 100 mg of GDH, and 50 mg of NADP were taken and reacted at 40°C and 220 RPM for 24 hours. A sample was taken to determine the conversion rate; 100% was achieved.
[0100] Example 7
[0101] The reaction was controlled at pH 6.5-8.0. 1.0 g of immobilized ketone reductase magnetic beads (from Example 2), 20.0 g of substrate, 140 ml of 0.1 M phosphate buffer solution, 60 ml of DMSO, 24.0 g of glucose, 200 mg of GDH, and 100 mg of NADP were used. The reaction temperature was 30-40℃, and the reaction was carried out at 220 RPM for 24 hours. Samples were taken to determine the conversion rate; 100% was achieved.
[0102] Example 8
[0103] Take 1.0 g of immobilized ketone reductase magnetic beads from Example 2, 20.0 g of substrate, 140 ml of 0.1 M phosphate buffer solution, 60 ml of DMSO, 24.0 g of glucose, 200 mg of GDH, and 100 mg of NADP. React at 30 °C with the pH controlled at 6.5-8.0 and at 220 RPM. The conversion rate is 100% after 18 h.
[0104] Example 9
[0105] Take 5.0 g of immobilized ketone reductase magnetic beads from Example 2, 100.0 g of substrate, 700 ml of 0.1 M phosphate buffer solution, 300 ml of DMSO, 120.0 g of glucose, 1.0 g of GDH, and 500 mg of NADP. React at 40 °C and 220 RPM. The conversion rate is 100% after 18 h.
[0106] Example 10: Continuous application experiment of immobilized ketone reductase magnetic beads.
[0107] Take 2.5g of immobilized ketoreductase magnetic beads from Example 2, 50.0g of substrate, 350ml of 0.1M pH 7.0 PBS buffer, 150ml of DMSO, 60.0g of glucose, 500mg of GDH, and 250mg of NADP. React at 40℃ and 220RPM. After the reaction, filter out the immobilized enzyme magnetic beads, wash with deionized water, and then repeat the feeding process. This process was repeated 20 times, and the conversion rate could still reach 98% after 24 hours. The number of times the enzyme was used and the conversion rate data are shown in Table 6 below.
[0108]
[0109] Table 6 shows the number of times the application was used.
Claims
1. A method for immobilizing ketone reductase expressed by Escherichia coli fermentation onto amino acid-targeting tag protein adsorption magnetic beads via covalent bonding, characterized in that... Includes the following steps: Step 1): The ketone reductase sequence was mutated by PCR, and an amino acid linker tag was added to the end of the amino acid sequence. After fermentation and post-treatment, wet cells were obtained. Step 2): Add 3-5 times the amount of 0.1M pH 7.0 phosphate buffer to the wet bacterial cells, stir well, homogenize in a homogenizer, and control the temperature at 10-15℃; Step 3): Centrifuge the broken wet bacterial cell solution at 8000-10000 rpm for 5-10 min to remove cell debris and obtain the centrifuged enzyme solution; Step 4): After centrifugation, add protein adsorption magnetic beads to the enzyme solution, react at 10-20℃, stir for 20-24 hours to complete the incubation. Step 5): The filter cake was washed with 0.1M pH 7.0 phosphate buffer to obtain immobilized ketone reductase magnetic beads.
2. The method for immobilizing ketone reductase expressed by Escherichia coli fermentation onto amino acid-Tag protein adsorption magnetic beads by covalent binding according to claim 1, characterized in that... In step 1), the amino acid tag is selected from histidine tag, lysine tag, and cysteine tag.
3. The method for immobilizing ketone reductase expressed by Escherichia coli fermentation onto amino acid-Tag protein adsorption magnetic beads by covalent binding according to claim 1, characterized in that... In step 4), the protein adsorption magnetic bead carriers are selected from agarose protein L magnetic beads, agarose protein G magnetic beads, agarose protein A magnetic beads, Strep-tag II protein purification magnetic beads, GST fusion protein purification agarose magnetic beads, and His-tag protein purification magnetic beads.
4. The method for immobilizing ketone reductase expressed by Escherichia coli fermentation onto amino acid-Tag protein adsorption magnetic beads by covalent binding according to claim 1, characterized in that... In step 4), the protein adsorption magnetic bead carrier is selected from histidine His-Tag protein adsorption magnetic beads.
5. The method for immobilizing ketone reductase expressed by Escherichia coli fermentation onto amino acid-Tag protein adsorption magnetic beads by covalent binding according to claim 1, characterized in that... In step 4), the mass ratio of wet bacterial cells to protein-adsorbing magnetic beads is 1g:1-2g.
6. A method for preparing ezetimibe intermediates using immobilized enzyme protein magnetic beads as described in any one of claims 1 to 4, characterized in that: Under the action of glucose dehydrogenase and immobilized enzyme magnetic beads prepared according to any one of claims 1-4, buffer and solubilizer are added, and (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone is used as substrate, NAD or NADP as coenzyme, and glucose as hydrogen donor, asymmetric reduction is carried out to obtain (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidinyl-2-one.
7. A method for preparing ezetimibe intermediates using the immobilized enzyme as described in any one of claims 1 to 4, as described in claim 5, characterized in that: The substrate was (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone, and the amount of NAD or NADP used was 0.05%-0.5% of the substrate mass.
8. A method for preparing ezetimibe intermediates using the immobilized enzyme as described in any one of claims 1 to 4, as described in claim 5, characterized in that: The substrate was (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone, and the amount of glucose used was 1.1-2 molar equivalents of the substrate.
9. A method for preparing ezetimibe intermediates using the immobilized enzyme as described in any one of claims 1 to 4, as described in claim 5, characterized in that: Using (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone as a substrate, glucose dehydrogenase is 0.1%-1% of the substrate mass.
10. A method for preparing ezetimibe intermediates using the immobilized enzyme as described in any one of claims 1 to 4, as described in claim 5, characterized in that: Using (4S)-3-[5-(4-fluorophenyl)-1,5-dioxopentyl]-4-phenyl-2-oxazolidinone as a substrate, immobilized ketone reductase magnetic beads account for 2%-10% of the substrate mass.
11. A method for preparing ezetimibe intermediates using the immobilized enzyme as described in any one of claims 1 to 4, as described in claim 5, characterized in that: The asymmetric reduction reaction is carried out at a temperature of 30-40℃, a pH of 6.5-8.0, and a reaction time of 18-24h.
12. A method for preparing ezetimibe intermediates using the immobilized enzyme as described in any one of claims 1 to 4, as described in claim 5, characterized in that: One or more of the following can be added as a co-solvent for the reaction: dimethyl sulfoxide, methyl tert-butyl ether, isopropyl acetate, N,N-dimethylformamide, ethanol, acetonitrile, methanol, isopropanol, or tetrahydrofuran.
13. A method for preparing ezetimibe intermediates using the immobilized enzyme as described in any one of claims 1 to 4, as described in claim 5, characterized in that: The buffer solution is selected from 0.1M pH 6-8 phosphate buffer or 0.1M pH 6-8 Tris-HCl buffer.