Halohydrin dehalogenase immobilization method
By modifying epoxy resin as the immobilized carrier of haloalcohol dehalogenase, covalent bonds and glycine reactions, the problem of poor improvement of enzyme activity in traditional carriers is solved, and the improvement of enzyme activity and production efficiency is achieved.
Patent Information
- Application Number
- CN202411529398.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-30
- Publication Date
- 2024-12-13
AI Technical Summary
The traditional haloalcohol dehalogenase immobilized carrier has poor effect on improving enzyme activity, resulting in a smaller increase in production efficiency during the production of drug intermediates.
By modifying epoxy resin as the immobilized carrier of halohydrin dehalogenase, it is adsorbed by hydrophobic and electrostatic effects, and reacts with amino residues on the surface of the enzyme through covalent bonds to reduce the activity of residual epoxy groups on the carrier by binding to glycine. , improve the binding force and enzyme activity of the enzyme to the carrier.
The improvement of enzyme activity and the chemical stability and temperature stability of the carrier are achieved, the number of reuses of enzymes and product yields are improved, the enzyme activity after immobilization is enhanced, and the efficiency of drug intermediate production is improved.
Abstract
Description
Technical Field
[0001] The invention relates to a method for immobilizing a halohydrin dehalogenase, and belongs to the field of enzyme immobilization. Background Art
[0002] Halohydrin dehalogenase (HHDH), also known as halohydrin hydrogen halide lyase, is a type of dehalogenase that catalyzes the conversion of ortho-halohydrins into epoxides through an intramolecular nucleophilic substitution mechanism and is one of the key enzymes for the degradation of organic halogen compounds by microorganisms in nature. Halohydrin dehalogenase can not only selectively catalyze the cleavage of the carbon-halogen bond of ortho-halohydrins to synthesize chiral epoxides through dehalogenation reactions, but also can highly selectively catalyze the acceptance of a series of non-natural nucleophiles other than halide ions to synthesize a series of optically pure chiral β-substituted alcohols.
[0003] In the common immobilization process of halohydrin dehalogenase, the use of epoxy resin as a carrier can provide better enzyme activity after immobilization, which is of great significance for the application of halohydrin dehalogenase. However, the improvement of enzyme activity by a simple epoxy resin carrier is relatively uniform and cannot achieve a higher enzyme activity intensity. When used in the production process of drug intermediates, the improvement in production efficiency is small. Summary of the invention
[0004] The present invention aims at the defects existing in the above prior art and provides a method for immobilizing a halohydrin dehalogenase, which solves the problem that the preparation method of the traditional halohydrin dehalogenase immobilization carrier has poor effect on improving the enzyme activity.
[0005] The objective of the present invention is achieved by the following technical scheme, the immobilization carrier of the halohydrin dehalogenase is an epoxy resin, and the halohydrin dehalogenase immobilization method comprises the following steps: S1: adsorbing the crude enzyme solution of halohydrin dehalogenase onto the epoxy resin carrier through hydrophobic and electrostatic effects; S2: The amino acid with an amino residue on the surface of the halohydrin dehalogenase forms a covalent bond with the epoxy group on the epoxy resin support, so that a covalent connection is formed between the halohydrin dehalogenase and the epoxy resin support; S3: adjusting the buffer around the halohydrin dehalogenase and the epoxy resin carrier to be alkaline, so that the enzyme molecules and the epoxy groups are multi-point covalent, thereby enhancing the binding force between the halohydrin dehalogenase and the epoxy resin carrier; S4: adding the halohydrin dehalogenase immobilized support obtained after step S3 into a buffer solution containing glycine, and reducing the activity of the remaining epoxy groups on the support by reacting the glycine with the unreacted epoxy groups in the halohydrin dehalogenase immobilized support.
[0006] The enzyme was derived from Escherichia coli, and the epoxy resin was SK ES-120.
[0007] Furthermore, the epoxy resin carrier is a modified epoxy resin, and the modification method is to copolymerize a monomer with a carboxyl group with an epoxy resin masterbatch to obtain the epoxy resin carrier.
[0008] Furthermore, the monomer having a carboxyl group is selected from one of acrylic acid and methacrylic acid.
[0009] Furthermore, in the copolymerization reaction, the reaction solvent is selected from one of acetone, DMF and THF.
[0010] Furthermore, in the copolymerization reaction, one of dibenzoyl peroxide and azobisisobutyronitrile is selected as an initiator.
[0011] Furthermore, in the copolymerization reaction, the reaction temperature is 25-60°C.
[0012] Furthermore, in the step S1, the adsorption reaction temperature is room temperature.
[0013] Furthermore, in the step S1, the adsorption reaction time is 1 to 4 hours.
[0014] Furthermore, in the S2 step, the covalent duration is 18 to 32 hours.
[0015] Furthermore, in the step S3, the pH of the buffer solution is 7-9.
[0016] In summary, compared with the prior art, the present invention has the following advantages: 1. In the scheme of the present invention, the epoxy resin is modified and used as an immobilized carrier of the halohydrin dehalogenase. The epoxy resin has good chemical stability, can remain stable in a wide range of pH values and in various solvents, is not easily corroded by chemical substances, has a good effect on maintaining the activity of the enzyme, has good temperature stability and operational stability, and can increase the number of enzymes reused to ensure the product yield; 2. In the scheme of the present invention, the roughness of the epoxy resin surface is increased by mechanical grinding, thereby increasing the active sites, and carboxyl functional groups are introduced on the epoxy resin surface to react with amino acid residues on the surface of the halohydrin dehalogenase enzyme through covalent bonds, thereby further improving the performance of the epoxy resin as a HHDH carrier to enhance the affinity with the enzyme; 3. In the scheme of the present invention, carboxyl functional groups are introduced on the surface of epoxy resin, and react with amino acid residues on the surface of halohydrin dehalogenase through covalent bonds, thereby improving the activity of the enzyme after immobilization. The enzyme activity after immobilization is improved by sacrificing a small amount of enzyme activity recovery rate, and the production efficiency can be improved when used in the production process of drug intermediates. DETAILED DESCRIPTION
[0017] The technical solution of the present invention is further specifically described below through specific embodiments, but the present invention is not limited to these embodiments.
[0018] Preparation of modified epoxy resin: Dissolve 5g of epoxy resin in 30ml of acetone solvent and put it into a magnetic stirrer. Weigh an appropriate amount of 1g of acrylic acid monomer and dissolve it in 20ml of acetone solvent to prepare a hydroxylation solution. Transfer the carboxylation reagent solution to a burette for standby use. Add 0.02g of dibenzoyl peroxide to the epoxy resin solution, drop the hydroxylation solution into the epoxy resin solution, and react at 35°C for 5h after the addition is complete. After the reaction is completed, stop stirring, pour the reaction solution into deionized water for dilution, and filter the modified epoxy resin to remove unreacted carboxylation reagent and by-products. Wash the modified epoxy resin with deionized water until the washing liquid is neutral and dry to obtain the modified epoxy resin.
[0019] Enzyme immobilization: Prepare 150mL sodium phosphate buffer (100mmol / L, pH7.0), add 20g modified epoxy resin and 10mL halohydrin dehalogenase crude enzyme solution, mix and place in a 25℃ water bath shaker, shake at 180r / min for 3h, filter, wash with distilled water 3 times, transfer to 150mL sodium phosphate buffer (100mmol / L, pH9.0), add 2ml glycerol, covalently incubate at 30℃ for 20h. Filter twice, wash with distilled water 3 times to obtain immobilized enzyme.
[0020] Enzyme activity detection: Add 10 mL of borate buffer (200 mM, pH 9.0) and 0.2 g of immobilized enzyme to a 50 mL transformation bottle, and place it in a 35 ° C water bath shaker for 10 minutes. Then take 38 μL of substrate and add it to the transformation bottle, the speed is 180 rpm / min, and the timing starts the reaction. After 2 minutes of reaction, take 0.4 mL of the reaction solution and place it in an EP tube with 0.8 mL of ethyl acetate for extraction, centrifuge at 12000 rpm for 10 minutes, and take the supernatant organic phase; The chromatographic column was an HP-5 capillary column, the column temperature was maintained at 60°C for 4 minutes, then increased to 160°C at 20°C / min and maintained for 1 minute; the front inlet temperature was 230°C, the detector FID temperature was 250°C, and the carrier gas (N2) flow rate was 54.0 mL / min. Under these conditions, the elution times of 1,3-DCP and ECH were 6.3 minutes and 4.1 minutes, respectively; The enzyme activity after fixation was: 527.4U / g; Enzyme activity recovery rate: 62.3%; Residual enzyme activity after 10 batches: 80.2%.
[0021] This example is a comparative example of Example 1, and the reaction conditions are modified as follows: the acrylic acid monomer is replaced with a methacrylic acid monomer, and other conditions are the same. The modified epoxy resin obtained by using the scheme of this example is used to immobilize the enzyme and perform enzyme activity detection, and the results are as follows: The enzyme activity after fixation was: 521.2U / g; Enzyme activity recovery rate: 61.8%; Residual enzyme activity after 10 batches: 78.5%.
[0022] This example is a comparative example of Example 1, and the reaction conditions are modified as follows: the initiator is replaced by azobisisobutyronitrile from dibenzoyl peroxide, and other conditions are the same. The modified epoxy resin obtained by the scheme of this example is used to immobilize the enzyme and perform enzyme activity detection. The results are as follows: The enzyme activity after fixation was: 518.6U / g; Enzyme activity recovery rate: 61.1%; Residual enzyme activity after 10 batches: 77.8%.
[0023] This embodiment is a comparative example of embodiment 1, and the reaction conditions are modified as follows: the temperature of the copolymerization reaction is changed to 25°C, 30°C, 40°C, 50°C, and 60°C in sequence, and other conditions are the same. The modified epoxy resin obtained by using the embodiment scheme is used for enzyme immobilization and enzyme activity detection. The enzyme activity detection results at 25°C and 30°C are very slightly different from those of embodiment 1. As the temperature increases, the grafting rate increases, but the enzyme activity results after immobilization do not increase. Instead, the enzyme activity recovery rate is reduced to less than 50%.
[0024] This embodiment is a comparative example of embodiment 2, and the reaction conditions are modified as follows: the pH of the buffer solution in the covalent insulation stage is 7.0, and other conditions are the same. The modified epoxy resin obtained by the embodiment scheme is used to immobilize the enzyme and then the enzyme activity is detected. The enzyme activity of the immobilized enzyme shows a significant downward trend and is only 502.7 U / g.
[0025] The implementation of the present invention is not limited to the above-mentioned embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and detail, and these are considered to fall within the protection scope of the present invention.
Claims
1. A method for immobilizing a halohydrin dehalogenase, characterized in that: The immobilization carrier of the halohydrin dehalogenase is an epoxy resin, and the halohydrin dehalogenase immobilization method comprises the following steps: S1: The crude enzyme solution of halohydrin dehalogenase is adsorbed onto the epoxy resin support through hydrophobic and electrostatic interactions; S2: The amino acid with an amino residue on the surface of the halohydrin dehalogenase forms a covalent bond with the epoxy group on the epoxy resin support, thereby forming a covalent connection between the halohydrin dehalogenase and the epoxy resin support; S3: Adjusting the buffer around the halohydrin dehalogenase and the epoxy resin support to be alkaline to allow the enzyme molecules to undergo multi-point covalent bonding with the epoxy groups, thereby enhancing the binding force between the halohydrin dehalogenase and the epoxy resin support; S4: adding the halohydrin dehalogenase immobilized support obtained after step S3 to a buffer solution containing glycine, and reacting the glycine with the unreacted epoxy groups in the halohydrin dehalogenase immobilized support to reduce the activity of the remaining epoxy groups on the support.
2. The method for immobilizing a halohydrin dehalogenase according to claim 1, wherein: The epoxy resin carrier is a modified epoxy resin, and the modification method is to copolymerize a monomer with a carboxyl group with an epoxy resin masterbatch to obtain the epoxy resin carrier.
3. A method for immobilizing a halohydrin dehalogenase according to claim 2, characterized in that: The monomer having a carboxyl group is selected from one of acrylic acid and methacrylic acid.
4. The method for immobilizing a halohydrin dehalogenase according to claim 3, wherein: In the copolymerization reaction, the reaction solvent is selected from acetone, DMF, and THF.
5. The method for immobilizing a halohydrin dehalogenase according to claim 4, wherein: In the copolymerization reaction, one of dibenzoyl peroxide and azobisisobutyronitrile is selected as the initiator.
6. The method for immobilizing a halohydrin dehalogenase according to claim 5, wherein: In the copolymerization reaction, the reaction temperature is 25-60°C.
7. The method for immobilizing a halohydrin dehalogenase according to claim 1, wherein: In the step S1, the adsorption reaction temperature is room temperature.
8. The method for immobilizing a halohydrin dehalogenase according to claim 7, wherein: In the step S1, the adsorption reaction time is 1 to 4 hours.
9. The method for immobilizing a halohydrin dehalogenase according to claim 8, wherein: In the S2 step, the covalent duration is 18 to 32 hours.
10. The method for immobilizing a halohydrin dehalogenase according to claim 9, wherein: In the step S3, the pH of the buffer solution is 7-9.