Carbonyl reductase immobilization process

Abstract

The invention discloses a carbonyl reductase immobilization process which comprises the following steps: carrying out coating crosslinking on carbonyl reductase and polyvinyl alcohol to obtain carbonyl reductase coated with a polyvinyl alcohol film, namely a carbonyl reductase-polyvinyl alcohol film compound; and 2, dissolving the carbonyl reductase-polyvinyl alcohol film compound obtained in the step 1 in a buffer solvent to obtain a mixed solution, adding a cross-linking agent glutaraldehyde, adding magnetic nanoparticles, carrying out low-frequency ultrasonic mixing for 10-15 minutes, filtering, taking out the nanoparticles, washing with the buffer solution, and drying to obtain the final product immobilized carbonyl reductase. The method disclosed by the invention has the beneficial effects that the mechanical stability and the chemical stability of the enzyme are enhanced through film formation and crosslinking of polyvinyl alcohol on the surface of the carbonyl reductase, and the inactivation of the enzyme in the operation process is reduced; the magnetic nanoparticles are introduced, so that the immobilized enzyme can be easily separated and recycled through a magnetic field, and the economical efficiency and reusability of the process are improved; pBS is used as a buffer solution and deionized water is used as a solvent.

Description

Technical Field

[0001] The invention relates to the technical field related to enzyme immobilization, in particular to a carbonyl reductase immobilization process. Background Art

[0002] Enzymes are widely used in many industries as a biocatalyst. Compared with chemical methods, they are more popular because of their mild reaction conditions, high selectivity, and no pollution. However, free enzyme catalysis also has many defects, such as low acid and alkalinity resistance, extreme temperature sensitivity, difficulty in separating from products, and high cost. In order to overcome the defects of free enzyme catalysis, a new method has been proposed, that is, enzyme immobilization.

[0003] Compared with free enzymes, immobilized enzymes have the following advantages: immobilized enzymes are easy to separate from products, thus improving product purity; immobilized enzymes can catalyze multiple times, thus creating profit margins; immobilized enzymes have strong structural toughness and can be used for continuous and automated production such as column packing; the reaction process can be controlled; and they can be used to immobilize multiple enzymes for synergistic catalysis of multi-enzyme systems. Summary of the invention

[0004] In view of the shortcomings of the above-mentioned free enzyme catalytic system, the present invention aims to provide a carbonyl reductase immobilization process, which overcomes the defects of free enzyme catalysis by enzyme immobilization. The technical solution of the present invention is as follows: A carbonyl reductase immobilization process comprises the following steps: The first step is to dissolve the freeze-dried carbonyl reductase enzyme powder in a buffer solution to obtain a carbonyl reductase solution, dissolve polyvinyl alcohol in a solvent to obtain a polyvinyl alcohol solution, add the polyvinyl alcohol solution to the carbonyl reductase solution to obtain a mixed solution, stir and mix evenly, gradually add cross-linking agent glutaraldehyde in batches, stir for 20 to 30 minutes for the first addition, and then gradually extend the stirring reaction time by 10 to 15 minutes. After the reaction is completed, centrifuge to obtain a polyvinyl alcohol film-coated carbonyl reductase, that is, a carbonyl reductase-polyvinyl alcohol film complex; In the second step, the carbonyl reductase-polyvinyl alcohol membrane complex obtained in the first step is dissolved in a buffer solvent to obtain a mixed solution, a cross-linking agent glutaraldehyde is added, magnetic nanoparticles are added, low-frequency ultrasonic mixing is performed for 10 to 15 minutes, the nanoparticles are filtered out, washed with a buffer solution, and dried to obtain the final product, immobilized carbonyl reductase.

[0005] Furthermore, the buffer solutions used in the first step and the second step are both PBS.

[0006] Furthermore, the solvent is deionized water.

[0007] Furthermore, the concentration of the carbonyl reductase in the buffer is 5-10 mg / mL.

[0008] Furthermore, the concentration of the polyvinyl alcohol in the solvent is 25 to 50 mg / mL.

[0009] Furthermore, in the first step, the amount of the cross-linking agent used is 0.5-1.2 wt % of the carbonyl reductase.

[0010] Furthermore, the cross-linking agent is a 1 mg / mL glutaraldehyde aqueous solution, and the cross-linking agent is added to the mixed solution in 3 to 5 times in the first step.

[0011] Furthermore, the second-step cross-linking agent is a 1 mg / mL glutaraldehyde aqueous solution, and the amount of the second-step cross-linking agent is 1 to 1.5 wt % of the carbonyl reductase-polyvinyl alcohol membrane complex.

[0012] Furthermore, in the second step, the concentration of the mixed solution of the carbonyl reductase-polyvinyl alcohol membrane complex is 20-30 mg / mL.

[0013] Furthermore, in the second step, the nanoparticles are PEI@Fe3O4 magnetic nanoparticles, the particle size of the nanoparticles is 20-30 nm, and the mass ratio of the nanoparticles to the carbonyl reductase-polyvinyl alcohol film complex is 1:5.

[0014] The beneficial effects of the present invention are as follows: 1. The mechanical stability and chemical stability of the enzyme are enhanced by film formation and cross-linking of polyvinyl alcohol on the surface of carbonyl reductase, and the inactivation of the enzyme during operation is reduced; 2. The introduction of magnetic nanoparticles allows the immobilized enzyme to be easily separated and recovered through a magnetic field, thereby improving the economy and reusability of the process; 3. Using PBS as a buffer and deionized water as a solvent helps to maintain the stability of the enzyme and other components, ensures that the immobilization process is carried out under a suitable environment, and adding glutaraldehyde in batches and gradually extending the stirring time helps to achieve uniform cross-linking, improves the binding effect of the membrane and the enzyme, and thus improves the immobilization efficiency; 4. By adjusting the amount of the cross-linking agent and the mass ratio of the nanoparticles, the immobilization process can be optimized to meet different application requirements, and the catalytic activity and reaction rate of the enzyme may be improved by optimizing the combination of the membrane and the nanoparticles. DETAILED DESCRIPTION

[0015] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] Example 1 The first step is to dissolve 0.5g of freeze-dried carbonyl reductase powder in 100ml of PBS buffer solution to obtain a carbonyl reductase solution, dissolve 2.5g of polyvinyl alcohol in 100ml of deionized water to obtain a polyvinyl alcohol solution, add the polyvinyl alcohol solution to the carbonyl reductase solution to obtain a mixed solution, stir and mix evenly, gradually add 2.5ml of 1mg / mL glutaraldehyde aqueous solution as a cross-linking agent in 3 batches, stir for 20 to 30min for the first time, keep the speed at 300r / min, and then gradually extend the stirring reaction time by 10 to 15min. After the reaction is completed, centrifuge, wash with buffer solution, and dry at low temperature to obtain 2.9g of carbonyl reductase product coated with polyvinyl alcohol film, i.e., carbonyl reductase-polyvinyl alcohol film complex, with an immobilization rate of 96.7%; In the second step, 2.0 g of the carbonyl reductase-polyvinyl alcohol membrane complex obtained in the first step was dissolved in 100 ml of PBS buffer solvent to obtain a mixed solution, 20 ml of 1 mg / mL cross-linking agent glutaraldehyde was added, 10 g of PEI@Fe3O4 magnetic nanoparticles with a particle size of 20-30 nm were added, low-frequency ultrasonic mixing was performed for 10-15 min, the nanoparticles were filtered out, washed with buffer for 2-3 times, and dried to obtain 11.5 g of the final product immobilized carbonyl reductase with an immobilization rate of 95.8%.

[0017] Example 2 The first step is to dissolve 1.0g of freeze-dried carbonyl reductase enzyme powder in 100ml of PBS buffer solution to obtain a carbonyl reductase solution, dissolve 5g of polyvinyl alcohol in 100ml of solvent deionized water to obtain a polyvinyl alcohol solution, add the polyvinyl alcohol solution to the carbonyl reductase solution to obtain a mixed solution, stir and mix evenly, gradually add 5ml of a 1mg / mL glutaraldehyde aqueous solution of a cross-linking agent in 3 batches, stir for 20 to 30min for the first addition, keep the rotation speed at 300r / min, and then gradually extend the stirring reaction time by 10 to 15min. After the reaction is completed, centrifuge, wash with a buffer solution, and dry at low temperature to obtain 5.8g of a polyvinyl alcohol film-coated carbonyl reductase product, i.e., a carbonyl reductase-polyvinyl alcohol film complex; the fixation rate is 96.7%.

[0018] Example 3 The first step is to dissolve 1.0g of freeze-dried carbonyl reductase enzyme powder in 100ml of PBS buffer solution to obtain a carbonyl reductase solution, dissolve 5g of polyvinyl alcohol in 100ml of solvent deionized water to obtain a polyvinyl alcohol solution, add the polyvinyl alcohol solution to the carbonyl reductase solution to obtain a mixed solution, stir and mix evenly, gradually add 12ml of a 1mg / mL glutaraldehyde aqueous solution of a cross-linking agent in 3 batches, stir for 20 to 30min for the first addition, keep the speed at 300r / min, and then gradually extend the stirring reaction time by 10 to 15min. After the reaction is completed, centrifuge, wash with buffer solution, and dry at low temperature to obtain 5.7g of a polyvinyl alcohol film-coated carbonyl reductase product, i.e., a carbonyl reductase-polyvinyl alcohol film complex, with a fixation rate of 95.0%; Example 4 In the second step, 3.0 g of the carbonyl reductase-polyvinyl alcohol membrane complex obtained in the first step was dissolved in 100 ml of PBS buffer solvent to obtain a mixed solution, 30 ml of 1 mg / mL cross-linking agent glutaraldehyde was added, 15 g of PEI@Fe3O4 magnetic nanoparticles with a particle size of 20-30 nm were added, low-frequency ultrasonic mixing was performed for 10-15 min, the nanoparticles were filtered out, washed with buffer 2-3 times, and dried to obtain 11.7 g of the final product immobilized carbonyl reductase, with an immobilization rate of 97.5%; Example 5 In the second step, 2.0 g of the carbonyl reductase-polyvinyl alcohol membrane complex obtained in the first step was dissolved in 100 ml of PBS buffer solvent to obtain a mixed solution, 30 ml of 1 mg / mL cross-linking agent glutaraldehyde was added, 10 g of PEI@Fe3O4 magnetic nanoparticles with a particle size of 20-30 nm were added, low-frequency ultrasonic mixing was performed for 10-15 min, the nanoparticles were filtered out, washed with buffer for 2-3 times, and dried to obtain 11.4 g of the final product immobilized carbonyl reductase with an immobilization rate of 95.0%.

[0019] The immobilization rates of the immobilized carbonyl reductases in the examples of the present invention were all higher than 90%, and the catalytic activity remained higher than 90% after repeated use for 5 times.

[0020] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0021] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A carbonyl reductase immobilization process, characterized in that The following steps are involved: The first step is to dissolve the freeze-dried carbonyl reductase enzyme powder in a buffer solution to obtain a carbonyl reductase solution, dissolve polyvinyl alcohol in a solvent to obtain a polyvinyl alcohol solution, add the polyvinyl alcohol solution to the carbonyl reductase solution to obtain a mixed solution, stir and mix evenly, gradually add cross-linking agent glutaraldehyde in batches, stir for 20 to 30 minutes for the first addition, and then gradually extend the stirring reaction time by 10 to 15 minutes. After the reaction is completed, centrifuge to obtain a polyvinyl alcohol film-coated carbonyl reductase, that is, a carbonyl reductase-polyvinyl alcohol film complex; In the second step, the carbonyl reductase-polyvinyl alcohol membrane complex obtained in the first step is dissolved in a buffer solvent to obtain a mixed solution, a cross-linking agent glutaraldehyde is added, magnetic nanoparticles are added, low-frequency ultrasonic mixing is performed for 10 to 15 minutes, the nanoparticles are filtered out, washed with a buffer solution, and dried to obtain the final product, immobilized carbonyl reductase.

2. A carbonyl reductase immobilization process according to claim 1, characterized in that: The buffer solutions used in the first step and the second step are both PBS.

3. A carbonyl reductase immobilization process according to claim 1, characterized in that: The solvent is deionized water.

4. The carbonyl reductase immobilization process according to claim 1, characterized in that: The concentration of the carbonyl reductase in the buffer is 5-10 mg / mL.

5. The carbonyl reductase immobilization process according to claim 1, characterized in that: The concentration of the polyvinyl alcohol in the solvent is 25-50 mg / mL.

6. A carbonyl reductase immobilization process according to claim 1, characterized in that: In the first step, the amount of the cross-linking agent used is 0.5-1.2 wt % of the carbonyl reductase.

7. A carbonyl reductase immobilization process according to claim 1, characterized in that: The cross-linking agent is a 1 mg / mL glutaraldehyde aqueous solution, and the cross-linking agent is added to the mixed solution in 3 to 5 times in the first step.

8. The carbonyl reductase immobilization process according to claim 1, characterized in that: The second-step cross-linking agent is a 1 mg / mL glutaraldehyde aqueous solution, and the amount of the second-step cross-linking agent is 1-1.5 wt % of the carbonyl reductase-polyvinyl alcohol membrane complex.

9. A carbonyl reductase immobilization process according to claim 1, characterized in that: In the second step, the concentration of the mixed solution of carbonyl reductase-polyvinyl alcohol membrane complex is 20-30 mg / mL.

10. The carbonyl reductase immobilization process according to claim 1, characterized in that: In the second step, the nanoparticles are PEI@Fe3O4 magnetic nanoparticles, the particle size of the nanoparticles is 20-30 nm, and the mass ratio of the nanoparticles to the carbonyl reductase-polyvinyl alcohol film complex is 1:5.