Lipase immobilization process

Abstract

The invention discloses a lipase immobilization process which comprises the following steps: step 1, dissolving lipase through a phosphate buffer solution 1 * PBS, adding an ionic liquid for mixing, adding a carbodiimide cross-linking agent, and selectively loading on the ionic liquid through carboxyl to obtain a lipase-ionic liquid compound; dissolving the substance obtained in the first step into a buffer solution, adding a cross-linking agent, adding nano-particles, stirring and mixing at room temperature, and carrying out ultrasonic treatment, so that the substance is loaded on the nano-particles. The method has the beneficial effects that the use of the ionic liquid can improve the thermal stability and chemical stability of the enzyme and prolong the service life of the enzyme, and the combination of the ionic liquid and the nanoparticles can improve the catalytic activity of the enzyme and improve the substrate conversion rate; the compounded enzyme can be repeatedly used in a plurality of reaction periods, the operation cost is reduced, the immobilized enzyme is easily separated from a reaction mixture through magnetic separation and other methods due to the existence of the nanoparticles, and the subsequent treatment is simplified.

Description

Technical Field

[0001] The invention relates to the technical field related to immobilized enzymes, in particular to a lipase immobilization process. Background Art

[0002] As an efficient and specific biocatalyst, enzymes play an important role in the development of industrial chemistry in the 21st century. This is attributed to the advantages of enzymes such as specificity, mild reaction conditions, low pollution, and high selectivity. Enzymes play a vital role in fields closely related to human life, such as medicine, energy, food, and organic synthesis. Traditional free enzymes have problems such as easy inactivation, poor dispersibility, difficulty in recycling, and difficulty in continuous use in applications. Enzyme immobilization technology is one of the effective measures to solve these problems. Immobilized enzymes not only retain the catalytic properties of enzymes, but also improve the dispersibility and operational stability of enzymes, and can also achieve enzyme recycling and continuous utilization, thereby reducing the input cost of enzymes in production.

[0003] In recent years, the study of enzyme catalysis in ionic liquids has gradually attracted attention. The stability, activity and selectivity of many enzymes in ionic liquids are higher than those in traditional organic solvents; the unique solubility of ionic liquids enables processes that were originally insoluble and unable to react in traditional organic solvents to proceed; the catalytic conditions can be controlled by changing the structure of ionic liquids; at the same time, ionic liquids have no vapor pressure, product separation is easier than traditional organic solvents, and the process is greener. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the traditional free enzyme, the present invention aims to provide a lipase immobilization process, which combines ionic liquids and nanoparticles to immobilize lipase, so that the stability, catalytic ability and preservation ability of lipase are greatly improved, the lipase can be reused, the separation is simple, and the reaction selectivity is improved. The technical solution of the present invention is as follows: A lipase immobilization process comprises the following steps: In the first step, the lipase is dissolved in phosphate buffer 1×PBS, an ionic liquid is added for mixing, a carbodiimide cross-linking agent is added, and the mixture is stirred at room temperature for 12 to 16 hours, and the lipase-ionic liquid complex is obtained by selectively loading the ionic liquid with the carboxyl group; In the second step, the lipase-ionic liquid complex obtained in the first step is dissolved in a buffer solution, nanoparticles are added, stirred and mixed at room temperature, and subjected to 20-30 kHz ultrasonic treatment for 5-10 min in an ice bath at 0°C to load the lipase-ionic liquid complex onto the nanoparticles.

[0005] Furthermore, the ionic liquid is 1-butyl-3-methylimidazolium tetrafluoroborate [Bmim] BF4 or 1-ethyl-3-methylimidazolium trifluoromethanesulfonate [EMIm][Tf2N].

[0006] Furthermore, the nanoparticles are ferroferric oxide nanoparticles, and the particle size of the ferroferric oxide nanoparticles is 20 to 30 nm.

[0007] Furthermore, the mass ratio of the lipase to the ionic liquid is 1:5-6.

[0008] Furthermore, the mass ratio of the ferrosoferric oxide nanoparticles to the ionic liquid immobilized enzyme obtained by the first step reaction is 1:5.

[0009] Furthermore, the carbodiimide is one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chloride (EDC-HCl), 1-ethyl-3-(4-fluorophenyl)-3-dimethylaminopropylcarbodiimide (such as EDC-F) or 1-ethyl-3-(4-nitrophenyl)-3-dimethylaminopropylcarbodiimide (such as EDC-NO2).

[0010] Furthermore, the amount of the cross-linking agent used in the first step is 5wt% of the amount of the lipase used.

[0011] Furthermore, the second step reaction and the buffer solution used in the second step reaction are both phosphate buffered saline 1×PBS.

[0012] Furthermore, the pH of the buffer solution used in the first step is 5.6-6.3.

[0013] Furthermore, the pH of the buffer solution used in the second step is 5.3-6.5.

[0014] The beneficial effects of the present invention are: 1. The use of ionic liquid in the present invention can improve the thermal stability and chemical stability of the enzyme and extend its service life. The combination of ionic liquid and nanoparticles may enhance the catalytic activity of the enzyme and improve the substrate conversion rate; 2. The composite enzyme can be reused in multiple reaction cycles, reducing operating costs. The presence of nanoparticles makes it easy to separate the immobilized enzyme from the reaction mixture by magnetic separation and other methods, simplifying subsequent treatment. 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 making creative work are within the scope of protection of the present invention.

[0016] The lipase activity used in the embodiment of the present invention is 2000-3000 U / g.

[0017] In the first step, 2 g of lipase was dissolved and diluted in 100 ml of phosphate buffer 1×PBS (adjusted with dilute hydrochloric acid to pH 5.6-6.3), 10 g of ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate [Bmim] BF4 was added for mixing, 0.1 g of carbodiimide crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was added, and the mixture was stirred and mixed at room temperature for 12-16 hours. The ionic liquid was selectively loaded with carboxyl groups, and centrifuged to obtain a lipase-ionic liquid complex. In the second step, 2 g of the lipase-ionic liquid complex obtained in the first step is dissolved in 100 ml of a buffer solution, phosphate buffer 1×PBS (pH adjusted to 5.3-6.5 with dilute hydrochloric acid), 10 g of ferroferric oxide nanoparticles with a particle size of 20-30 nm are added, the mixture is stirred at room temperature, and ultrasonic treatment is performed at 20-30 kHz for 5-10 min in an ice bath at 0°C to load the lipase-ionic liquid complex onto the nanoparticles. The mixture is centrifuged, the loaded nanoparticles are washed with a buffer solution, and 11.9 g of a final product, a lipase-ionic liquid complex-loaded ferroferric oxide nanoparticles, is obtained after drying.

[0018] In the first step, 2 g of lipase was dissolved and diluted in 100 ml of phosphate buffer 1×PBS (pH adjusted to 5.6-6.3 with dilute hydrochloric acid), 12 g of ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate [Bmim] BF4 was added for mixing, 0.1 g of carbodiimide crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was added, and the mixture was stirred and mixed at room temperature for 12-16 h. The ionic liquid was selectively loaded with carboxyl groups, and centrifuged to obtain a lipase-ionic liquid complex. In the second step, 2 g of the lipase-ionic liquid complex obtained in the first step is dissolved in 100 ml of a buffer solution, phosphate buffer 1×PBS (pH adjusted to 5.3-6.5 with dilute hydrochloric acid), 10 g of ferroferric oxide nanoparticles with a particle size of 20-30 nm are added, the mixture is stirred at room temperature, and ultrasonic treatment is performed at 20-30 kHz for 5-10 min in a 0°C ice bath to load the lipase-ionic liquid complex onto the nanoparticles. The nanoparticles are then centrifuged and washed with a buffer solution. After drying, 11.7 g of a final product, a lipase-ionic liquid complex-loaded ferroferric oxide nanoparticles, is obtained.

[0019] In the first step, 2 g of lipase is dissolved and diluted in 100 ml of phosphate buffer 1×PBS (dilute hydrochloric acid is used to adjust the pH to 5.6-6.3), 10 g of ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate [Bmim] BF4 is added for mixing, 0.1 g of carbodiimide crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide chloride (EDC-HCl) is added, and the mixture is stirred and mixed at room temperature for 12-16 hours. The ionic liquid is selectively loaded with carboxyl groups, and centrifuged to obtain a lipase-ionic liquid complex. In the second step, 2 g of the lipase-ionic liquid complex obtained in the first step is dissolved in 100 ml of a buffer solution, phosphate buffer 1×PBS (pH adjusted to 5.3-6.5 with dilute hydrochloric acid), 10 g of ferroferric oxide nanoparticles with a particle size of 20-30 nm are added, the mixture is stirred at room temperature, and ultrasonic treatment is performed at 20-30 kHz for 5-10 min in a 0°C ice bath to load the lipase-ionic liquid complex onto the nanoparticles. The nanoparticles are then centrifuged and washed with a buffer solution. After drying, 11.6 g of a final product, a lipase-ionic liquid complex-loaded ferroferric oxide nanoparticles, is obtained.

[0020] In the first step, 2 g of lipase was dissolved and diluted in 100 ml of phosphate buffer 1×PBS (pH adjusted to 5.6-6.3 with dilute hydrochloric acid), 10 g of ionic liquid 1-ethyl-3-methylimidazolium trifluoromethanesulfonate [EMIm][Tf2N] was added for mixing, 0.1 g of carbodiimide crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was added, and the mixture was stirred and mixed at room temperature for 12-16 h. The ionic liquid was selectively loaded with carboxyl groups, and centrifuged to obtain a lipase-ionic liquid complex. In the second step, 2 g of the lipase-ionic liquid complex obtained in the first step is dissolved in 100 ml of a buffer solution, phosphate buffer 1×PBS (pH adjusted to 5.3-6.5 with dilute hydrochloric acid), 10 g of ferroferric oxide nanoparticles with a particle size of 20-30 nm are added, the mixture is stirred at room temperature, and ultrasonic treatment is performed at 20-30 kHz for 5-10 min in an ice bath at 0°C to load the lipase-ionic liquid complex onto the nanoparticles. The nanoparticles are then centrifuged and washed with a buffer solution. After drying, 11.8 g of a final product, a lipase-ionic liquid complex-loaded ferroferric oxide nanoparticles, is obtained.

[0021] After testing, it was measured under the conditions of 37°C and pH 7.0-8.0 with octyl octanoate as the substrate. The affinity of the lipase-ionic liquid complex loaded with ferrosoferric oxide nanoparticles prepared in the embodiment of the present application to the reaction substrate was 3 to 4 times that of the free enzyme system, and the reaction activity was still maintained above 85% after repeated use for 5 times; after testing again one week later, the reaction activity was higher than 90%, there was no obvious precipitation or color change, the pHp was maintained within the initial setting range, fluctuated within ±0.2 pH units, and the total protein content should be close to the initial value, so it was determined that the product of the present invention had good stability.

[0022] 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.

[0023] 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 lipase immobilization process, characterized in that The following steps are involved: In the first step, the lipase is dissolved in phosphate buffer 1×PBS, an ionic liquid is added for mixing, a carbodiimide cross-linking agent is added, and the mixture is stirred at room temperature for 12 to 16 hours, and the lipase-ionic liquid complex is obtained by selectively loading the ionic liquid with the carboxyl group; In the second step, the lipase-ionic liquid complex obtained in the first step is dissolved in a buffer solution, nanoparticles are added, stirred and mixed at room temperature, and subjected to 20-30 kHz ultrasonic treatment for 5-10 min in an ice bath at 0°C to load the lipase-ionic liquid complex onto the nanoparticles.

2. A lipase immobilization process according to claim 1, characterized in that: The ionic liquid is 1-butyl-3-methylimidazolium tetrafluoroborate [Bmim] BF4 or 1-ethyl-3-methylimidazolium trifluoromethanesulfonate [EMIm][Tf2N].

3. A lipase immobilization process according to claim 1, characterized in that: The nanoparticles are ferroferric oxide nanoparticles, and the particle size of the ferroferric oxide nanoparticles is 20-30 nm.

4. A lipase immobilization process according to claim 1, characterized in that: The mass ratio of the lipase to the ionic liquid is 1:5-6.

5. A lipase immobilization process according to claim 1, characterized in that: The mass ratio of the ferrosoferric oxide nanoparticles to the ionic liquid immobilized enzyme obtained by the first step reaction is 1:

5.

6. A lipase immobilization process according to claim 1, characterized in that: The carbodiimide is one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chloride (EDC-HCl), 1-ethyl-3-(4-fluorophenyl)-3-dimethylaminopropylcarbodiimide (such as EDC-F) or 1-ethyl-3-(4-nitrophenyl)-3-dimethylaminopropylcarbodiimide (such as EDC-NO2).

7. A lipase immobilization process according to claim 1, characterized in that: The amount of the cross-linking agent used in the first step is 5wt% of the amount of the lipase used.

8. The lipase immobilization process according to claim 1, characterized in that: The buffer solution used in the second step reaction and the second step reaction is phosphate buffer 1×PBS.

9. A lipase immobilization process according to claim 1, characterized in that: The pH of the buffer solution used in the first step is 5.6-6.

3.

10. A lipase immobilization process according to claim 1, characterized in that: The pH of the buffer solution used in the second step is 5.3-6.5.