A method for synthesizing protein backbone nanomaterials and its application in immobilized enzymes
By synthesizing protein skeleton nanomaterials as immobilized enzyme carriers, the problems of easy inactivation and poor reusability of enzymes in organic phase reactions are solved, and the efficient solid loading and stability of enzymes are improved, with environmental protection and low cost advantages.
Patent Information
- Application Number
- CN202111649442.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-12-30
- Publication Date
- 2025-06-20
- Estimated Expiration
- 2041-12-30
AI Technical Summary
In the prior art, enzymes are difficult to play a role in organic phase reactions, are prone to inactivation, have poor reusability, and the rigid structure of traditional nanomaterials inhibits the activity of enzymes.
Synthetic protein skeleton nanomaterials were used as carriers for immobilizing enzymes, and nanomaterials were obtained by reaction at room temperature and gradient centrifugation were washed. Combined with the use of Tris-HCl buffer solution and organic reagents, a stable enzyme carrier was formed.
It improves the solid loading and stability of the enzyme, reduces the inactivation rate of the enzyme, enhances the reusability of the enzyme, and avoids the generation of by-products in traditional methods, and has the advantages of environmental protection and low cost.
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Figure CN114214309B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of nano material preparation, and specifically relates to a method for synthesizing protein skeleton nano material and application of the method on immobilized enzyme. Background Art
[0002] Lipase is one of the important industrial enzyme preparations. It can catalyze reactions such as lipolysis, ester exchange, and ester synthesis. It is widely used in oil processing, food, medicine, daily chemical and other industries. Lipases from different sources have different catalytic characteristics and catalytic activities. Among them, the large-scale production of lipases with transesterification or esterification functions used for organic phase synthesis is of great significance for the enzyme-catalyzed synthesis of chiral compounds of fine chemicals. At present, most esterification reactions are high-temperature acid-base catalysis, with toxic by-products, harsh reaction conditions, high energy consumption, and environmental pollution. Enzymatic synthesis can overcome the above shortcomings, with mild reaction conditions, low energy consumption, strong catalytic reaction specificity, and is not easy to produce by-products. It is environmentally friendly and is the development direction of the green chemical industry. The common problems of free enzymes are that they are difficult to play a role in organic phase reactions, are easily inactivated, and have poor reusability. The common solution is to immobilize the enzyme. After the enzyme is immobilized, the stability is generally increased, it is easy to separate from the reaction system, and it is easy to control. It can be used repeatedly, easy to transport and store, and is conducive to automated production.
[0003] Nanomaterials have a large specific surface area. As immobilized materials, nanomaterials have great advantages. They are easy to immobilize enzymes, increase the enzyme loading capacity, can be quickly separated for reuse, and have good biocompatibility, such as MOF and COF. However, the traditional nanomaterial structure is rigid and has a certain inhibitory effect on enzyme activity. Summary of the invention
[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for synthesizing protein skeleton nanomaterials and application thereof in immobilized enzymes.
[0005] A method for synthesizing a protein skeleton nanomaterial comprises the following steps:
[0006] Step 1: Take 1 mL of 10-100 mg / mL protein solution at room temperature, add 6 mL of DMSO at 50-300 rpm, and then add a pH of 7, 50-75 mmol / L Tris-HCl buffer solution. After a white precipitate is formed, increase the speed to 300-600 rpm and react for 30 min. After the reaction is completed, place the reaction system at 4 ° C for 12 h to obtain a white suspension, wherein the volume of the Tris-HCl buffer solution must be greater than 3% of the total volume of the reaction system;
[0007] Step 2: Centrifuge the white suspension at a speed gradient of 4000 - 8000 rpm for 10 min. After discarding the supernatant, wash it 3 times with an equal volume of organic reagent or water. Each washing is centrifuged and washed at 4000 - 8000 rpm for 5 min to obtain the protein skeleton nanomaterial.
[0008] As an improvement, the concentration of the protein solution in Step 1 is 60 mg / mL, and the concentration of the Tris-HCl buffer solution in Step 1 is 50 mmol / L. The Tris-HCl buffer solution plays a role in initiating the reaction. As an improvement, the initial rotation speed in Step 1 is 200 rpm and is increased to 400 rpm.
[0009] As an improvement, the rotation speed used for centrifugation in Step 2 is 6000 rpm. If the speed is too high, it is difficult to wash.
[0010] As an improvement, the organic reagent used for washing in Step 2 is methanol, DMF, dimethyl sulfoxide, DMI, or hexamethyl phthalic triamide.
[0011] As an improvement, the protein in Step 1 is ovalbumin, whey protein, or bovine serum albumin.
[0012] Based on the above application of the protein skeleton nanomaterial in immobilized enzymes, the enzyme of the immobilized enzyme is lipase, L-threonine aldehyde carboxylase, or glucose oxidase.
[0013] As an improvement, the concentration of the immobilized enzyme is 0.05 - 2.0 g / L in the whole system.
[0014] Beneficial effects:
[0015] Compared with the prior art, the method for synthesizing a protein skeleton nanomaterial and its application in immobilized enzymes of the present invention have the following advantages:
[0016] (1) The carrier preparation process is simple and the cost is low. The reaction process basically only requires simple stirring, and the reactants can be bought on the market and the prices are low;
[0017] (2) It can have a certain immobilization effect on a variety of enzymes. It does not contain metal ions and will not inhibit the activity of enzymes such as decarboxylase, which has certain significance for production.
[0018] (3) No toxic and harmful by-products are generated during the preparation process, and the preparation process is environmentally friendly;
[0019] (4) The protein skeleton nanomaterial can be separated from the product after centrifugation, which is convenient for product separation and detection and is conducive to the reuse of enzymes. Description of the drawings
[0020] Figure 1 Enzymatic properties of immobilized lipase obtained by different treatment methods, where (a) is the immobilization rate curve of lipase, (b) is the effect of pH of Tris-HCl buffer on lipase activity, (c) is the effect of the percentage of organic reagent in the total system on it, (d) is the reusability of the enzyme; (e) is the effect of the concentration of Tris-HCl buffer solution on enzyme activity;
[0021] Figure 2 SEM characterization of protein framework nanomaterials from 0 to 30 min starting from the addition of Tris buffer in the synthesis of Example 1;
[0022] Figure 3 Infrared spectra of ovalbumin and protein framework nanomaterials in Example 1 of the present invention;
[0023] Figure 4 TEM image of the immobilized lipase in Example 5 of the present invention;
[0024] Figure 5 TGA-DTA curve of the protein framework nanomaterials in Example 1 of the present invention;
[0025] Figure 6 Effect of different immobilization materials on the production of L-TA. Detailed implementation manners
[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.
[0027] A method for synthesizing protein framework nanomaterials, comprising the following steps:
[0028] Step 1, at room temperature, take 1 mL of a protein solution at 10 - 100 mg / mL, add 6 mL of DMSO at 50 - 300 rpm, and then add a Tris-HCl buffer solution with a pH of 7 and a concentration of 50 - 75 mmol / L. After forming a white precipitate, increase the rotation speed to 300 - 600 rpm and react for 30 min. After the reaction is completed, place the reaction system at 4°C and let it stand for 12 hours to obtain a white suspension. Among them, the volume of the Tris-HCl buffer solution needs to be greater than 3% of the total volume of the reaction system;
[0029] Step 2: Centrifuge the white suspension at a speed gradient of 4000 - 8000 rpm for 10 min. After discarding the supernatant, wash it 3 times with an equal volume of organic reagent or water. Each wash is centrifuged and cleaned at 4000 - 8000 rpm for 5 min to obtain the protein framework nanomaterial.
[0030] Among them, the concentration of the protein solution in Step 1 is 60 mg / mL, the concentration of the Tris-HCl buffer solution in Step 1 is 50 mmol / L, and the Tris-HCl buffer solution plays a role in initiating the reaction.
[0031] The initial rotation speed in Step 1 is 200 rpm and is increased to 400 rpm.
[0032] The rotation speed used for centrifugation in Step 2 is 6000 rpm. If the speed is too high, it is difficult to wash.
[0033] The organic reagent used for washing in Step 2 is methanol, DMF, dimethyl sulfoxide, DMI, or hexamethylphthalimide.
[0034] The protein mentioned in Step 1 is ovalbumin, whey protein, or bovine serum albumin. Example 1
[0035] A method for synthesizing a protein framework nanomaterial, comprising the following steps:
[0036] Step 1: At room temperature, take 1 mL of ovalbumin solution at 60 mg / mL, add 6 mL of DMSO at 200 rpm, and finally add 4 mL of Tris-HCl buffer solution with a concentration of 50 mmol / L and pH = 7. After forming a white precipitate, increase the rotation speed to 400 rpm and react for 30 min. After the reaction is completed, let the reaction system stand at 4°C for 12 h to obtain a white suspension. The Tris-HCl buffer solution plays a role in initiating the reaction.
[0037] Step 2: Centrifuge the white suspension at a speed gradient of 6000 rpm for 10 min. After discarding the supernatant, wash it 3 times with an equal volume of water. Each wash is centrifuged and cleaned at 4000 - 8000 rpm for 5 min to obtain the protein framework nanomaterial. If the speed is too high, it is difficult to wash.
[0038] During the preparation process, SEM characterization of the protein framework nanomaterial was carried out from 0 to 30 min starting from the addition of tris buffer solution. The results are as Figure 2 shown, and the time line is recorded from left to right and top to bottom in the figure.
[0039] Through infrared determination of ovalbumin and the protein framework nanomaterial, the results are as Figure 3 shown.
[0040] The protein backbone nanomaterials were analyzed, and the TGA-DTA curves are as Figure 5 shown (detected by Juanke Testing Company).
[0041] In order to better characterize the protein backbone nanomaterials of Example 1 of the present invention, a series of determinations were specifically carried out.
[0042] Take the protein backbone nanomaterials prepared in Example 1, disperse them with an equal volume of water, and then add lipase to make the final concentration of the system 0.1 g / l, and then measure the following data.
[0043] The adsorption and immobilization rate of lipase over time is as Figure 1 (a) shown. The adsorption and immobilization rate increases over time until 4 h and then remains unchanged.
[0044] With other conditions unchanged, change the pH of the Tris-HCl buffer used to prepare the material, and set the pH gradient to 3-10. The influence on enzyme activity is as Figure 1 (b) shown. The Tris-HCl buffer with pH = 7 is preferred.
[0045] The recycling rate of the lipase immobilized with this material is as Figure 1 (c) shown. Take 1 mmol / L, 0.8 ml PNPP substrate solution, and then add 0.2 ml of the immobilized lipase dispersed with water to a concentration of 0.1 g / L to form a 1 mmol / L catalytic system. React at 45 °C for 15 minutes, then centrifuge at 6000 rpm to remove the supernatant. Re-add 0.2 ml of the dispersion with water, add 0.8 ml of the PNPP substrate solution with a concentration of 1 mmol / L for the second reaction, and centrifuge again. Repeat the operations of centrifuging and adding the substrate 6 times, and then test the enzyme activity of the immobilized enzyme. The remaining enzyme activity after six repeated uses is 58.32%. Example 2
[0046] In order to compare the effects of Tris-HCl buffer solutions with different concentrations on the formation of the material, the concentration of the Tris-HCl buffer solution in Step 1 was specifically set to 25 mmol / L, 50 mmol / L, 75 mmol / L, 100 mmol / L, 125 mmol / L, and other conditions were the same as in Example 1.
[0047] From the experimental results, it can be seen that when the concentration of Tris-HCl buffer solution is too low, it cannot be formed smoothly. Although the amount of prepared material increases at high concentration, a large part of the material is empty material, and too high concentration affects the activity of the enzyme itself. When the concentration is 50 mmol / L-75 mmol / L, the synthesis of protein skeleton nanomaterials is more stable. Among them, when the Tris-HCl buffer solution is 50 mmol / L, the performance of the protein skeleton nanomaterial is more excellent. The results are as follows Figure 1 (d) as shown. Example 3
[0048] The above contents are the same as those in Example 1 except that “after the reaction is completed, the reaction system is allowed to stand at 4° C. for 12 h”.
[0049] We found that the protein skeleton nanomaterial prepared in Example 3 is unstable in the aqueous phase and will decompose by itself. Methanol is often used for preservation, but methanol preservation will reduce the enzyme activity of the immobilized enzyme after application.
[0050] In the synthesis process of Example 1 of the present invention, the original system was allowed to stand for 12 h before use. The protein content of the solution after washing was measured by the Coomassie Brilliant Blue method. The protein content in the supernatant was 0.05 mg / ml, and the protein concentration of the total system before synthesis was 5.5 mg / ml, with a loss rate of less than 1%. Therefore, the synthesis method of the present invention can ensure the stability of the material, which is not affected by water and has a high stability in the aqueous phase. Example 4
[0051] Free enzyme reaction system: add 400 μL of 2.5 mol / L glycine, 60 μL of 1 mol / L 3,4-dihydroxybenzaldehyde, 20 μL of 1 mmol / L PLP, add 500 μL of 2 g / L L-TA, and finally add 20 μL PBS buffer to supplement the system to 1 ml.
[0052] Immobilized enzyme reaction system: add 400 μL of 2.5 mol / L glycine, 60 μL of 1 mol / L 3,4-dihydroxybenzaldehyde, 20 μL of 1 mmol / L PLP, and 500 μL of immobilized L-threonine aldolase (the preparation method of immobilized L-threonine aldolase, the specific steps are as follows: after the protein skeleton nanomaterial is centrifuged and washed, the same volume of PBS is added to disperse it, and then an equal volume of 4 g / L L-threonine aldolase is added. It is stirred at a constant temperature of 25 ° C for 12 h at a speed of 300 rpm, and then washed three times with PBS buffer and dispersed with an equal volume of PBS. The final enzyme concentration is 2 mg / ml), and finally 20 μL of PBS buffer is added to supplement the system to 1 ml.
[0053] The biocatalytic method performs aldol condensation through L-threonine aldolase to generate L-threo-DOPS (droxidopa) and L-erythro-DOPS. After the synthetic material in Example 1 is washed, the enzyme is immobilized on this material by the direct adsorption method (stirring at room temperature for 12 h). In the 4-hour reaction, the yield of droxidopa is increased by 56% compared with the free enzyme, and it has better effects than other immobilized materials with the same enzyme concentration (such as ZIF-8, ZIF-67, ZIF-Cu, sodium deoxycholate), which has certain significance for production. (As Figure 6 shown) Example 5
[0054] After the protein skeleton nanomaterial is centrifuged and washed, the same volume of water is added for dispersion, and then 1.2 g / l lipase is added. It is evenly stirred on a magnetic stirrer at 25 °C for 4 hours at a rotation speed of 400 rpm, and then centrifuged and washed 3 times with the same volume of methanol. The final enzyme concentration is 0.1 mg / mL. The immobilized lipase is characterized, and the results are as Figure 4 shown.
[0055] The above is only the preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any simple change or equivalent replacement of the technical solution that can be obviously obtained by those skilled in the art within the technical scope disclosed by the present invention falls within the protection scope of the present invention.
Claims
1. A method for synthesizing a protein scaffold nanomaterial, characterized in that, It includes the following steps: Step 1: At room temperature, take 1 mL of a protein solution with a concentration of 10 - 100 mg / mL, add 6 mL of DMSO at 50 - 300 rpm, and then add a Tris-HCl buffer solution with a pH of 7 and a concentration of 50 - 75 mmol / L. After a white precipitate forms, increase the rotation speed to 300 - 600 rpm and react for 30 min. After the reaction is completed, let the reaction system stand at 4 °C for 12 h to obtain a white suspension. Among them, the volume of the Tris-HCl buffer solution should be greater than 3% of the total volume of the reaction system. The protein is ovalbumin; Step 2: Centrifuge the white suspension at a speed gradient of 4000 - 8000 rpm for 10 min. After discarding the supernatant, wash it 3 times with an equal volume of an organic reagent or water. Each washing is centrifuged and cleaned at 4000 - 8000 rpm for 5 min to obtain a protein framework nanomaterial.
2. The method for synthesizing a protein scaffold nanomaterial according to claim 1, characterized in that, In Step 1, the concentration of the protein solution is 60 mg / mL, and the concentration of the Tris-HCl buffer solution described in Step 1 is 50 mmol / L.
3. The method for synthesizing a protein scaffold nanomaterial according to claim 1, characterized in that, In Step 1, the initial rotation speed is 200 rpm, which is increased to 400 rpm.
4. The method for synthesizing a protein scaffold nanomaterial according to claim 1, characterized in that, In Step 2, the rotation speed used for centrifugation is 6000 rpm.
5. The method for synthesizing a protein scaffold nanomaterial according to claim 1, characterized in that, The organic reagent used for washing in Step 2 is methanol, DMF, dimethyl sulfoxide, DMI, or hexamethyl phthalimide.
6. The application of the protein scaffold nanomaterial prepared based on claim 1 in immobilized enzymes, characterized in that, The enzyme of the immobilized enzyme is L-threonine aldolase.
7. The application according to claim 6, characterized in that, The concentration of the immobilized enzyme is 0.05 - 2.0 g / L in the whole system.