Method for directionally immobilizing proteolytic enzyme through biomimetic ligand
By constructing the nano-film bioimitative ligand g-C3N4 and using the alkali-soluble-immobilized complex enzyme combination method, the problems of low enzymatic efficiency and high production cost in walnut peptide production were solved, and efficient directed immobilization of enzymes and the improvement of biocatalytic efficiency were achieved.
Patent Information
- Application Number
- CN202411874582.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-03-28
AI Technical Summary
In the production process of walnut peptides, the enzymatic lysis efficiency is limited by the low solubility of walnut protein, the hydrolysis degree is difficult to accurately control, and the free enzyme is difficult to recover, resulting in an increase in production costs.
By constructing the nano-film biosimilar ligand g-C3N4, multiple forces are generated with the functional groups of the protein, and the directional immobilization of the proteolytic enzyme is achieved, and the crude protein of walnut meal is hydrolyzed by the combined alkali-soluble-immobilized complex enzyme.
It achieves better chemical and thermal stability of enzymes, enhanced enzyme activity and reusability, reduces industrial application costs, and improves biocatalytic efficiency.
Smart Images
Figure CN119709721A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of bioengineering, relates to a method for synthesizing a biomimetic ligand and co-immobilizing multiple enzymes, and in particular to a method for directional immobilization of a proteolytic enzyme with a biomimetic ligand. Background Art
[0003] In the production process of walnut peptides, the enzymatic hydrolysis process plays a core role, but it encounters a series of technical challenges in actual operation. These challenges include the low solubility of walnut protein, which limits the efficiency of enzymatic hydrolysis, the difficulty in accurately controlling the degree of hydrolysis, and the difficulty in recovering free enzymes, which leads to increased production costs. The application of immobilized enzyme technology has been proven to reduce production costs while enhancing the thermal stability, acid-base stability and storage stability of the enzyme, which is of great significance for the industrial production of walnut peptides.
[0004] Common immobilization techniques include physical adsorption, covalent bonding, embedding and copolymer methods. These methods have their own advantages, but also limitations. In practice, it is often necessary to use a variety of immobilization techniques in combination to achieve the best results. Through the synergistic effect of this method, the limitations of a single method can be overcome, and the enzymatic efficiency in the production process of walnut peptides can be maximized, while reducing costs and improving product quality. The optimization of this comprehensive application strategy will further promote the development of the walnut peptide industry and make it more widely used in the field of functional foods.
[0005] The present invention relates to the design and synthesis of an innovative nano-film bionic ligand g-C3N4. Relying on the uniqueness of its triazine ring structure, the material realizes efficient directional immobilization of proteins through multiple interaction forces such as hydrogen bonding, π-π stacking, and hydrophobic interaction. As a carrier of immobilized enzyme technology, g-C3N4 exhibits multiple advantages such as excellent chemical and thermal stability, excellent biocompatibility, significantly enhanced enzyme activity, convenient recycling and reuse, improved enzyme stability, optimized orientation, enhanced substrate affinity, regulatable enzyme activity, and environmental friendliness. These characteristics indicate that g-C3N4 is expected to become a powerful tool for improving biocatalytic efficiency and reducing industrial application costs, bringing innovative solutions to the field of bioengineering. Summary of the invention
[0006] In view of the shortcomings of the existing preparation process, the present invention proposes a method for directional immobilization of proteolytic enzymes by biomimetic ligands. By constructing a nano-film biomimetic ligand g-C3N4, the multiple forces generated between the biomimetic ligand and the functional groups of the protein are used to achieve directional immobilization of the proteolytic enzyme, giving the immobilized enzyme better chemical and thermal stability, enhanced enzyme activity and reusability, effectively reducing the cost of industrial applications and improving the efficiency of biocatalysis.
[0007] In order to achieve the above object, the technical solution adopted by the present invention is:
[0008] A method for immobilizing a biomimetic ligand directed proteolytic enzyme, wherein the method firstly prepares a biomimetic ligand g-C3N4, and immobilizes the enzyme molecule in a buffer solution with a suitable pH by means of adaptive assembly; secondly, hydrolyzing walnut cake crude protein by means of an alkali-soluble-immobilized composite enzyme combination to prepare walnut peptides; specifically comprises the following steps:
[0009] The first step is to prepare the biomimetic ligand g-C3N4;
[0010] The crucible containing urea is placed in a tubular muffle furnace, heated to 500-600°C at a heating rate of 5-8°C / min, and the temperature is maintained for 2-5 hours; then, the obtained solid product is ground and washed alternately with deionized water and anhydrous ethanol and dried to obtain a bionic ligand g-C3N4 nanofilm material as a biomimetic ligand g-C3N4.
[0011] The second step is to prepare the immobilized enzyme;
[0012] 2.1) adding the proteolytic enzyme and the g-C3N4 material into a buffer solution to obtain a mixed solution, wherein the mass ratio of the proteolytic enzyme to the g-C3N4 material is 1:3 to 5:1;
[0013] Furthermore, the proteolytic enzyme is alkaline protease, neutral protease or a mixture of alkaline protease and neutral protease.
[0014] When the proteolytic enzyme is alkaline protease, the pH of the buffer solution is 9.0-10.0, and the solvent for preparing the buffer solution in step 2.1 includes sodium bicarbonate-sodium carbonate solution, glycine-sodium hydroxide, boric acid-borax solution;
[0015] When the proteolytic enzyme is a neutral protease, the pH of the buffer solution is 6.5-7.5, and the solvent for preparing the buffer solution in step 2.1 includes potassium dihydrogen phosphate-sodium hydroxide solution and disodium hydrogen phosphate-dipotassium hydrogen phosphate solution.
[0016] 2.2) Stir and evenly mix the mixed solution of proteolytic enzyme and g-C3N4 material at 15-30°C; then centrifuge at 0-4°C and 8000-12000 rpm for 5-15 min, collect the precipitate and wash it several times with the corresponding buffer to remove the unbound enzyme; finally, collect the precipitate and freeze-dry it to obtain the immobilized enzyme.
[0017] Furthermore, the stirring and shaking time is 30 to 120 minutes, and the stirring speed is 100 to 200 rpm / min.
[0018] The third step is to use immobilized enzyme to hydrolyze walnut cake protein;
[0019] 3.1) First, the walnut cake is defatted, homogenized and dried to prepare defatted walnut powder;
[0020] 3.2) Then, the defatted walnut powder is dispersed in deionized water, the mass of the defatted walnut powder and the deionized water is 1g:10mL~1g:15mL, and the pH value is adjusted to 9.5 to remove insoluble impurities;
[0021] 3.3) Then, the immobilized enzyme prepared in the second step is added to the filtrate of step 3.2), the mass ratio of the immobilized enzyme to the walnut protein is 1:10-20, and the mixture is stirred and hydrolyzed at 45-55°C for 2-8h;
[0022] 3.4) Finally, after the hydrolysis is completed, the enzyme activity is inactivated and the degree of protein hydrolysis is measured.
[0023] The effects and benefits of the present invention are:
[0024] (1) The present invention constructs a nano g-C3N4 film and utilizes the directional force of its unique triazine ring structural unit to achieve rapid capture and efficient directional immobilization of target protease molecules, expand the optimal pH and temperature range of the enzyme, and improve the overall reaction efficiency and product selectivity; by combining the simultaneous hydrolysis of alkali-soluble walnut cake crude protein with immobilized composite enzymes, the hydrolysis efficiency is further improved, the functional properties of the protein are improved, the anti-nutritional factors are reduced, and the process flow is simplified.
[0025] (2) The biomimetic ligand g-C3N4 used in the present invention is used as a carrier of the immobilized enzyme. g-C3N4 has good thermal stability and chemical stability, excellent biocompatibility, is non-toxic and harmless, has a simple preparation process, and has low production cost. In the process of hydrolyzing walnut cake protein with immobilized enzyme, g-C3N4 can achieve efficient directional immobilization of protein through multiple interaction forces such as hydrogen bonding, π-π stacking, and hydrophobic interaction, and at the same time has high affinity for the substrate, which can significantly enhance the enzyme activity and stability.
[0026] (3) The present invention uses the biomimetic ligand g-C3N4 as an immobilized enzyme carrier to achieve dual enzyme co-immobilization of alkaline protease and neutral protease, which not only reduces the distance between enzymes, reduces mass transfer resistance, shortens enzymatic hydrolysis time, and improves the catalytic efficiency of the enzyme, but also can obtain a higher oligopeptide yield, improve the flavor of the hydrolyzate, and enhance the biological activity of the product, which has important application value in the food industry. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1is the SEM spectrum of the nano-film biomimetic ligand g-C3N4 obtained in Example 1;
[0028] Figure 2 This is the TEM spectrum of the biomimetic ligand g-C3N4 immobilized alkaline protease obtained in Example 1.
[0029] Figure 3 This is the TEM spectrum of the biomimetic ligand g-C3N4 immobilized dual enzyme obtained in Example 5. DETAILED DESCRIPTION
[0030] The specific implementation of the present invention is described in detail below in conjunction with the technical solution (and drawings).
[0031] Example 1
[0032] The crucible containing urea was placed in a tubular muffle furnace, heated to 550°C at a heating rate of 5°C / min, and maintained at this temperature for 2h. After heating, it was naturally cooled to room temperature. The obtained solid product was ground. It was washed alternately with deionized water and anhydrous ethanol three times to remove possible impurities. The washed solid was dried to obtain the biomimetic ligand g-C3N4 nanofilm material, see attached Figure 1 .
[0033] Add 0.3g g-C3N4 material, 50ml carbonate buffer with pH 9.0 and 0.1g alkaline protease in the shake flask. Place the centrifuge tube in a shaker, set the temperature to 25℃, the speed to 200rpm, and the shake time to 120min. Then centrifuge at 4℃ and 10000rpm for 15min, collect the precipitate and wash it 3 times with the same pH 9.0 buffer to remove the unbound enzyme. Collect the precipitate and freeze-dry it to obtain the immobilized enzyme, see attached Figure 2 .
[0034] Weigh 1g of defatted walnut protein, stir it in 15mL of deionized water, and adjust the pH value to 9.5. Add 0.1g of g-C3N4 immobilized alkaline protease to the filtrate, stir and hydrolyze at 50℃ for 5h, inactivate the enzyme to terminate the hydrolysis reaction, and the degree of hydrolysis of walnut protein is 30.6% as determined by pH-stat method.
[0035] Example 2
[0036] The crucible containing urea was placed in a tubular muffle furnace, heated to 500°C at a heating rate of 8°C / min, and maintained at this temperature for 5 hours. After heating, it was naturally cooled to room temperature. The obtained solid product was ground. Deionized water and anhydrous ethanol were used to wash three times alternately to remove possible impurities. The washed solid was dried to obtain the biomimetic ligand g-C3N4 nanofilm material.
[0037] Add 0.3g g-C3N4 material, 50ml glycine buffer with pH value of 10.0 and 1.5g alkaline protease in a shake flask. Place the centrifuge tube in a shaker, set the temperature to 25℃, the speed to 200rpm, and the shake time to 30min. Then centrifuge at 4℃ and 10000rpm for 10min, collect the precipitate and wash it 3 times with the same pH value of 10.0 buffer to remove the unbound enzyme. Collect the precipitate and freeze-dry it to obtain the immobilized enzyme.
[0038] Weigh 1g of defatted walnut protein, stir it evenly in 10mL of deionized water, and adjust the pH value to 9.5. Add 0.05g of g-C3N4 immobilized alkaline protease to the filtrate, stir and hydrolyze at 45℃ for 8h, inactivate the enzyme to terminate the hydrolysis reaction, and the degree of hydrolysis of walnut protein is 27.3% as determined by pH-stat method.
[0039] Example 3
[0040] The crucible containing urea was placed in a tubular muffle furnace, heated to 600°C at a heating rate of 5°C / min, and maintained at this temperature for 2 hours. After heating, it was naturally cooled to room temperature. The obtained solid product was ground. Deionized water and anhydrous ethanol were used to wash three times alternately to remove possible impurities. The washed solid was dried to obtain the biomimetic ligand g-C3N4 nanofilm material.
[0041] Add 0.3g g-C3N4 material, 50ml phosphate buffer with pH value of 6.5 and 0.3g neutral protease in the shake flask. Place the centrifuge tube in a shaker, set the temperature to 25℃, the speed to 200rpm, and the shake time to 60min. Then centrifuge at 4℃ and 10000rpm for 10min, collect the precipitate and wash it 3 times with the same pH value of 6.5 buffer to remove the unbound enzyme. Collect the precipitate and freeze-dry it to obtain the immobilized enzyme.
[0042] Weigh 1g of defatted walnut protein, stir it evenly in 10mL of deionized water, and adjust the pH value to 6.5. Add 0.1g of g-C3N4 immobilized neutral protease to the filtrate, stir and hydrolyze at 50℃ for 3h, inactivate the enzyme to terminate the hydrolysis reaction, and the degree of hydrolysis of walnut protein is 7.8% as determined by pH-stat method.
[0043] Example 4
[0044] 0.3 g of g-C3N4 material prepared by the method of Example 1, 50 ml of phosphate buffer with a pH value of 7.5, and 0.6 g of neutral protease were added to the shaking flask. The centrifuge tube was placed in a shaking table, the temperature was set to 25 ° C, the speed was set to 200 rpm, and the shaking time was 30 min. After that, centrifugation was performed at 4 ° C and 10000 rpm for 5 min, and the precipitate was collected and washed 3 times with a buffer with a pH value of 7.5 to remove unbound enzyme. The precipitate was collected and freeze-dried to obtain an immobilized enzyme.
[0045] Weigh 1g of defatted walnut protein, stir it in 10mL of deionized water, and adjust the pH value to 7.5. Add 0.08g of g-C3N4 immobilized neutral protease to the filtrate, stir and hydrolyze at 55℃ for 5h, inactivate the enzyme to terminate the hydrolysis reaction, and the degree of hydrolysis of walnut protein is 7.2% as determined by pH-stat method.
[0046] Example 5
[0047] In a shaking flask, add 0.3g of g-C3N4 material prepared by the method of Example 1, 50ml of boric acid buffer with a pH value of 9.0, and 0.6g of alkaline protease. Place the centrifuge tube in a shaker, set the temperature to 25°C, the speed to 200rpm, and the shaking time to 60min. Then adjust the pH to 7.5, add 0.3g of neutral protease, and shake the flask at 25°C and 200rpm for 6min. Centrifuge at 4°C and 10000rpm for 10min, collect the precipitate and wash it 3 times with a phosphate buffer with a pH value of 7.5 to remove unbound enzymes. Collect the precipitate and freeze-dry it to obtain the immobilized dual enzyme, see attached. Figure 3 .
[0048] Weigh 1g of defatted walnut protein, stir it evenly in 10mL of deionized water, and adjust the pH value to 9.0. Add 0.03g of g-C3N4 immobilized dual enzyme to the filtrate, stir and hydrolyze at 55℃ for 8h, inactivate the enzyme to terminate the hydrolysis reaction, and the degree of hydrolysis of walnut protein is 40.9% as determined by pH-stat method.
[0049] The above-described embodiments merely express the implementation methods of the present invention, but they cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.
Claims
1. A method for directional immobilization of proteolytic enzymes by biomimetic ligands, characterized in that: The method for immobilizing proteolytic enzyme with biomimetic ligand in a directional manner utilizes nano-film biomimetic ligand g-C3N4 to immobilize proteolytic enzyme in a directional manner. First, the biomimetic ligand g-C3N4 is prepared; second, the enzyme molecules are immobilized in a buffer solution with a suitable pH value by means of adaptive assembly; finally, walnut cake crude protein is hydrolyzed by means of an alkali-solution-immobilized composite enzyme combination to prepare walnut peptides; The following steps are involved: The first step is to prepare the biomimetic ligand g-C3N4; The second step is to prepare the immobilized enzyme; The third step is to use immobilized enzyme to hydrolyze walnut cake protein.
2. The method for directional immobilization of proteolytic enzymes with biomimetic ligands according to claim 1, characterized in that: The specific steps are: The first step is to prepare the biomimetic ligand g-C3N4, specifically: The crucible containing urea is placed in a tubular muffle furnace and maintained at 500-600° C. for 2-5 hours; then, the obtained solid product is ground, washed and dried to obtain a biomimetic ligand g-C3N4 nanofilm material as a biomimetic ligand g-C3N4; The second step is to prepare the immobilized enzyme, specifically: Step 2.1) adding the proteolytic enzyme and the g-C3N4 material into a buffer solution to obtain a mixed solution, wherein the mass ratio of the proteolytic enzyme to the g-C3N4 material is 1:3 to 5:1; Step 2.2) stirring and shaking the mixed solution of the proteolytic enzyme and the g-C3N4 material; then centrifuging, collecting the precipitate, washing, and freeze-drying to obtain the immobilized enzyme; The third step is to use immobilized enzyme to hydrolyze walnut cake protein; Step 3.1) defatting, homogenizing and drying the walnut cake to prepare defatted walnut powder; Step 3.2) dispersing defatted walnut powder in deionized water; Step 3.3) adding the immobilized enzyme prepared in the second step to the filtrate of step 3.2), wherein the mass ratio of the immobilized enzyme to the walnut protein is 1:10-20, and stirring and hydrolyzing at a certain temperature; Step 3.4) After the hydrolysis is completed, the enzyme is inactivated and the degree of protein hydrolysis is determined.
3. The method for directional immobilization of proteolytic enzymes with biomimetic ligands according to claim 2, characterized in that: In the step 2.1), the proteolytic enzyme is alkaline protease, neutral protease or a mixture of alkaline protease and neutral protease.
4. The method for directional immobilization of proteolytic enzymes with biomimetic ligands according to claim 3, characterized in that: In the step 2.1), the proteolytic enzyme is preferably a mixture of alkaline protease and neutral protease.
5. The method for directional immobilization of proteolytic enzymes with biomimetic ligands according to claim 3, characterized in that: When the proteolytic enzyme is alkaline protease, the pH of the buffer solution is 9.0-10.0; when the proteolytic enzyme is neutral protease, the pH of the buffer solution is 6.5-7.
5.
6. The method for directional immobilization of proteolytic enzymes with biomimetic ligands according to claim 4, characterized in that: When the proteolytic enzyme is an alkaline protease, the buffer solution includes sodium bicarbonate-sodium carbonate solution, glycine-sodium hydroxide, and boric acid-borax solution; when the proteolytic enzyme is a neutral protease, the buffer solution includes potassium dihydrogen phosphate-sodium hydroxide solution, disodium hydrogen phosphate-dipotassium hydrogen phosphate solution.
7. The method for directional immobilization of proteolytic enzymes with biomimetic ligands according to claim 2, characterized in that: The step 2.2) is specifically as follows: stirring and evenly mixing the mixed solution of the proteolytic enzyme and the g-C3N4 material at 15 to 30°C; then centrifuging at 0 to 4°C and 8000 to 12000 rpm for 5 to 15 minutes, collecting the precipitate and washing it several times with the corresponding buffer, and finally freeze-drying the precipitate to obtain the immobilized enzyme.
8. The method for directional immobilization of proteolytic enzymes with biomimetic ligands according to claim 2, characterized in that: In the step 2.2), the proteinase and the g-C3N4 material are mixed and stirred for 30 to 120 minutes at a stirring speed of 100 to 200 rpm / min.
9. The method for directional immobilization of proteolytic enzymes with biomimetic ligands according to claim 2, characterized in that: In the step 3.2), the mass of defatted walnut powder and deionized water is 1 g:10 mL to 1 g:15 mL, and the pH value is adjusted to 9.5 to remove insoluble impurities.
10. The method for directional immobilization of proteolytic enzymes with biomimetic ligands according to claim 2, characterized in that: In the step 3.3), the stirring hydrolysis temperature is 45 to 55° C. and the time is 2 to 8 hours.