Potato starch nano-carrier immobilized lipase and preparation method thereof

Abstract

The invention discloses potato starch nano-carrier immobilized lipase and a preparation method thereof, and relates to the technical field of biological catalysis. The immobilized lipase comprises a lipase and a carrier, and the lipase is immobilized on the carrier; the carrier is potato starch modified magnetic nanoparticles. The immobilized lipase provided by the invention takes the magnetic potato starch nanoparticles as a carrier and has the advantages of being green and environment-friendly and easily available in materials, and the lipase is successfully immobilized on the magnetic nanoparticles by using the potato starch and the traditional glutaraldehyde as cross-linking agents. The method not only improves the activity and stability of the immobilized enzyme, but also has good biocompatibility and safety. By optimizing parameters such as potato starch dosage, enzyme concentration and immobilization time, the obtained immobilized enzyme shows excellent storage stability and repeated utilization rate. In addition, the method provides reference for preparation of other high-performance immobilized enzymes.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme immobilization, and relates to potato starch nano-carrier immobilized lipase and a preparation method thereof. Background Art

[0002] Enzymes are green and efficient biomolecular catalysts with high chemical selectivity and catalytic activity. Enzyme-catalyzed reactions have the advantages of mild conditions, high catalytic efficiency, non-toxicity, biocompatibility, biodegradability and specificity. Enzymes are widely used in chemical synthesis, biotransformation, food processing, pharmaceutical research and other fields due to their high selectivity, high catalytic rate and environmental friendliness. However, the instability of enzymes limits their wide application. In addition, in industrial production, enzymes are difficult to separate from substrate products and cannot be recycled, which greatly increases costs.

[0003] Lipase is a type of biological enzyme. Lipase without carrier immobilization has poor stability. Lipase immobilization may change the spatial structure outside the catalytic active center of the enzyme through interaction with the immobilization material, thereby improving the stability of the enzyme in an industrial environment. It is also convenient to separate the target product from the reaction system and recover the enzyme preparation. The traditional methods for immobilizing lipase mainly include encapsulation, adsorption, cross-linking, covalent bonding, etc. However, traditional immobilization methods have certain defects. The adsorption method has weak action force and the enzyme is easy to escape. The encapsulation method is easy to leak. The cross-linking agent used in the cross-linking method will destroy the structure of the enzyme and cause the enzyme activity to decrease. The reaction conditions in the covalent bonding method are complex and will change the structure of the enzyme and affect the activity of the enzyme.

[0004] Since the catalytic activity of enzymes depends on the complete primary structure and spatial structure of enzyme molecules, the enzyme immobilization technology can improve the stability of enzymes by immobilizing enzymes on nanomaterial carriers, and the enzyme can be recovered and reused by carrier recovery methods. Enzyme immobilization carriers and methods have an important influence on the catalytic activity, stability and reusability of enzymes. The development of suitable enzyme immobilization carriers and methods with high comprehensive performance is the key to the development of enzyme immobilization technology.

[0005] Potato starch is one of the most abundant polysaccharides in nature and has many unique properties, such as low cost, renewability and biodegradability. Due to its biocompatibility, biodegradability and non-toxicity, it has been considered as a potential candidate biomaterial for drug carriers and coating agents for MR. As a green renewable resource, potato starch has good biocompatibility and non-immunogenicity and has been widely used in the pharmaceutical field. Glutaraldehyde, as a traditional immobilized enzyme cross-linking agent, has many advantages, such as mild reaction conditions, high immobilization efficiency and good stability. Summary of the invention

[0006] In view of the deficiencies in the above-mentioned prior art, the present invention provides a method for preparing potato starch nanocarrier-immobilized lipase. The method for preparing potato starch nanocarrier-immobilized lipase of the present invention can improve the loading rate of lipase while ensuring the activity of the enzyme, and form temperature tolerance, pH tolerance, catalytic stability and reusability.

[0007] The technical solution adopted by the present invention is: The present invention provides a method for preparing a potato starch nanocarrier immobilized enzyme, the method comprising: (1) Under nitrogen atmosphere, FeCl 3 6H 2 O and FeCl 2 ·4H 2 After mixing, add deionized water and stir evenly; after heating, add ammonia water, adjust the pH to 10, continue stirring to form a black suspension; separate, wash and dry with a magnet to obtain magnetic nanoparticles; (2) dissolving potato starch in deionized water, heating and continuously stirring until the starch is completely dissolved to form a transparent potato starch solution; slowly adding the magnetic nanoparticles to the potato starch solution, ultrasonically treating until the magnetic nanoparticles are uniformly dispersed in the potato starch solution, adding N,N'-methylenebisacrylamide, and continuously stirring; separating with a magnet, washing, and freeze-drying to obtain magnetic potato starch nanocarriers; (3) The magnetic potato starch nanocarrier is added to phosphate buffer A, glutaraldehyde is added, and the mixture is cross-linked at room temperature for 0.5 to 4.0 hours and then washed multiple times to obtain a precipitate. Then, phosphate buffer B and free enzyme solution are added, and the mixture is placed in an air shaker for shaking to obtain lipase immobilized on the magnetic potato starch nanocarrier.

[0008] Preferably, the FeCl 3 6H 2 O and FeCl 2 ·4H 2 The molar ratio of O is 2:1, and the mass ratio of ammonia water to deionized water is 1:(6.9-7.0).

[0009] Preferably, after adjusting the pH to 10, stirring is continued at a temperature of 60 to 80° C. for 0.8 to 1 h.

[0010] Preferably, the concentration of the potato starch solution is 3-5%.

[0011] Preferably, the potato starch is dissolved in deionized water and heated at 60-70° C. with continuous stirring, and the ultrasonic time is 5-15 min.

[0012] Preferably, the concentration of the N,N'-methylenebisacrylamide is 0.1-1%, and the stirring time is 1-1.5 hours.

[0013] Preferably, the mass of the magnetic potato starch nanocarrier added to the phosphate buffer is 0.1-1 g, and the concentration of the glutaraldehyde is 0.5-4%.

[0014] Preferably, the concentration of the lipase solution is 5-25 mg / mL, and the pH of the phosphate buffer A and the phosphate buffer B are both 6.0-9.0.

[0015] Preferably, the fixed rotation speed of the air shaker is 150-200 rpm, the temperature is 30-70° C., and the time is 3-6 hours.

[0016] The invention also provides a potato starch nano-carrier immobilized lipase, which is prepared by the above method.

[0017] The present invention has the following beneficial effects: (1) The present invention improves the storage stability and durability of the immobilized enzyme. The present invention improves the storage stability and durability of the immobilized enzyme by adding potato starch to the magnetic Fe 3 O 4 The surface of the nanoparticles is modified to obtain a magnetic potato starch carrier containing a large number of hydroxyl groups for TLL immobilization, and then the TLL is immobilized by glutaraldehyde cross-linking. The immobilized TLL prepared by the present invention has a small particle size, a high enzyme activity recovery rate, better thermal stability and pH stability than free enzymes, and strong organic solvent tolerance. The storage stability of the immobilized enzyme is 80%, and the reuse rate reaches 50% to 60%.

[0018] (2) The present invention improves the acid-base tolerance and thermal stability of the immobilized enzyme and has good enzymatic properties. The magnetic potato starch nanocarrier immobilized lipase provides a reference for the preparation of other immobilized enzymes with excellent performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure is a schematic diagram of the preparation principle of potato starch nanocarrier immobilized lipase in the present invention. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0021] Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples of the present invention are all conventional products that can be purchased from the market. The lipase used in the present invention is Thermomyces lanuginosus lipase TLL. Figure 1 This is a schematic diagram of the preparation principle of potato starch nanocarrier immobilized lipase in this application.

[0022] Method for determining the activity of immobilized enzymes in the examples and comparative examples of the present invention: Refer to GB / T23535--2009 "Lipase Preparation" and make some improvements. The principle is that lipase hydrolyzes natural oils into fatty acids and glycerol. The enzyme activity unit is defined as the amount of enzyme required to hydrolyze the substrate to produce 1μmol of titratable fatty acids per minute at pH 7.5 and 40°C.

[0023] Determination of the loading amount of immobilized enzyme protein in the examples and comparative examples of the present invention: The protein loading of the immobilized enzyme was determined by the Bradford method, and a standard curve was drawn using 1.0 mg / mL bovine serum albumin solution prepared in 0.2 mol / L pH 7.0 phosphate buffer as the reaction model substrate.

[0024] The calculation formula for the recovery rate of immobilized enzyme activity in the examples and comparative examples of the present invention is: Enzyme activity recovery rate = [(enzyme activity of immobilized enzyme - protein loading of immobilized enzyme) / (enzyme activity of free enzyme - protein content of free enzyme)] × 100%.

[0025] Example 1 This embodiment provides a method for preparing potato starch nanocarrier-immobilized lipase, and the specific preparation steps are as follows: (1) 5.2 g FeCl 3 6H 2 O and 2.0 g FeCl 2 ·4H 2 O was dissolved in 160 mL of deionized water. Then 30 mL of ammonia water was quickly added and stirred vigorously for 50 min under nitrogen protection. Then the temperature was raised to 80 ° C. After the system was stable, the black precipitate was separated with a magnet and washed several times with ultrapure water. Then, the obtained Fe 3 O 4 The nanoparticles were dried under vacuum at 60 °C for 12 h; (2) Weigh 5g of potato starch and dissolve it in 100mL of deionized water. Heat to 70°C and continue stirring until the starch is completely dissolved to form a transparent starch solution. Slowly add 1g of the prepared magnetic nanoparticles to the potato starch solution and ultrasonicate for 10min. When the magnetic nanoparticles are evenly dispersed in the starch solution, add 1% N,N'-methylenebisacrylamide and continue stirring. Use a magnet to separate, wash, and freeze-dry to obtain magnetic potato starch nanoparticles. After freeze-drying, seal and store at 4°C. (3) 0.5 g of magnetic potato starch nanocarrier was added to 15 mL of phosphate buffer A (0.1 M, pH 7.0), and 1% glutaraldehyde was added. After cross-linking at room temperature for 1 h, the mixture was washed several times to obtain a precipitate. Then, 15 mL of phosphate buffer B (0.1 M, pH 7.0) and 5 mL of free TLL enzyme solution were added. The mixture was placed in an air shaker at 40 °C and shaken at 200 rpm for 4 h. After the immobilization was completed, the system was cooled to room temperature, and the precipitate was obtained by using its magnetism. The mixture was washed several times until the upper liquid was transparent, freeze-dried, and stored in a sealed container at 4 °C to obtain lipase immobilized on the magnetic potato starch nanocarrier.

[0026] Example 2 This embodiment provides a method for preparing potato starch nanocarrier-immobilized lipase, and the specific preparation steps are as follows: (1) 5.2 g FeCl 3 6H 2 O and 2.0 g FeCl 2 ·4H 2 O was dissolved in 160 mL of deionized water. Then 30 mL of ammonia water was quickly added and stirred vigorously for 50 min under nitrogen protection. Then the temperature was raised to 80 ° C, the black precipitate was separated with a magnet and washed several times with ultrapure water. Then, the obtained Fe 3 O 4 The nanoparticles were dried under vacuum at 60 °C for 12 h; (2) Weigh 3g of potato starch and dissolve it in 100mL of deionized water. Heat to 70°C and continue stirring until the starch is completely dissolved to form a transparent starch solution. Slowly add 1g of the prepared magnetic nanoparticles to the potato starch solution and ultrasonicate for 10min. When the magnetic nanoparticles are evenly dispersed in the starch solution, add 1% N,N'-methylenebisacrylamide and continue stirring. Use a magnet to separate, wash, and freeze-dry to obtain magnetic potato starch nanoparticles. After freeze-drying, seal and store at 4°C. (3) 1 g of magnetic potato starch nanocarrier was added to 15 mL of phosphate buffer A (0.1 M, pH 7.0), and 1% glutaraldehyde was added. After cross-linking at room temperature for 1 h, the mixture was washed several times to obtain a precipitate. Then, 15 mL of phosphate buffer B (0.1 M, pH 8.0) and 5 mL of free TLL enzyme solution were added. The mixture was placed in an air shaker at 50 °C and shaken at 200 rpm for 4 h. After the immobilization was completed, the system was cooled to room temperature, and the precipitate was obtained by using its magnetism. The mixture was washed several times until the upper liquid was transparent, and then freeze-dried and stored in a sealed container at 4 °C to obtain lipase immobilized on the magnetic potato starch nanocarrier.

[0027] Example 3 This embodiment provides a method for preparing potato starch nanocarrier-immobilized lipase, and the specific preparation steps are as follows: (1) 5.2 g FeCl 3 6H 2 O and 2.0 g FeCl 2 ·4H 2 O was dissolved in 160 mL of deionized water. Then 30 mL of ammonia water was quickly added and stirred vigorously for 50 min under nitrogen protection. Then the temperature was raised to 80 ° C, the black precipitate was separated with a magnet and washed several times with ultrapure water. Then, the obtained Fe 3 O 4 The nanoparticles were dried under vacuum at 60 °C for 12 h; (2) Weigh 4 g of potato starch and dissolve it in 100 mL of deionized water. Heat to 80 °C and continue stirring until the starch is completely dissolved to form a transparent starch solution. Slowly add 1 g of the prepared magnetic nanoparticles to the potato starch solution and ultrasonicate for 10 min. When the magnetic nanoparticles are evenly dispersed in the starch solution, add 1% N,N'-methylenebisacrylamide and continue stirring. Use a magnet to separate, wash, and freeze-dry to obtain magnetic potato starch nanoparticles. After freeze-drying, seal and store at 4 °C. (3) 0.5 g of magnetic potato starch nanocarrier was added to 15 mL of phosphate buffer A (0.1 M, pH 7.0), and 1% glutaraldehyde was added. After cross-linking at room temperature for 1 h, the mixture was washed several times to obtain a precipitate. Then, 15 mL of phosphate buffer B (0.1 M, pH 9.0) and 5 mL of free TLL enzyme solution were added. The mixture was placed in an air shaker at 40 °C and shaken at 200 rpm for 4 h. After the immobilization was completed, the system was cooled to room temperature, and the precipitate was obtained by using its magnetism. The mixture was washed several times until the upper liquid was transparent, and then freeze-dried and stored in a sealed container at 4 °C to obtain lipase immobilized on the magnetic potato starch nanocarrier.

[0028] Example 4 This embodiment provides a method for preparing potato starch nanocarrier-immobilized lipase, and the specific preparation steps are as follows: (1) 5.2 g FeCl 3 6H 2 O and 2.0 g FeCl 2 ·4H 2 O was dissolved in 160 mL of deionized water. Then 30 mL of ammonia water was quickly added and stirred vigorously for 50 min under nitrogen protection. Then the temperature was raised to 80 ° C, the black precipitate was separated with a magnet and washed several times with ultrapure water. Then, the obtained Fe 3 O 4 The nanoparticles were dried under vacuum at 60 °C for 12 h; (2) Weigh 5g of potato starch and dissolve it in 100mL of deionized water. Heat to 80°C and continue stirring until the starch is completely dissolved to form a transparent starch solution. Slowly add 1g of the prepared magnetic nanoparticles to the potato starch solution and ultrasonicate for 10min. When the magnetic nanoparticles are evenly dispersed in the starch solution, add 1% N,N'-methylenebisacrylamide and continue stirring. Use a magnet to separate, wash, and freeze-dry to obtain magnetic potato starch nanoparticles. After freeze-drying, seal and store at 4°C. (3) 0.5 g of magnetic potato starch nanocarrier was added to 15 mL of phosphate buffer A (0.1 M, pH 7.0), and 1% glutaraldehyde was added. After cross-linking at room temperature for 1 h, the mixture was washed several times to obtain a precipitate. Then, 15 mL of phosphate buffer B (0.1 M, pH 6.0) and 5 mL of free TLL enzyme solution were added. The mixture was placed in an air shaker at 60 °C and shaken at 200 rpm for 4 h. After the immobilization was completed, the system was cooled to room temperature, and the precipitate was obtained by using its magnetism. The mixture was washed several times until the upper liquid was transparent, and then freeze-dried and stored in a sealed container at 4 °C to obtain lipase immobilized on the magnetic potato starch nanocarrier.

[0029] Comparative Example 1 (1) 5.2 g FeCl 3 6H 2 O and 2.0 g FeCl 2 ·4H 2 O was dissolved in 160 mL of deionized water. Then 30 mL of ammonia water was quickly added and stirred vigorously for 50 min under nitrogen protection. Then the temperature was raised to 80 ° C, the black precipitate was separated with a magnet and washed several times with ultrapure water. Then, the obtained Fe 3 O 4 The nanoparticles were dried under vacuum at 60 °C for 12 h; (2) Weigh 5g of potato starch and dissolve it in 100mL of deionized water. Heat to 70°C and continue stirring until the starch is completely dissolved to form a transparent starch solution. Slowly add 1g of the prepared magnetic nanoparticles to the potato starch solution and ultrasonicate for 10min. When the magnetic nanoparticles are evenly dispersed in the starch solution, add 1% N,N'-methylenebisacrylamide and continue stirring. Use a magnet to separate, wash, and freeze-dry to obtain magnetic potato starch nanoparticles. After freeze-drying, seal and store at 4°C. (3) 0.5 g of magnetic potato starch nanocarrier was added to 15 mL of phosphate buffer A (0.1 M, pH 7.0), and 1% glutaraldehyde was added. After cross-linking at room temperature for 1 h, the mixture was washed several times to obtain a precipitate. Then, 15 mL of phosphate buffer B (0.1 M, pH 8.0) and 5 mL of free TLL enzyme solution were added. The mixture was placed in an air shaker at 40 °C and shaken at 200 rpm for 4 h. After the immobilization was completed, the system was cooled to room temperature, and the precipitate was obtained by using its magnetism. The mixture was washed several times until the upper liquid was transparent, and then freeze-dried and stored in a sealed container at 4 °C to obtain lipase immobilized on the magnetic potato starch nanocarrier.

[0030] Comparative Example 2 (1) 5.2 g FeCl 3 6H 2 O and 2.0 g FeCl 2 ·4H 2 O was dissolved in 160 mL of deionized water. Then 30 mL of ammonia water was quickly added and stirred vigorously for 50 min under nitrogen protection. Then the temperature was raised to 80 ° C, the black precipitate was separated with a magnet and washed several times with ultrapure water. Then, the obtained Fe 3 O 4 The nanoparticles were dried under vacuum at 60 °C for 12 h; (2) Weigh 3g of potato starch and dissolve it in 100mL of deionized water. Heat to 80°C and continue stirring until the starch is completely dissolved to form a transparent starch solution. Slowly add 1g of the prepared magnetic nanoparticles to the potato starch solution and ultrasonically treat for 10 minutes. When the magnetic nanoparticles are evenly dispersed in the starch solution, add 1% N,N'-methylenebisacrylamide and continue stirring. Use a magnet to separate, wash, and freeze-dry to obtain magnetic potato starch nanoparticles. After freeze-drying, seal and store at 4°C. (3) 0.5 g of magnetic potato starch nanocarrier was added to 15 mL of phosphate buffer A (0.1 M, pH 7.0), and 1% glutaraldehyde was added. After cross-linking at room temperature for 1 h, the mixture was washed several times to obtain a precipitate. Then, 15 mL of phosphate buffer B (0.1 M, pH 7.0) and 5 mL of free TLL enzyme solution were added. The mixture was placed in an air shaker at 40 °C and shaken at 200 rpm for 3 h. After the immobilization was completed, the system was cooled to room temperature, and the precipitate was obtained by using its magnetism. The mixture was washed several times until the upper liquid was transparent, and then freeze-dried and stored in a sealed container at 4 °C to obtain lipase immobilized on the magnetic potato starch nanocarrier.

[0031] Comparative Example 3 (1) 5.2 g FeCl 3 6H 2 O and 2.0 g FeCl 2 ·4H 2O was dissolved in 160 mL of deionized water. Then 30 mL of ammonia water was quickly added and stirred vigorously for 50 min under nitrogen protection. Then the temperature was raised to 80 ° C, the black precipitate was separated with a magnet and washed several times with ultrapure water. Then, the obtained Fe 3 O 4 The nanoparticles were dried under vacuum at 60 °C for 12 h; (2) Weigh 5g of potato starch and dissolve it in 100mL of deionized water. Heat to 70°C and continue stirring until the starch is completely dissolved to form a transparent starch solution. Slowly add 1g of the prepared magnetic nanoparticles to the potato starch solution and ultrasonicate for 10min. When the magnetic nanoparticles are evenly dispersed in the starch solution, add 1% N,N'-methylenebisacrylamide and continue stirring. Use a magnet to separate, wash, and freeze-dry to obtain magnetic potato starch nanoparticles. After freeze-drying, seal and store at 4°C. (3) 0.5 g of magnetic potato starch nanocarrier was added to 15 mL of phosphate buffer A (0.1 M, pH 7.0), and 1% glutaraldehyde was added. After cross-linking at room temperature for 1 h, the mixture was washed several times to obtain a precipitate. Then, 15 mL of phosphate buffer B (0.1 M, pH 7.0) and 5 mL of free TLL enzyme solution were added. The mixture was placed in an air shaker at 60 °C and shaken at 200 rpm for 5 h. After the immobilization was completed, the system was cooled to room temperature, and the precipitate was obtained by using its magnetism. The mixture was washed several times until the upper liquid was transparent, and then freeze-dried and stored in a sealed container at 4 °C to obtain lipase immobilized on the magnetic potato starch nanocarrier.

[0032] Example 5 Study on pH stability of immobilized enzymes: Take 0.1 mL of free enzyme and 0.5 g of immobilized enzyme of Examples 1 to 4 and Comparative Examples 1 to 3, add them into 10 mL of phosphate buffer with pH values ​​of 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 and 9, respectively, and place them at 4°C for 12 hours, and detect the enzyme activity according to the method. The initial enzyme activity of the free enzyme and the immobilized enzyme is defined as 100%, and the enzyme activity recovery rate under other reaction conditions is calculated.

[0033] Example 6 Study on operational stability of immobilized enzymes: Take 0.1 mL of free enzyme and 0.5 g of immobilized enzyme of Examples 1 to 4 and Comparative Examples 1 to 3, add them into 10 mL of phosphate buffer of the optimum pH of the enzyme, store them at 4°C for 180 days, and detect the enzyme activity every 10 days according to the method. The initial enzyme activity of the free enzyme and the immobilized enzyme is defined as 100%, and the enzyme activity recovery rate under other reaction conditions is calculated.

[0034] Example 7 Study on organic solvent tolerance of immobilized enzymes: Take 0.1 mL of free enzyme and 0.5 g of immobilized enzyme of Examples 1 to 4 and Comparative Examples 1 to 3, add them into 10 mL of organic solvent (acetone, tetrahydrofuran (THF), 2-methyltetrahydrofuran (2-THF), dimethyl sulfoxide (DMSO), respectively, store them at 4 ° C for 12 h, and detect the enzyme activity according to the method. The initial enzyme activity of the free enzyme and the immobilized enzyme is defined as 100%, and the enzyme activity recovery rate under other reaction conditions is calculated.

[0035] Example 8 Study on thermal stability of immobilized enzyme: Take 0.1 mL of free enzyme and 0.5 g of immobilized enzyme of Examples 1 to 4 and Comparative Examples 1 to 3, and set the reaction temperature to 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C. and 70° C. respectively. Define the enzyme activity at the optimum reaction temperature as 100%, and calculate the enzyme activity recovery rate under other conditions.

[0036] By comparing the comparative examples, the immobilized enzyme activity, immobilized enzyme protein loading and immobilized enzyme activity recovery rate of each example are measured and calculated, and it can be known that the present invention improves the tolerance of the immobilized enzyme in terms of temperature and time. The relative enzyme activity of the magnetic potato starch nanocarrier immobilized enzyme reaches the maximum at 40°C, and it still retains 80% at 60°C, which is significantly higher than the free enzyme; the immobilization rate of the magnetic potato starch nanocarrier immobilized enzyme is above 66.1% at 4h, which indicates that the immobilized enzyme on the surface of the magnetic carrier is saturated, and the enzyme is partially inactivated as time goes by.

[0037] By comparing the comparative examples, the enzymatic activity of the immobilized enzyme, the enzymatic protein loading of the immobilized enzyme and the enzymatic activity recovery rate of the immobilized enzyme of each example are measured and calculated. The present invention improves the acid-base tolerance of the immobilized enzyme. The relative enzymatic activity of the magnetic potato starch nanocarrier immobilized enzyme reaches the maximum at pH 7.0, and at pH 8.0, it is significantly greater than the relative enzymatic activity of the free enzyme; the present invention improves the organic solvent tolerance of the immobilized enzyme, and the enzymatic activity of the immobilized enzyme is higher than that of the free enzyme in any organic solvent, and the enzymatic activity recovery rate of 2-THF is the best among the four organic solvents. It shows that immobilization can improve the stability of the enzyme, and the immobilized enzyme can maintain high catalysis in most organic solvents.

[0038] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing potato starch nanocarrier immobilized lipase, characterized in that: The following steps are involved: (1) Under a nitrogen atmosphere, FeCl3·6H2O and FeCl2·4H2O were mixed, deionized water was added, and the mixture was stirred evenly; after heating, ammonia water was added, the pH was adjusted to 10, and stirring was continued to form a black suspension; the mixture was separated by a magnet, washed, and dried to obtain magnetic nanoparticles; (2) dissolving potato starch in deionized water, heating and continuously stirring until the starch is completely dissolved to form a transparent potato starch solution; slowly adding the magnetic nanoparticles to the potato starch solution, ultrasonically treating until the magnetic nanoparticles are uniformly dispersed in the potato starch solution, adding N,N'-methylenebisacrylamide, and continuously stirring; separating with a magnet, washing, and freeze-drying to obtain magnetic potato starch nanocarriers; (3) The magnetic potato starch nanocarrier is added to phosphate buffer A, glutaraldehyde is added, and the mixture is cross-linked at room temperature for 0.5 to 4.0 hours and then washed multiple times to obtain a precipitate. Then, phosphate buffer B and free enzyme solution are added, and the mixture is placed in an air shaker for shaking to obtain lipase immobilized on the magnetic potato starch nanocarrier.

2. The method for preparing potato starch nanocarrier-immobilized lipase according to claim 1, characterized in that: The molar ratio of the FeCl3·6H2O to FeCl2·4H2O is 2:1, and the mass ratio of the ammonia water to the deionized water is 1:(6.9-7.0).

3. The method for preparing potato starch nanocarrier-immobilized lipase according to claim 1, characterized in that: After adjusting the pH to 10, stirring was continued at 60 to 80°C for 0.8 to 1 h.

4. The method for preparing potato starch nanocarrier-immobilized lipase according to claim 1, characterized in that: The concentration of the potato starch solution is 3-5%.

5. The method for preparing potato starch nanocarrier-immobilized lipase according to claim 1, characterized in that: The potato starch is dissolved in deionized water and heated at 60-70° C. with continuous stirring. The ultrasonic time is 5-15 minutes.

6. The method for preparing potato starch nanocarrier-immobilized lipase according to claim 1, characterized in that: The concentration of the N,N'-methylenebisacrylamide is 0.1-1%, and the stirring time is 1-1.5 hours.

7. The method for preparing potato starch nanocarrier-immobilized lipase according to claim 1, characterized in that: The mass of the magnetic potato starch nanocarrier added to the phosphate buffer is 0.1-1 g, and the concentration of the glutaraldehyde is 0.5-4%.

8. The method for preparing potato starch nanocarrier-immobilized lipase according to claim 1, characterized in that: The concentration of the lipase solution is 5-25 mg / mL, and the pH of the phosphate buffer A and the phosphate buffer B are both 6.0-9.

0.

9. The method for preparing potato starch nanocarrier-immobilized lipase according to claim 1, characterized in that: The fixed rotation speed of the air shaker is 150-200 rpm, the temperature is 30-70° C., and the time is 3-6 hours.

10. Potato starch nanocarrier-immobilized lipase prepared by the method for preparing potato starch nanocarrier-immobilized lipase according to any one of claims 1 to 9.

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