A metal framework material for adjustable lipase selective catalysis, preparation method and application

Abstract

The present invention relates to a metal framework material for adjustable lipase selective catalysis, a preparation method and an application. The preparation method includes: (1) immobilizing lipase; (2) amino modification; (3) polyisopropylacrylamide modification. The metal framework material is polyisopropylacrylamide modified amino-modified immobilized lipase CRL@ZIF-8-PNIPAM. Among them, CRL@ZIF-8-PNIPAM selectively catalyzes different guest molecules at different temperatures. Its advantages are that the pore size of CRL@ZIF-8-PNIPAM is regulated by the reaction temperature, temporarily enhancing the binding of lipase and different substrates, and endowing CRL@ZIF-8-PNIPAM with a novel and adjustable substrate selective catalysis function.

Description

Technical Field

[0001] The present invention relates to the technical field of biocatalysis, and particularly to a metal framework material capable of regulating the selective catalysis of lipase, a preparation method and an application thereof. Background Art

[0002] Candida rugosa lipase (CRL) is a biocatalyst widely used in organic chemistry and can catalyze reactions such as esterification, transesterification, enantioselective hydrolysis, etc. It has extensive applications in industries such as food, energy, and medicine. However, due to the relatively high cost, unstable structure and difficult recovery of enzyme catalysts, the further application of lipase is limited.

[0003] Enzyme immobilization can not only solve the problem of difficult recovery, but also maintain the conformational stability of the enzyme and enhance its tolerance to extreme environments. Strategies for improving the catalytic stability of lipase in harsh environments include protein engineering techniques and chemical modification techniques. In recent years, lipase encapsulated in nanostructured gels can increase the surface area and mass transfer resistance of the enzyme, thereby enhancing its catalytic stability in harsh environments.

[0004] Metal-organic frameworks (MOFs) are materials with a periodic network structure formed by the self-assembly of inorganic metal centers and organic ligands. They have a multi-level ordered porous structure and size, making them have a high specific surface area and porosity, and are suitable for the immobilization of lipase. There are many types of MOFs, such as IRMOF, MILS, ZIF, UIO, PCN-based MOFs, etc. They usually have a regular crystal morphology of micropores (<2 nm) and mesopores (2 - 50 nm), a large specific surface area (even up to 6000 m2 / g), high thermal stability / mechanical stability, high aqueous phase stability and good optoelectronic properties. And MOFs are composed of metal ions or clusters and organic molecules. Due to the large number of choices of metal nodes and organic ligands, MOFs have rich functions and diverse structures, so they are suitable as new carriers for enzyme immobilization.

[0005] Although immobilized enzymes reduce cost waste and achieve the purpose of enzyme recycling, the existing immobilized enzyme technology cannot control the selective catalysis of substrates by adjusting the temperature.

[0006] Chinese Invention Patent CN105498694A discloses a thermosensitive polymer - encapsulated metal - organic framework magnetic material and its application. This patent introduces a thermosensitive polymer and a magnetic material into the metal - organic framework to achieve the enrichment and separation of target pollutants. The introduction of the thermosensitive polymer endows the adsorbent material with high enrichment efficiency for target pollutants at low temperatures due to the unique temperature - responsive property of the polymer, and at the same time avoids the volatilization of organic solvents during the extraction and elution processes, reducing secondary pollution to the environment. However, although this patent separates target pollutants by adjusting temperature changes, it does not modify the thermosensitive material on the surface of the metal - organic framework for catalytic reactions and cannot solve the problem of selectively catalyzing substrates by regulating temperature through MOF.

[0007] Currently, in view of the problems in the related technologies, such as the inability to adjust temperature to control the selective catalysis of MOF for substrates, no effective solution has been proposed. Summary of the Invention

[0008] The purpose of the present invention is to provide a metal framework material, a preparation method and an application thereof for adjustable lipase - selective catalysis in view of the deficiencies in the prior art, so as to solve the problems in the related technologies, such as the inability to adjust temperature to control the selective catalysis of MOF for substrates.

[0009] To achieve the above - mentioned purpose, the technical solution adopted by the present invention is as follows:

[0010] In the first aspect, the present invention provides a preparation method of a metal framework material for adjustable lipase - selective catalysis, including:

[0011] (1) Immobilizing lipase

[0012] Add lipase to a solution containing Zn(NO3)2·6H2O and mix to form a first mixed solution;

[0013] Add an organic ligand to the first mixed solution and react to form a second mixed solution;

[0014] Perform solid - liquid separation on the second mixed solution to obtain a first crude product;

[0015] Wash and dry the first crude product to obtain immobilized lipase CRL@ZIF - 8;

[0016] (2) Amination modification

[0017] Mix and react the immobilized lipase with ammonia water to form a third mixed solution;

[0018] Perform solid - liquid separation on the third mixed solution to obtain a second crude product;

[0019] Dry the second crude product to obtain the immobilized lipase CRL@ZIF-NH2 modified by amination;

[0020] (III) Modification with polyisopropylacrylamide

[0021] Mix and react the solution containing polyisopropylacrylamide with the solution containing the immobilized lipase modified by amination to form a fourth mixed solution;

[0022] Perform solid-liquid separation on the fourth mixed solution to obtain a third crude product;

[0023] Wash and dry the third crude product to obtain a metal framework material, wherein the metal framework material is the immobilized lipase CRL@ZIF-8-PNIPAM modified by polyisopropylacrylamide and amination;

[0024] Among them, CRL@ZIF-8-PNIPAM selectively catalyzes different guest molecules at different temperatures.

[0025] In some of the embodiments, the mass ratio of Zn(NO3)2·6H2O to the lipase is 1.97 - 5.9:1.

[0026] In some of the embodiments, the mass ratio of the organic ligand to the lipase is 36.75 - 113.5:1.

[0027] In some of the embodiments, the organic ligand is 2-methylimidazole.

[0028] In some of the embodiments, mix and react the immobilized lipase, ammonia water, and deionized water.

[0029] In some of the embodiments, add the lipase to the solution containing Zn(NO3)2·6H2O, stir at room temperature for 5 min - 45 min, and the rotation speed is 120 rpm - 300 rpm.

[0030] In some of the embodiments, add the organic ligand to the first mixed solution, react at room temperature for 30 min - 1440 min, and the rotation speed is 120 rpm - 300 rpm.

[0031] In some of the embodiments, centrifuge the second mixed solution for 10 min - 15 min, and the rotation speed is 8000 rpm - 10000 rpm.

[0032] In some of the embodiments, wash the first crude product with deionized water at least 2 times.

[0033] In some of these embodiments, the washed first crude product is vacuum dried at 55°C for at least 6 h.

[0034] In some of these embodiments, the added mass of the immobilized lipase is 0.1 mg to 1 g.

[0035] In some of these embodiments, the added volume of the ammonia water is 6.25 mL to 62.5 mL.

[0036] In some of these embodiments, the immobilized lipase is mixed with ammonia water and reacted at 27°C to 29°C for 22 h to 24 h, with a rotation speed of 120 rpm to 300 rpm.

[0037] In some of these embodiments, the third mixed solution is allowed to stand for 5 min to 20 min, and the upper layer solution is removed.

[0038] In some of these embodiments, the washed second crude product is vacuum dried at 55°C for at least 6 h.

[0039] In some of these embodiments, the mass ratio of polyisopropylacrylamide to CRL@ZIF-NH2 is 1 to 2:1.

[0040] In some of these embodiments, the solvent of the solution containing polyisopropylacrylamide is chloroform.

[0041] In some of these embodiments, the solvent of the solution containing CRL@ZIF-NH2 is chloroform.

[0042] In some of these embodiments, the solution containing polyisopropylacrylamide is mixed with the solution containing the amino-modified immobilized lipase and reacted at 50°C to 55°C for 24 h to 26 h.

[0043] In some of these embodiments, the fourth mixed solution is allowed to stand for 5 min to 20 min, and the upper layer solution is removed.

[0044] In some of these embodiments, the third crude product is washed with chloroform at least 2 times.

[0045] In some of these embodiments, the washed third crude product is vacuum dried at 55°C for at least 6 h.

[0046] In some of these embodiments, it includes:

[0047] (I) Immobilized lipase

[0048] Add lipase to a solution containing Zn(NO3)2·6H2O and stir at 120 rpm to 300 rpm for 5 min to 45 min at room temperature to form a first mixed solution, wherein the mass ratio of Zn(NO3)2·6H2O to the lipase is 1.97 to 5.9:1;

[0049] Add 2-methylimidazole to the first mixed solution and react at 120 rpm to 300 rpm for 30 min to 1440 min at room temperature to form a second mixed solution, wherein the mass ratio of 2-methylimidazole to the lipase is 36.75 to 113.5:1;

[0050] Centrifuge the second mixed solution at 8000 rpm to 10000 rpm for 10 min to 15 min to obtain a first crude product;

[0051] Wash the first crude product with deionized water at least 2 times;

[0052] Vacuum dry the washed first crude product at 55 °C for at least 6 h to obtain immobilized lipase CRL@ZIF-8;

[0053] (II) Amination modification

[0054] Mix the immobilized lipase with ammonia water and react at 27 °C to 29 °C for 22 h to 24 h at a rotation speed of 120 rpm to 300 rpm to form a third mixed solution, wherein the added mass of the immobilized lipase is 0.1 mg to 1 g, and the added volume of the ammonia water is 6.25 mL to 62.5 mL;

[0055] Let the third mixed solution stand for 5 min to 20 min and remove the upper layer solution to obtain a second crude product;

[0056] Vacuum dry the second crude product at 55 °C for at least 6 h to obtain aminated modified immobilized lipase CRL@ZIF-8-NH2;

[0057] (III) Polyisopropylacrylamide modification

[0058] Mix a chloroform solution containing polyisopropylacrylamide with a chloroform solution containing the aminated modified immobilized lipase and react at 50 °C to 55 °C for 24 h to 26 h to form a fourth mixed solution, wherein the mass ratio of polyisopropylacrylamide to CRL@ZIF-NH2 is 1 to 2:1;

[0059] Let the fourth mixed solution stand for 5 min to 20 min and remove the upper layer solution to obtain a third crude product;

[0060] Wash the third crude product with chloroform at least twice;

[0061] Vacuum dry the washed third crude product at 55 °C for at least 6 h to obtain a metal framework material, wherein the metal framework material is immobilized lipase CRL@ZIF-8-PNIPAM modified with polyisopropylacrylamide and amino modification;

[0062] Among them, CRL@ZIF-8-PNIPAM selectively catalyzes different guest molecules at different temperatures.

[0063] In a second aspect, the present invention provides a metal framework material capable of regulating the selective catalysis of lipase, which is prepared by the preparation method described in the first aspect.

[0064] In a third aspect, the present invention provides an application of the metal framework material capable of regulating the selective catalysis of lipase described in the second aspect in biocatalysis

[0065] The present invention adopts the above technical solutions, and compared with the prior art, has the following technical effects:

[0066] The metal framework material, preparation method and application capable of regulating the selective catalysis of lipase of the present invention regulate the pore size of CRL@ZIF-8-PNIPAM by the reaction temperature, temporarily enhance the binding of lipase and different substrates, and endow CRL@ZIF-8-PNIPAM with a novel and regulable substrate selective catalysis function. Description of the Drawings

[0067] Figure 1 It is a schematic diagram of the selective catalysis result of CRL@ZIF-8-PNIPAM with p-nitrophenyl palmitate as the substrate;

[0068] Figure 2 It is a schematic diagram of the selective catalysis result of CRL@ZIF-8-PNIPAM with 4-nitrophenyl laurate as the substrate;

[0069] Figure 3 It is a schematic diagram of the selective catalysis result of CRL@ZIF-8-PNIPAM with 4-nitrophenyl butyrate as the substrate;

[0070] Figure 4 It is a schematic diagram of the catalytic result of free lipase with p-nitrophenyl palmitate as the substrate;

[0071] Figure 5 It is a schematic diagram of the catalytic result of free lipase with 4-nitrophenyl laurate as the substrate;

[0072] Figure 6Schematic diagram of the catalytic result of free lipase with 4-nitrobutyrate as the substrate;

[0073] Figure 7 Schematic diagram of the effect of changing external temperature on the catalytic activity of CRL@ZIF-8-PNIPAM;

[0074] Figure 8 Comparison chart of the catalytic activities of free lipase and CRL@ZIF-8-PNIPAM at 27 °C and 40 °C. Detailed implementation manners

[0075] In order to make the objectives, technical solutions and advantages of this application clearer, the following describes and explains this application in combination with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application. Based on the embodiments provided in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope protected by this application.

[0076] Obviously, the accompanying drawings in the following description are only some examples or embodiments of this application. For those of ordinary skill in the art, without creative efforts, this application can also be applied to other similar scenarios based on these drawings. In addition, it can also be understood that although the efforts made in this development process may be complex and lengthy, for those of ordinary skill in the art related to the content disclosed in this application, some design, manufacturing or production changes based on the technical content disclosed in this application are only conventional technical means and should not be understood as the content disclosed in this application being insufficient.

[0077] Referring to "embodiments" in this application means that specific features, structures or characteristics described in combination with the embodiments can be included in at least one embodiment of this application. The appearance of this phrase at various positions in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those of ordinary skill in the art explicitly and implicitly understand that the embodiments described in this application can be combined with other embodiments without conflict.

[0078] Unless otherwise defined, the technical terms or scientific terms involved in this application shall have the ordinary meanings understood by those with ordinary skills in the technical field to which this application belongs. The words such as "a", "an", "one", "the" and the like involved in this application do not indicate a limitation in quantity and may represent a singular or plural number. The terms "comprise", "include", "have" and any variations thereof involved in this application are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device that includes a series of steps or units (units) is not limited to the listed steps or units, but may further include unlisted steps or units, or may further include other steps or units inherent to these processes, methods, products or devices. The words such as "connect", "be connected", "couple" and the like involved in this application are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. The "plurality" / "several" involved in this application refers to two or more. "And / or" describes the association relationship of associated objects and indicates that three relationships may exist. For example, "A and / or B" may represent: A exists alone, A and B exist simultaneously, and B exists alone. The character " / " generally represents an "or" relationship between the front and back associated objects. The terms "first", "second", "third" and the like involved in this application are only used to distinguish similar objects and do not represent a specific order for the objects.

[0079] Example 1

[0080] This example is a schematic example of the present invention.

[0081] A preparation method of a metal framework material for adjustable lipase selective catalysis, comprising:

[0082] (1) Immobilized lipase

[0083] Add lipase to a solution containing Zn(NO3)2·6H2O and mix to form a first mixed solution;

[0084] Add an organic ligand to the first mixed solution and mix and react to form a second mixed solution;

[0085] Perform solid-liquid separation on the second mixed solution to obtain a first crude product;

[0086] Wash and dry the first crude product to obtain immobilized lipase CRL@ZIF-8;

[0087] (2) Amination modification

[0088] Mix and react the immobilized lipase with ammonia water to form a third mixed solution;

[0089] Perform solid-liquid separation on the third mixed solution to obtain the second crude product;

[0090] Dry the second crude product to obtain the immobilized lipase CRL@ZIF-NH2 modified with an amino group;

[0091] (III) Modification with polyisopropylacrylamide

[0092] Mix and react the solution containing polyisopropylacrylamide with the solution containing the immobilized lipase modified with an amino group to form a fourth mixed solution;

[0093] Perform solid-liquid separation on the fourth mixed solution to obtain the third crude product;

[0094] Wash and dry the third crude product to obtain a metal framework material, wherein the metal framework material is the immobilized lipase CRL@ZIF-8-PNIPAM modified with polyisopropylacrylamide and an amino group;

[0095] Among them, CRL@ZIF-8-PNIPAM selectively catalyzes different guest molecules at different temperatures.

[0096] Among them, the lipase is Candida rugosa lipase (CRL).

[0097] Among them, the protein concentration of CRL@ZIF is measured by a BCA protein assay kit. The protein content is calculated by the following formula:

[0098] m = c1v1 - c2v2

[0099] Among them, m is the protein content, c1 is the protein concentration of CRL used in the immobilization process, v1 is the volume of the enzyme solution used in the immobilization process, c2 is the protein concentration in the supernatant obtained after the washing process, and v2 is the volume of the supernatant obtained after the washing process.

[0100] In some of these embodiments, the mass ratio of Zn(NO3)2·6H2O to the lipase is 1.97 - 5.9:1.

[0101] Preferably, the mass ratio of Zn(NO3)2·6H2O to the lipase is 1.97:1, 2.95:1, 3.95:1, 5.9:1.

[0102] In some of these embodiments, the mass ratio of the organic ligand to the lipase is 36.75 - 113.5:1.

[0103] Preferably, the mass ratio of the organic ligand to the lipase is 36.75:1, 37.8:1, 75.6:1, 113.5:1.

[0104] In some of these embodiments, the organic ligand is 2-methylimidazole.

[0105] In some of these embodiments, immobilized lipase, ammonia water, and deionized water are mixed and reacted.

[0106] In some of these embodiments, lipase is added to a solution containing Zn(NO3)2·6H2O and stirred at room temperature for 5 min to 45 min at a rotation speed of 120 rpm to 300 rpm.

[0107] Preferably, the stirring time is 5 min, 15 min, 25 min, 30 min, or 45 min.

[0108] Preferably, the rotation speed is 120 rpm, 150 rpm, 200 rpm, 240 rpm, or 300 rpm.

[0109] In some of these embodiments, the organic ligand is added to the first mixed solution and reacted at room temperature for 30 min to 1440 min at a rotation speed of 120 rpm to 300 rpm.

[0110] Preferably, the reaction time is 30 min, 48 min, 60 min, 75 min, 90 min, 120 min, 240 min, 480 min, 600 min, 720 min, 900 min, 1080 min, 1200 min, or 1440 min.

[0111] In some of these embodiments, the second mixed solution is centrifuged for 10 min to 15 min at a rotation speed of 8000 rpm to 10000 rpm.

[0112] Preferably, the centrifugation time is 10 min, 12 min, or 15 min.

[0113] In some of these embodiments, the first crude product is washed with deionized water at least 2 times.

[0114] Preferably, the first crude product is washed with deionized water 3 times.

[0115] In some of these embodiments, the washed first crude product is vacuum dried at 55°C for at least 6 h.

[0116] Preferably, the vacuum drying time is 8 h to 12 h.

[0117] In some of these embodiments, the added mass of the immobilized lipase is 0.1 mg to 1 g.

[0118] In some of these embodiments, the added volume of ammonia water is 6.25 mL to 62.5 mL.

[0119] In some of these embodiments, the immobilized lipase is mixed with ammonia water and reacted at 27 °C to 29 °C for 22 h to 24 h, and the rotation speed is 120 rpm to 300 rpm.

[0120] Preferably, the rotation speeds are 120 rpm, 150 rpm, 200 rpm, 240 rpm, and 300 rpm.

[0121] In some of these embodiments, the third mixed solution is allowed to stand for 5 min to 20 min, and the upper layer solution is removed.

[0122] Preferably, the standing times are 5 min, 10 min, 12 min, 15 min, 18 min, and 20 min.

[0123] In some of these embodiments, the second crude product is vacuum dried at 55 °C for at least 6 h.

[0124] Preferably, the vacuum drying time is 8 h to 12 h.

[0125] In some of these embodiments, the mass ratio of polyisopropylacrylamide to CRL@ZIF-NH2 is 1 to 2:1.

[0126] Preferably, the mass ratio of polyisopropylacrylamide to CRL@ZIF-NH2 is 1:1, 1.5:1, and 2:1.

[0127] In some of these embodiments, the solvent of the solution containing polyisopropylacrylamide is chloroform.

[0128] In some of these embodiments, the solvent of the solution containing CRL@ZIF-NH2 is chloroform.

[0129] In some of these embodiments, the solution containing polyisopropylacrylamide is mixed with the solution containing the immobilized lipase modified with amino groups and reacted at 50 °C to 55 °C for 24 h to 26 h.

[0130] In some of these embodiments, the fourth mixed solution is allowed to stand for 5 min to 20 min, and the upper layer solution is removed.

[0131] Preferably, the standing times are 5 min, 10 min, 12 min, 15 min, 18 min, and 20 min.

[0132] In some of these embodiments, the third crude product is washed with chloroform at least 2 times.

[0133] Preferably, the third crude product is washed with chloroform 3 times.

[0134] In some of these embodiments, the washed third crude product is vacuum dried at 55 °C for at least 6 h.

[0135] Preferably, vacuum drying is carried out for 8 h to 12 h.

[0136] The metal-organic framework material prepared by the above preparation method and selectively catalyzed by the adjustable lipase can be applied to biocatalysis.

[0137] Specifically, the metal-organic framework material can selectively catalyze guest molecules at different temperatures (27 °C, 40 °C), and convenient switching between these two states can be achieved simply by changing the temperature. These characteristics stem from the rapid conformational change (coiling-globular transition) of the thermosensitive polymer attached to the MOF surface.

[0138] The advantages of the present invention are that the pore size of CRL@ZIF-8-PNIPAM is regulated by the reaction temperature, temporarily enhancing the binding of lipase to different substrates, endowing CRL@ZIF-8-PNIPAM with a novel and adjustable substrate-selective catalytic function; the selective catalytic behavior of the immobilized lipase towards substrates is regulated by adjusting the reaction temperature, that is, different substrates can be catalyzed at different reaction temperatures to obtain the target product.

[0139] Example 2

[0140] This example is a specific embodiment of the present invention.

[0141] A preparation method of a metal-organic framework material selectively catalyzed by an adjustable lipase, comprising:

[0142] (1) Immobilized lipase

[0143] Add 50 mg of CRL to 50 mL of a solution containing 0.1475 g of Zn(NO3)2·6H2O, and stir at 300 rpm at room temperature for 5 min to form a first mixed solution;

[0144] Add 1.8375 g of 2-methylimidazole to the first mixed solution, and react at 300 rpm at room temperature for 30 min to form a second mixed solution;

[0145] After the reaction is completed, transfer it to a 100 mL centrifuge tube,

[0146] Centrifuge the second mixed solution at 10000 rpm for 10 min to obtain a first crude product;

[0147] Wash the first crude product 3 times with deionized water;

[0148] The first crude product after washing was dried overnight under vacuum at 55 °C to obtain immobilized lipase CRL@ZIF-8;

[0149] (II) Amination modification

[0150] 0.1 mg of CRL@ZIF-8 was added to a mixture of 6.25 mL of NH₃·H₂O and 2.5 mL of deionized water, and the reaction was carried out at 28 °C at 200 rpm for 24 h to form a third mixed solution;

[0151] After the reaction was completed, the third mixed solution was allowed to stand for 10 min, and the upper layer solution was removed to obtain a second crude product;

[0152] The second crude product was dried overnight under vacuum at 55 °C to obtain amination-modified immobilized lipase CRL@ZIF-8-NH₂;

[0153] (III) Poly(N-isopropylacrylamide) modification

[0154] 20 mg of PNIPAM was dissolved in 1 mL of chloroform;

[0155] 20 mg of CRL@ZIF-8-NH₂ was dissolved in 1.5 mL of chloroform;

[0156] The above two were added to a reaction kettle and mixed, and the reaction was carried out at 55 °C for 24 h to form a fourth mixed solution;

[0157] The fourth mixed solution was allowed to stand for 10 min, and the upper layer solution was removed to obtain a third crude product;

[0158] The third crude product was washed 3 times with chloroform;

[0159] The washed third crude product was dried overnight under vacuum at 55 °C to obtain a metal framework material, wherein the metal framework material is poly(N-isopropylacrylamide)-modified amination-modified immobilized lipase CRL@ZIF-8-PNIPAM;

[0160] Among them, CRL@ZIF-8-PNIPAM selectively catalyzes different guest molecules at different temperatures.

[0161] Example 3

[0162] This example is a specific example of the present invention.

[0163] A preparation method of a metal framework material for adjustable lipase selective catalysis, comprising:

[0164] (I) Immobilized lipase

[0165] Add 50 mg of CRL to 50 mL of a solution containing 0.295 g of Zn(NO3)2·6H2O, and stir at 180 rpm for 20 min at room temperature to form a first mixed solution;

[0166] Add 5.675 g of 2-methylimidazole to the first mixed solution, and react at 180 rpm for 90 min at room temperature to form a second mixed solution;

[0167] After the reaction is completed, transfer it to a 100 mL centrifuge tube,

[0168] Centrifuge the second mixed solution at 10000 rpm for 15 min to obtain a first crude product;

[0169] Wash the first crude product 3 times with deionized water;

[0170] Vacuum dry the washed first crude product at 55 °C overnight to obtain immobilized lipase CRL@ZIF-8;

[0171] (II) Amination modification

[0172] Add 1 g of CRL@ZIF-8 to a mixture of 62.5 mL of NH3·H2O and 25 mL of deionized water, and react at 300 rpm for 23 h at 27 °C to form a third mixed solution;

[0173] After the reaction is completed, let the third mixed solution stand for 15 min, and remove the upper layer solution to obtain a second crude product;

[0174] Vacuum dry the second crude product at 55 °C overnight to obtain aminated modified immobilized lipase CRL@ZIF-8-NH2;

[0175] (III) Polyisopropylacrylamide modification

[0176] Dissolve 40 mg of PNIPAM in 2 mL of chloroform;

[0177] Dissolve 20 mg of CRL@ZIF-8-NH2 in 1.5 mL of chloroform;

[0178] Add the above two to the reaction kettle and mix, and react at 52 °C for 26 h to form a fourth mixed solution;

[0179] Let the fourth mixed solution stand for 15 min, and remove the upper layer solution to obtain a third crude product;

[0180] Wash the third crude product 3 times with chloroform;

[0181] The third crude product after washing was dried overnight under vacuum at 55 °C to obtain a metal-organic framework material, wherein the metal-organic framework material was immobilized lipase CRL@ZIF-8-PNIPAM modified with polyisopropylacrylamide and amino groups;

[0182] Among them, CRL@ZIF-8-PNIPAM selectively catalyzes different guest molecules at different temperatures.

[0183] Example 4

[0184] This example is a specific example of the present invention.

[0185] A preparation method of a metal-organic framework material for regulating the selective catalysis of lipase includes:

[0186] (1) Immobilized lipase

[0187] 50 mg of CRL was added to 50 mL of a solution containing 0.0985 g of Zn(NO3)2·6H2O and stirred at 250 rpm for 15 min at room temperature to form a first mixed solution;

[0188] 1.89 g of 2-methylimidazole was added to the first mixed solution and reacted at 250 rpm for 60 min at room temperature to form a second mixed solution;

[0189] After the reaction was completed, it was transferred to a 100 mL centrifuge tube.

[0190] The second mixed solution was centrifuged at 8000 rpm for 15 min to obtain a first crude product;

[0191] The first crude product was washed 3 times with deionized water;

[0192] The washed first crude product was dried overnight under vacuum at 55 °C to obtain immobilized lipase CRL@ZIF-8;

[0193] (2) Amino modification

[0194] 0.5 g of CRL@ZIF-8 was added to a mixture of 31.25 mL of NH3·H2O and 12.5 mL of deionized water and reacted at 200 rpm for 24 h at 29 °C to form a third mixed solution;

[0195] After the reaction was completed, the third mixed solution was allowed to stand for 20 min and the upper layer solution was removed to obtain a second crude product;

[0196] The second crude product was dried overnight under vacuum at 55 °C to obtain amino-modified immobilized lipase CRL@ZIF-8-NH2;

[0197] (3) Modification with polyisopropylacrylamide

[0198] Dissolve 30 mg of PNIPAM in 2.25 mL of chloroform;

[0199] Dissolve 20 mg of CRL@ZIF-8-NH2 in 1.5 mL of chloroform;

[0200] Add the above two to a reaction kettle and mix them, and react at 50 °C for 26 h to form a fourth mixed solution;

[0201] Let the fourth mixed solution stand for 10 min and remove the upper layer solution to obtain the third crude product;

[0202] Wash the third crude product with chloroform three times;

[0203] Vacuum dry the washed third crude product at 55 °C overnight to obtain a metal framework material, where the metal framework material is immobilized lipase CRL@ZIF-8-PNIPAM modified with polyisopropylacrylamide;

[0204] Among them, CRL@ZIF-8-PNIPAM selectively catalyzes different guest molecules at different temperatures.

[0205] Example 5

[0206] This example is a specific example of the present invention.

[0207] A preparation method of a metal framework material for adjustable lipase selective catalysis includes:

[0208] (1) Immobilized lipase

[0209] Add 50 mg of CRL to 50 mL of a solution containing 0.1975 g of Zn(NO3)2·6H2O and stir at 180 rpm at room temperature for 45 min to form a first mixed solution;

[0210] Add 2.8375 g of 2-methylimidazole to the first mixed solution and react at 180 rpm at room temperature for 240 min to form a second mixed solution;

[0211] After the reaction is completed, transfer it to a 100 mL centrifuge tube,

[0212] Centrifuge the second mixed solution at 9000 rpm for 12 min to obtain the first crude product;

[0213] Wash the first crude product with deionized water three times;

[0214] The washed first crude product was dried overnight under vacuum at 55 °C to obtain immobilized lipase CRL@ZIF-8;

[0215] (II) Amination modification

[0216] 0.8 g of CRL@ZIF-8 was added to a mixture of 50 mL of NH3·H2O and 20 mL of deionized water and reacted at 27 °C at 180 rpm for 24 h to form a third mixed solution;

[0217] After the reaction was completed, the third mixed solution was allowed to stand for 10 min and the upper layer solution was removed to obtain a second crude product;

[0218] The second crude product was dried overnight under vacuum at 55 °C to obtain amination-modified immobilized lipase CRL@ZIF-8-NH2;

[0219] (III) Polyisopropylacrylamide modification

[0220] 25 mg of PNIPAM was dissolved in 1.875 mL of chloroform;

[0221] 20 mg of CRL@ZIF-8-NH2 was dissolved in 1.5 mL of chloroform;

[0222] The above two were added to a reaction kettle and mixed, and reacted at 54 °C for 25 h to form a fourth mixed solution;

[0223] The fourth mixed solution was allowed to stand for 15 min and the upper layer solution was removed to obtain a third crude product;

[0224] The third crude product was washed 3 times with chloroform;

[0225] The washed third crude product was dried overnight under vacuum at 55 °C to obtain a metal framework material, wherein the metal framework material is polyisopropylacrylamide-modified amination-modified immobilized lipase CRL@ZIF-8-PNIPAM;

[0226] Among them, CRL@ZIF-8-PNIPAM selectively catalyzes different guest molecules at different temperatures.

[0227] Example 6

[0228] This example is a specific example of the present invention.

[0229] A preparation method of a metal framework material capable of regulating the selective catalysis of lipase includes:

[0230] (I) Immobilized lipase

[0231] Add 50 mg of CRL to 50 mL of a solution containing 0.15 g of Zn(NO3)2·6H2O, and stir at 150 rpm for 35 min at room temperature to form a first mixed solution;

[0232] Add 2.5 g of 2-methylimidazole to the first mixed solution, and react at 150 rpm for 600 min at room temperature to form a second mixed solution;

[0233] After the reaction is completed, transfer it to a 100 mL centrifuge tube,

[0234] Centrifuge the second mixed solution at 10000 rpm for 14 min to obtain a first crude product;

[0235] Wash the first crude product with deionized water 3 times;

[0236] Vacuum dry the washed first crude product at 55 °C overnight to obtain immobilized lipase CRL@ZIF-8;

[0237] (II) Amination modification

[0238] Add 0.2 g of CRL@ZIF-8 to a mixture of 12.5 mL of NH3·H2O and 5 mL of deionized water, and react at 240 rpm for 22 h at 28 °C to form a third mixed solution;

[0239] After the reaction is completed, let the third mixed solution stand for 15 min, and remove the upper layer solution to obtain a second crude product;

[0240] Vacuum dry the second crude product at 55 °C overnight to obtain aminated modified immobilized lipase CRL@ZIF-8-NH2;

[0241] (III) Polyisopropylacrylamide modification

[0242] Dissolve 35 mg of PNIPAM in 2.625 mL of chloroform;

[0243] Dissolve 20 mg of CRL@ZIF-8-NH2 in 1.5 mL of chloroform;

[0244] Add the above two to the reaction kettle and mix, and react at 54 °C for 25 h to form a fourth mixed solution;

[0245] Let the fourth mixed solution stand for 15 min, and remove the upper layer solution to obtain a third crude product;

[0246] Wash the third crude product with chloroform 3 times;

[0247] The third crude product after washing was dried overnight under vacuum at 55 °C to obtain a metal framework material, wherein the metal framework material was immobilized lipase CRL@ZIF-8-PNIPAM modified with polyisopropylacrylamide and amino groups;

[0248] Among them, CRL@ZIF-8-PNIPAM selectively catalyzes different guest molecules at different temperatures.

[0249] Example 7

[0250] This example is a specific example of the present invention.

[0251] A preparation method of a metal framework material capable of regulating the selective catalysis of lipase includes:

[0252] (1) Immobilized lipase

[0253] 50 mg of CRL was added to 50 mL of a solution containing 0.25 g of Zn(NO3)2·6H2O and stirred at 270 rpm at room temperature for 25 min to form a first mixed solution;

[0254] 3.5 g of 2-methylimidazole was added to the first mixed solution and reacted at 270 rpm at room temperature for 1440 min to form a second mixed solution;

[0255] After the reaction, it was transferred to a 100 mL centrifuge tube.

[0256] The second mixed solution was centrifuged at 8000 rpm for 12 min to obtain a first crude product;

[0257] The first crude product was washed 3 times with deionized water;

[0258] The washed first crude product was dried overnight under vacuum at 55 °C to obtain immobilized lipase CRL@ZIF-8;

[0259] (2) Amino modification

[0260] 0.3 g of CRL@ZIF-8 was added to a mixture of 18.75 mL of NH3·H2O and 7.5 mL of deionized water and reacted at 270 rpm at 28 °C for 24 h to form a third mixed solution;

[0261] After the reaction, the third mixed solution was allowed to stand for 10 min and the upper layer solution was removed to obtain a second crude product;

[0262] The second crude product was dried overnight under vacuum at 55 °C to obtain amino-modified immobilized lipase CRL@ZIF-8-NH2;

[0263] (3) Polyisopropylacrylamide modification

[0264] Dissolve 36 mg of PNIPAM in 2.7 mL of chloroform;

[0265] Dissolve 20 mg of CRL@ZIF-8-NH2 in 1.5 mL of chloroform;

[0266] Add the above two to the reaction kettle and mix, and react at 53 °C for 25 h to form the fourth mixed solution;

[0267] Let the fourth mixed solution stand for 10 min, and remove the upper layer solution to obtain the third crude product;

[0268] Wash the third crude product with chloroform 3 times;

[0269] Vacuum dry the washed third crude product at 55 °C overnight to obtain the metal framework material, where the metal framework material is immobilized lipase CRL@ZIF-8-PNIPAM modified with polyisopropylacrylamide;

[0270] Among them, CRL@ZIF-8-PNIPAM selectively catalyzes different guest molecules at different temperatures.

[0271] Example 8

[0272] This example is a specific example of the present invention.

[0273] A preparation method of a metal framework material for adjustable lipase selective catalysis, including:

[0274] (1) Immobilized lipase

[0275] Add 50 mg of CRL to 50 mL of a solution containing 0.125 g of Zn(NO3)2·6H2O, and stir at 200 rpm at room temperature for 25 min to form the first mixed solution;

[0276] Add 2.5 g of 2-methylimidazole to the first mixed solution, and react at 200 rpm at room temperature for 1200 min to form the second mixed solution;

[0277] After the reaction is completed, transfer it to a 100 mL centrifuge tube,

[0278] Centrifuge the second mixed solution at 9000 rpm for 10 min to obtain the first crude product;

[0279] Wash the first crude product with deionized water 3 times;

[0280] Vacuum dry the washed first crude product at 55 °C overnight to obtain immobilized lipase CRL@ZIF-8;

[0281] (II) Amination modification

[0282] Add 0.6 g of CRL@ZIF-8 to the mixture of 37.5 mL of NH₃·H₂O and 15 mL of deionized water, and react at 28 °C at 250 rpm for 23 h to form the third mixed solution;

[0283] After the reaction, let the third mixed solution stand for 5 min, and remove the upper layer solution to obtain the second crude product;

[0284] Vacuum dry the second crude product at 55 °C overnight to obtain amination-modified immobilized lipase CRL@ZIF-8-NH₂;

[0285] (III) Polyisopropylacrylamide modification

[0286] Dissolve 24 mg of PNIPAM in 1.8 mL of chloroform;

[0287] Dissolve 20 mg of CRL@ZIF-8-NH₂ in 1.5 mL of chloroform;

[0288] Add the above two to the reaction kettle and mix, and react at 53 °C for 24 h to form the fourth mixed solution;

[0289] Let the fourth mixed solution stand for 5 min, and remove the upper layer solution to obtain the third crude product;

[0290] Wash the third crude product with chloroform three times;

[0291] Vacuum dry the washed third crude product at 55 °C overnight to obtain the metal framework material, where the metal framework material is polyisopropylacrylamide-modified amination-modified immobilized lipase CRL@ZIF-8-PNIPAM;

[0292] Among them, CRL@ZIF-8-PNIPAM selectively catalyzes different guest molecules at different temperatures.

[0293] Example 9

[0294] This example relates to the verification of the selective catalysis of CRL@ZIF-8-PNIPAM of the present invention.

[0295] Take 54.4 mg of phenyl p-nitropalmitate (PNP-P) and dissolve it in 10 mL of absolute ethanol to obtain a 14.4 mM PNP-P solution;

[0296] Take 8 mg of CRL@ZIF-8-PNIPAM in a reactor, add 4 mL of phosphate buffer solution (pH = 7.5, 0.5 mM), and preheat at 27 °C and 40 °C for 30 min respectively;

[0297] After 30 min, add 4 mL of PNP-P (14.4 mM) to the reactor and react at 200 rpm and 27 °C for 30 min;

[0298] After 30 min, take the supernatant and measure the OD value at 410 nm using an enzyme-labeling instrument;

[0299] After 30 min, react at 40 °C for half an hour, i.e., the 60 min node, and measure the OD value at 410 nm using an enzyme-labeling instrument;

[0300] Then react at 27 °C for half an hour, i.e., the 90 min node, and measure the OD value at 410 nm using an enzyme-labeling instrument;

[0301] Finally, react at 40 °C for half an hour, i.e., the 120 min node, and measure the OD value at 410 nm using an enzyme-labeling instrument;

[0302] Determine the concentration of p-NP according to the p-NP standard curve (Y = 1.2189X + 0.0336, R 2 = 0.9945).

[0303] As Figure 1 shown, according to the previously obtained immobilized protein content, the calculated enzyme activity results at the four nodes of 30 min, 60 min, 90 min, and 120 min are 0.25619 mM pnp / mg protein / min, 0.27667 mM pnp / mg protein / min, 0.25519 mM pnp / mg protein / min, and 0.32779 mM pnp / mg protein / min respectively.

[0304] Example 10

[0305] This example relates to the selective catalysis verification of the CRL@ZIF-8-PNIPAM of the present invention.

[0306] Take 46.28 mg of 4-nitrophenyl laurate (PNP-M) and dissolve it in 10 mL of absolute ethanol to obtain a 14.4 mM PNP-M solution;

[0307] Take 8 mg of CRL@ZIF-8-PNIPAM in a reactor, add 4 mL of phosphate buffer solution (pH = 7.5, 0.5 mM), and preheat at 27 °C and 40 °C for 30 min respectively;

[0308] After 30 min, 4 mL of PNP-M (14.4 mM) was added to the reactor, and the reaction was carried out at 200 rpm and 27 °C for 30 min;

[0309] After 30 min, the supernatant was taken, and the OD value was measured at 410 nm by an enzyme-labeled instrument;

[0310] After 30 min, the reaction was carried out at 40 °C for half an hour, i.e., the 60 min node, and the OD value was measured at 410 nm by an enzyme-labeled instrument;

[0311] After that, the reaction was carried out at 27 °C for half an hour, i.e., the 90 min node, and the OD value was measured at 410 nm by an enzyme-labeled instrument;

[0312] Finally, the reaction was carried out at 40 °C for half an hour, i.e., the 120 min node, and the OD value was measured at 410 nm by an enzyme-labeled instrument;

[0313] According to the p-NP standard curve, the concentration of p-NP was determined (Y = 1.2189X + 0.0336, R 2 = 0.9945).

[0314] As Figure 2 shown, according to the previously obtained immobilized protein content, the enzyme activity results at the four nodes of 30 min, 60 min, 90 min, and 120 min were 3.10987 mM pnp / mg protein / min, 10.13301 mM pnp / mg protein / min, 21.26167 mM pnp / mg protein / min, and 19.00615 mM pnp / mg protein / min, respectively.

[0315] Example 11

[0316] This example relates to the selective catalysis verification of CRL@ZIF-8-PNIPAM of the present invention.

[0317] 25 μL of 4-nitrophenyl butyrate (PNP-Be) was taken and dissolved in 10 mL of absolute ethanol to obtain a 14.4 mM PNP-Be solution;

[0318] 8 mg of CRL@ZIF-8-PNIPAM was taken in a reactor, and 4 mL of phosphate buffer solution (pH = 7.5, 0.5 mM) was added, and it was preheated at 27 °C and 40 °C for 30 min, respectively;

[0319] After 30 min, 4 mL of PNP-Be (14.4 mM) was added to the reactor, and the reaction was carried out at 200 rpm and 27 °C for 30 min;

[0320] After 30 min, the supernatant was taken, and the OD value was measured at 410 nm by an enzyme-labeled instrument;

[0321] After 30 min, the reaction was carried out at 40 °C for half an hour, i.e., the 60 min node, and the OD value was measured at 410 nm by an enzyme-labeled instrument;

[0322] Then, the reaction was carried out at 27 °C for half an hour, i.e., the 90 min node, and the OD value was measured at 410 nm by an enzyme-labeled instrument;

[0323] Finally, the reaction was carried out at 40 °C for half an hour, i.e., the 120 min node, and the OD value was measured at 410 nm by an enzyme-labeled instrument;

[0324] According to the p-NP standard curve, the concentration of p-NP was determined (Y = 1.2189X + 0.0336, R 2 = 0.9945).

[0325] [[ID=】] Figure 3 Figure 3 As shown, according to the previously obtained content of immobilized protein, the enzyme activity results at the four nodes of 30 min, 60 min, 90 min, and 120 min were calculated to be 23.4840 mM pnp / mg protein / min, 30.8947 mM pnp / mg protein / min, 26.2340 mM pnp / mg protein / min, and 30.1357 mM pnp / mg protein / min, respectively.

[0326] Example 12

[0327] This example relates to the selective catalysis verification of CRL@ZIF-8-PNIPAM of the present invention.

[0328] 54.4 mg of phenyl p-nitropalmitate (PNP-P) was taken and dissolved in 10 mL of absolute ethanol to obtain a 14.4 mM PNP-P solution;

[0329] 2 mg of CRL@ZIF-8-PNIPAM was taken in a reactor, and 1 mL of phosphate buffer solution (pH = 7.5, 0.5 mM) was added, and it was preheated at 27 °C and 40 °C for 30 min respectively;

[0330] After 30 min, 1 mL of PNP-P (14.4 mM) was added to the reactor, and the reaction was carried out at 200 rpm at 27 °C / 40 °C for 5 min each;

[0331] After 5 min, 2 mL of Na2CO3 was added to terminate the reaction;

[0332] Centrifugation was carried out at 10000 rpm at 25 °C for 10 min, the supernatant was taken, and the OD value was measured at 410 nm by an enzyme-labeled instrument;

[0333] Determine the concentration of p-NP according to the p-NP standard curve (Y = 1.2189X + 0.0336, R 2 = 0.9945).

[0334] The enzyme activity results are 1.0668 mM pnp / mg protein / min and 1.3697 mM pnp / mg protein / min at 27 °C and 40 °C, respectively.

[0335] Example 13

[0336] This example relates to the verification of the selective catalysis of CRL@ZIF-8-PNIPAM of the present invention.

[0337] Take 46.28 mg of 4-nitrophenyl laurate (PNP-M) and dissolve it in 10 mL of absolute ethanol to obtain a 14.4 mM PNP-M solution;

[0338] Take 2 mg of CRL@ZIF-8-PNIPAM in a reactor, and add 1 mL of phosphate buffer solution (pH = 7.5, 0.5 mM), and preheat it at 27 °C and 40 °C for 30 min respectively;

[0339] After 30 min, add 1 mL of PNP-M (14.4 mM) to the reactor, and react at 200 rpm, 27 °C / 40 °C for 5 min each;

[0340] After 5 min, add 2 mL of Na2CO3 to terminate the reaction;

[0341] Centrifuge at 10000 rpm and 25 °C for 10 min, take the supernatant, and measure the OD value at 410 nm with an enzyme-labeled instrument;

[0342] Determine the concentration of p-NP according to the p-NP standard curve (Y = 1.2189X + 0.0336, R 2 = 0.9945).

[0343] The enzyme activity results are 9.2904 mM pnp / mg protein / min and 13.2811 mM pnp / mg protein / min at 27 °C and 40 °C, respectively.

[0344] Example 14

[0345] This example relates to the verification of the selective catalysis of CRL@ZIF-8-PNIPAM of the present invention.

[0346] Dissolve 25 μL of 4-nitrophenyl butyrate (PNP-Be) in 10 mL of absolute ethanol to obtain a 14.4 mM PNP-Be solution;

[0347] Take 2 mg of CRL@ZIF-8-PNIPAM in a reactor, and add 1 mL of phosphate buffer solution (pH = 7.5, 0.5 mM), and preheat it at 27 °C and 40 °C for 30 min respectively;

[0348] After 30 min, add 1 mL of PNP-Be (14.4 mM) to the reactor, and react at 200 rpm, 27 °C / 40 °C for 5 min each;

[0349] After 5 min, add 2 mL of Na2CO3 to terminate the reaction;

[0350] Centrifuge at 10000 rpm and 25 °C for 10 min, take the supernatant, and measure the OD value at 410 nm with an enzyme-labeled instrument;

[0351] Determine the concentration of p-NP according to the p-NP standard curve (Y = 1.2189X + 0.0336, R 2 = 0.9945).

[0352] The enzyme activity results are 107.2912 mM pnp / mg protein / min and 114.5924 mM pnp / mg protein / min at 27 °C and 40 °C respectively.

[0353] Comparative Example 1

[0354] This comparative example is compared with Example 9.

[0355] The free enzyme CRL is used as a substrate for a cycling experiment with 14.4 mM phenyl p-nitropalmitate (PNP-P).

[0356] React at 200 rpm and 27 °C for 30 min;

[0357] After 30 min, take the supernatant and measure the OD value at 410 nm with an enzyme-labeled instrument;

[0358] After 30 min, react at 40 °C for half an hour, that is, at the 60 min node, measure the OD value at 410 nm with an enzyme-labeled instrument;

[0359] Then react at 27 °C for half an hour, that is, at the 90 min node, measure the OD value at 410 nm with an enzyme-labeled instrument;

[0360] Finally, react at 40 °C for half an hour, that is, at the 120 min node, measure the OD value at 410 nm with an enzyme-labeled instrument;

[0361] The concentration of p-NP was determined according to the p-NP standard curve (Y = 1.2189X + 0.0336, R 2 = 0.9945).

[0362] As Figure 4 shown, based on the previously obtained immobilized protein content, the calculated enzyme activity results at the four time points of 30 min, 60 min, 90 min, and 120 min were 25.45903 mM pnp / mg protein / min, 25.44362 mM pnp / mg protein / min, 12.96717 mM pnp / mg protein / min, and 12.99929 mM pnp / mg protein / min, respectively.

[0363] Comparative Example 2

[0364] This comparative example was compared with Example 10.

[0365] The free enzyme CRL was subjected to a recycling experiment with 14.4 mM lauric acid 4-nitrophenyl ester (PNP-M) as the substrate.

[0366] The reaction was carried out at 200 rpm and 27 °C for 30 min;

[0367] After 30 min, the supernatant was taken, and the OD value was measured at 410 nm by an enzyme-labeled instrument;

[0368] After 30 min, the reaction was carried out at 40 °C for half an hour (i.e., the 60-min time point), and the OD value was measured at 410 nm by an enzyme-labeled instrument;

[0369] Then, the reaction was carried out at 27 °C for half an hour (i.e., the 90-min time point), and the OD value was measured at 410 nm by an enzyme-labeled instrument;

[0370] Finally, the reaction was carried out at 40 °C for half an hour (i.e., the 120-min time point), and the OD value was measured at 410 nm by an enzyme-labeled instrument;

[0371] The concentration of p-NP was determined according to the p-NP standard curve (Y = 1.2189X + 0.0336, R 2 = 0.9945).

[0372] As Figure 5As shown, based on the previously obtained immobilized protein content, the calculated enzyme activity results at the four time points of 30 min, 60 min, 90 min, and 120 min are 28.94549 mM pnp / mg protein / min, 28.23638 mM pnp / mg protein / min, 13.99229 mM pnp / mg protein / min, and 14.17343 mM pnp / mg protein / min, respectively.

[0373] Comparative Example 3

[0374] This comparative example was compared with Example 11.

[0375] The free enzyme CRL was subjected to a cycling experiment with 14.4 mM 4-nitrophenyl butyrate (PNP-Be) as the substrate.

[0376] The reaction was carried out at 200 rpm and 27 °C for 30 min;

[0377] After 30 min, the supernatant was taken, and the OD value was measured at 410 nm by an enzyme-labeling instrument;

[0378] After 30 min, the reaction was carried out at 40 °C for half an hour (i.e., the 60-min time point), and the OD value was measured at 410 nm by an enzyme-labeling instrument;

[0379] Then, the reaction was carried out at 27 °C for half an hour (i.e., the 90-min time point), and the OD value was measured at 410 nm by an enzyme-labeling instrument;

[0380] Finally, the reaction was carried out at 40 °C for half an hour (i.e., the 120-min time point), and the OD value was measured at 410 nm by an enzyme-labeling instrument;

[0381] According to the p-NP standard curve, the concentration of p-NP was determined (Y = 1.2189X + 0.0336, R 2 = 0.9945).

[0382] As Figure 6 shown, based on the previously obtained immobilized protein content, the calculated enzyme activity results at the four time points of 30 min, 60 min, 90 min, and 120 min are 28.88639 mM pnp / mg protein / min, 28.50101 mM pnp / mg protein / min, 14.22609 mM pnp / mg protein / min, and 14.16058 mM pnp / mg protein / min, respectively.

[0383] Comparative Example 4

[0384] This comparative example was compared with Example 12.

[0385] The free enzyme CRL was subjected to a selective catalysis experiment with 14.4 mM phenyl p-nitropalmitate (PNP-P) as the substrate.

[0386] Take 1 mL of the free enzyme and place it in a reactor, and add 1 mL of phosphate buffer solution (pH = 7.5, 0.5 mM). Preheat at 27 °C and 40 °C for 30 min respectively;

[0387] After 30 min, add 1 mL of PNP-P (14.4 mM) to the reactor and react at 200 rpm, 27 °C / 40 °C for 5 min each;

[0388] After 5 min, add 2 mL of Na2CO3 to terminate the reaction respectively;

[0389] Centrifuge at 10000 rpm and 25 °C for 10 min, take the supernatant, and measure the OD value at 410 nm with an enzyme-labeling instrument;

[0390] Determine the concentration of p-NP according to the p-NP standard curve (Y = 1.2189X + 0.0336, R 2 = 0.9945).

[0391] The enzyme activity results were 52.3937 mM pnp / mg protein / min and 21.3144 mM pnp / mg protein / min at 27 °C and 40 °C respectively.

[0392] Comparative Example 5

[0393] This comparative example was compared with Example 13.

[0394] The free enzyme CRL was subjected to a selective catalysis experiment with 14.4 mM 4-nitrophenyl laurate (PNP-M) as the substrate.

[0395] Take 1 mL of the free enzyme and place it in a reactor, and add 1 mL of phosphate buffer solution (pH = 7.5, 0.5 mM). Preheat at 27 °C and 40 °C for 30 min respectively;

[0396] After 30 min, add 1 mL of PNP-M (14.4 mM) to the reactor and react at 200 rpm, 27 °C / 40 °C for 5 min each;

[0397] After 5 min, add 2 mL of Na2CO3 to terminate the reaction respectively;

[0398] Centrifuge at 10000 rpm and 25 °C for 10 min, take the supernatant, and measure the OD value at 410 nm with an enzyme-labeling instrument;

[0399] The concentration of p-NP was determined according to the p-NP standard curve (Y = 1.2189X + 0.0336, R 2 = 0.9945).

[0400] The enzyme activity results were 65.4571 mM pnp / mg protein / min and 27.3160 mM pnp / mg protein / min at 27 °C and 40 °C, respectively.

[0401] Comparative Example 6

[0402] This comparative example was compared with Example 14.

[0403] The free enzyme CRL was used for a selective catalytic experiment with 14.4 mM 4-nitrophenyl butyrate (PNP-Be) as the substrate.

[0404] Take 1 mL of the free enzyme in a reactor, and add 1 mL of phosphate buffer solution (pH = 7.5, 0.5 mM), and preheat at 27 °C and 40 °C for 30 min respectively;

[0405] After 30 min, add 1 mL of PNP-M (14.4 mM) to the reactor, and react at 200 rpm, 27 °C / 40 °C for 5 min each;

[0406] After 5 min, add 2 mL of Na2CO3 to terminate the reaction respectively;

[0407] Centrifuge at 10000 rpm and 25 °C for 10 min, take the supernatant, and measure the OD value at 410 nm with an enzyme-labeled instrument;

[0408] The concentration of p-NP was determined according to the p-NP standard curve (Y = 1.2189X + 0.0336, R 2 = 0.9945).

[0409] The enzyme activity results were 70.9423 mM pnp / mg protein / min and 34.4859 mM pnp / mg protein / min at 27 °C and 40 °C, respectively.

[0410] Verification results

[0411] Figure 7 are the verification results of Examples 9 to 11. As Figure 7As shown, all three substrates showed a trend of increasing product, indicating that CRL@ZIF-8-PNIPAM can catalyze the reaction of the substrates. Among the three substrates, CRL@ZIF-8-PNIPAM showed the most obvious selective catalytic behavior for PNP Be. In the temperature cycle verification, the enzyme activity at 40 °C twice was higher than that at 27 °C, probably because the space in the microreactor was small and the generated products were not exchanged out of the reactor in time. The same trend was shown for the catalysis of PNP M, verifying the selective catalytic behavior of CRL@ZIF-8-PNIPAM for the substrates. Compared with the pore size of the MOF, the PNP M molecule is larger and difficult to enter the substrate for selective catalysis, resulting in lower enzyme activity and making it difficult to observe the selective catalytic behavior of CRL@ZIF-8-PNIPAM.

[0412] Figure 8 are the verification results of Examples 12 to 14 and Comparative Examples 4 to 6. As Figure 8 shown, the activity of the free lipase increased after being immobilized. Comparing the enzyme activities at 27 °C and 40 °C, the lower enzyme activity at 27 °C compared to 40 °C was probably because the space in the microreactor was small and the generated products were not exchanged out of the reactor in time, resulting in the reaction not proceeding continuously rapidly. At 40 °C, it may be because the entry of the substrate was limited due to the "off" state.

[0413] The above are only the preferred embodiments of the present invention, and do not limit the implementation manners and protection scope of the present invention. For those skilled in the art, it should be realized that all equivalent substitutions and obvious changes made by using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a metal framework material capable of regulating the selective catalysis of lipase, characterized in that: include: (1) Immobilized lipase adding lipase to a solution containing Zn(NO3)2·6H2O and mixing the mixture to form a first mixed solution; adding an organic ligand to the first mixed solution for a mixing reaction to form a second mixed solution, wherein the organic ligand is 2-methylimidazole; performing solid-liquid separation on the second mixed solution to obtain a first crude product; Washing and drying the first crude product to obtain immobilized lipase CRL@ZIF-8; (II) Amino modification mixing the immobilized lipase with aqueous ammonia to react to form a third mixed solution; performing solid-liquid separation on the third mixed solution to obtain a second crude product; The second crude product is dried to obtain amino-modified immobilized lipase CRL@ZIF-NH2; (3) Polyisopropylacrylamide modification Mixing a solution containing polyisopropylacrylamide with a solution containing the amino-modified immobilized lipase to form a fourth mixed solution, wherein the solvent of the solution containing polyisopropylacrylamide is chloroform, and the solvent of the solution containing CRL@ZIF-NH2 is chloroform; performing solid-liquid separation on the fourth mixed solution to obtain a third crude product; Washing and drying the third crude product to obtain a metal skeleton material, wherein the metal skeleton material is an amino-modified immobilized lipase CRL@ZIF-8-PNIPAM modified with polyisopropylacrylamide; Among them, CRL@ZIF-8-PNIPAM selectively catalyzes different guest molecules at different temperatures.

2. The preparation method according to claim 1, characterized in that In (a) Immobilized lipase: The mass ratio of the Zn(NO3)2·6H2O to the lipase is 1.97-5.9:1; and / or The mass ratio of the organic ligand to the lipase is 36.75-113.5:

1.

3. The preparation method according to claim 1 or 2, characterized in that In (a) Immobilized lipase: Add lipase to a solution containing Zn(NO3)2·6H2O and stir at room temperature for 5 min to 45 min at a speed of 120 rpm to 300 rpm; and / or Adding the organic ligand to the first mixed solution, reacting at room temperature for 30 min to 1440 min at a rotation speed of 120 rpm to 300 rpm; and / or Centrifuging the second mixed solution for 10 min to 15 min at a speed of 8000 rpm to 10000 rpm; and / or Washing the first crude product with deionized water at least twice; and / or The washed first crude product was dried under vacuum at 55° C. for at least 6 h.

4. The preparation method according to claim 1, characterized in that In (di)amination modification: The added mass of the immobilized lipase is 0.1 mg to 1 g; and / or The added volume of the ammonia water is 6.25 mL to 62.5 mL.

5. The preparation method according to claim 1 or 4, characterized in that In (di)amination modification: Mixing the immobilized lipase with aqueous ammonia, reacting at 27° C. to 29° C. for 22 h to 24 h at a rotation speed of 120 rpm to 300 rpm; and / or The third mixed solution is allowed to stand for 5 to 20 minutes, and the upper layer of the solution is removed; and / or The second crude product was dried under vacuum at 55°C for at least 6 h.

6. The preparation method according to claim 1, characterized in that In (iii) polyisopropylacrylamide modification: The mass ratio of the polyisopropylacrylamide to the CRL@ZIF-NH2 is 1-2:

1.

7. The preparation method according to claim 1 or 6, characterized in that In (iii) polyisopropylacrylamide modification: Mixing a solution containing polyisopropylacrylamide with a solution containing the amino-modified immobilized lipase, and reacting at 50° C. to 55° C. for 24 h to 26 h; and / or The fourth mixed solution is allowed to stand for 5 to 20 minutes, and the upper layer of the solution is removed; and / or Washing the third crude product with chloroform at least twice; and / or The washed third crude product was dried under vacuum at 55° C. for at least 6 hours.

8. The preparation method according to claim 1, characterized in that include: (1) Immobilized lipase Adding lipase to a solution containing Zn(NO3)2·6H2O and stirring at 120 rpm to 300 rpm for 5 min to 45 min at room temperature to form a first mixed solution, wherein the mass ratio of the Zn(NO3)2·6H2O to the lipase is 1.97 to 5.9:1; 2-methylimidazole is added to the first mixed solution, and reacted at room temperature at 120 rpm to 300 rpm for 30 min to 1440 min to form a second mixed solution, wherein the mass ratio of the 2-methylimidazole to the lipase is 36.75 to 113.5:1; Centrifuging the second mixed solution at 8000 rpm to 10000 rpm for 10 min to 15 min to obtain a first crude product; Washing the first crude product with deionized water at least twice; The washed first crude product was vacuum dried at 55° C. for at least 6 h to obtain immobilized lipase CRL@ZIF-8; (II) Amino modification Mixing the immobilized lipase with aqueous ammonia, reacting at 27° C. to 29° C. for 22 h to 24 h at a rotation speed of 120 rpm to 300 rpm to form a third mixed solution, wherein the added mass of the immobilized lipase is 0.1 mg to 1 g, and the added volume of the aqueous ammonia is 6.25 mL to 62.5 mL; The third mixed solution is allowed to stand for 5 to 20 minutes, and the upper layer of the solution is removed to obtain a second crude product; The second crude product was vacuum dried at 55° C. for at least 6 h to obtain amino-modified immobilized lipase CRL@ZIF-8-NH2; (3) Polyisopropylacrylamide modification Mixing a chloroform solution containing polyisopropylacrylamide with a chloroform solution containing the amino-modified immobilized lipase, and reacting at 50° C. to 55° C. for 24 h to 26 h to form a fourth mixed solution, wherein the mass ratio of the polyisopropylacrylamide to the CRL@ZIF-NH2 is 1 to 2:1; The fourth mixed solution is allowed to stand for 5 min to 20 min, and the upper layer of the solution is removed to obtain a third crude product; Washing the third crude product with chloroform at least twice; The washed third crude product is vacuum-dried at 55° C. for at least 6 hours to obtain a metal skeleton material, wherein the metal skeleton material is an amino-modified immobilized lipase CRL@ZIF-8-PNIPAM modified with polyisopropylacrylamide; Among them, CRL@ZIF-8-PNIPAM selectively catalyzes different guest molecules at different temperatures.

9. A metal skeleton material capable of regulating the selective catalysis of lipase, prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the metal framework material capable of regulating lipase selective catalysis as claimed in claim 9 in lipase biocatalysis.

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