Carrier for immobilized enzyme, preparation method and application of carrier, immobilized enzyme and preparation method of immobilized enzyme

Abstract

The invention relates to the technical field of biology, and discloses a carrier for immobilized enzyme, a preparation method and application of the carrier, the immobilized enzyme and a preparation method of the immobilized enzyme. The carrier for immobilizing the enzyme takes amino-modified cellulose acetate porous balls as a carrier. The carrier for immobilizing the enzyme can realize enzyme immobilization and multi-batch reutilization, has high storage stability and use stability, is biodegradable, reduces the use cost of the enzyme, and has significant industrial application potential and application prospects.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, for example, to a carrier for immobilized enzymes and a preparation method and application thereof, and to immobilized enzymes and a preparation method thereof. Background Art

[0002] Enzymes are high-molecular-weight substances with biocatalytic properties. Their chemical nature is protein. Their catalytic action is characterized by high selectivity, high catalytic activity, mild reaction conditions, and environmental friendliness. However, as biocatalysts, enzymes are highly sensitive to their environment and easily inactivated by physical, chemical, and biological factors. Furthermore, they can be incorporated into the product after reaction, making purification difficult and preventing continuous operation difficult.

[0003] Immobilized enzymes are free enzymes that are physically or chemically bound to a water-insoluble polymer carrier, or embedded in a water-insoluble gel or semipermeable membrane microcapsule to maintain catalytic activity and allow for repeated use. Immobilized enzymes generally have increased stability, allowing for smoother separation from the reaction system, easier transport and storage, and easier handling, allowing for multiple uses.

[0004] The most important issue in the research and application of immobilized enzyme technology is improving the activity and stability of the immobilized enzyme. At the same time, considerations must also be given to increasing the enzyme loading capacity and the degree of immobilization. When selecting a carrier, considerations should also include the compatibility of the carrier material with the enzyme molecule, the reactivity of the carrier surface groups and the chemical binding type with the enzyme, the carrier shape, and its specific surface area.

[0005] Immobilized enzyme carriers include natural materials and chemically synthesized materials. Natural materials include diatomaceous earth, gelatin, chitosan, and calcium alginate; chemically synthesized materials include resins. Diatomaceous earth and resins have poor biocompatibility, while gelatin and chitosan have poor mechanical properties. Calcium alginate is soluble in common buffers, resulting in various drawbacks. Chemically synthesized materials, such as resins, are generally non-biodegradable and have poor biocompatibility.

[0006] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of this application, and therefore may include information that does not constitute prior art known to ordinary technicians in this field. Summary of the Invention

[0007] In order to provide a basic understanding of some aspects of the disclosed embodiments, a brief summary is given below. The summary is not an extensive review, nor is it intended to identify key / critical elements or delineate the scope of protection of these embodiments, but rather serves as a prelude to the detailed description that follows.

[0008] The embodiments of the present disclosure provide a carrier for immobilized enzyme, a preparation method thereof, and an application thereof, as well as an immobilized enzyme and a preparation method thereof, so as to improve the stability and usage times of the immobilized enzyme.

[0009] In some embodiments, a carrier for immobilizing an enzyme is provided, wherein amino-modified cellulose acetate porous spheres are used as the carrier.

[0010] In some embodiments, a method for preparing a carrier for immobilizing an enzyme is provided, comprising: modifying cellulose acetate with a modifier to obtain cellulose acetate carbamate; dissolving the cellulose acetate carbamate in a first organic solvent and then dripping the solvent into water to form cellulose acetate carbamate porous balls.

[0011] In some embodiments, a method for preparing an immobilized enzyme is provided, wherein a carrier prepared by the method for preparing a carrier for immobilized enzyme described in any one of the above embodiments is combined with an enzyme to obtain an immobilized enzyme.

[0012] In some embodiments, an immobilized enzyme is provided, comprising an enzyme and a carrier, wherein the carrier comprises the carrier for immobilizing an enzyme as described in any of the above embodiments, or the carrier is prepared using the method for preparing the carrier for immobilizing an enzyme as described in any of the above embodiments.

[0013] In some embodiments, a method for preparing a carrier for immobilizing an enzyme according to any one of the above embodiments is provided for use in the biological field; or a method for preparing a carrier for immobilizing an enzyme according to any one of the above embodiments is provided for use in the biological field.

[0014] The carrier for immobilized enzyme, its preparation method, and application, and the immobilized enzyme, its preparation method, and application provided in the embodiments of the present disclosure can achieve the following technical effects:

[0015] The immobilized enzyme carrier provided in the embodiments of the present disclosure utilizes amino-modified porous cellulose acetate spheres as the carrier, which exhibits good compatibility with the enzyme. Cellulose acetate is low-cost, readily soluble in organic solvents, and easy to process. Furthermore, cellulose acetate exhibits high strength, high toughness, and is non-flammable. It also degrades in the environment, making it an environmentally friendly material, and the immobilized enzyme prepared is reusable.

[0016] Furthermore, cellulose acetate itself carries many unreacted hydroxyl groups. Using amino-modified cellulose acetate porous spheres as carriers for immobilized enzymes can produce porous spheres with amino groups on their surfaces that are active for enzyme binding. These active groups can covalently bond with chemical groups on the enzyme surface, significantly improving enzyme immobilization efficiency and increasing enzyme loading capacity. The enzyme covalently binds to the carrier, making it less likely to fall off. Furthermore, the porous structure of cellulose acetate increases the carrier's internal surface area and enzyme loading capacity, while also imparting high mechanical strength to the carrier, making it suitable for industrial operations.

[0017] The above general description and the following description are exemplary and explanatory only and are not intended to limit the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] One or more embodiments are exemplarily described by corresponding drawings. These exemplary descriptions and drawings do not limit the embodiments. Elements with the same reference numerals in the drawings are shown as similar elements. The drawings do not constitute a scale limitation. In addition,

[0019] Figure 1 Schematic diagram of a method for preparing a carrier for immobilizing an enzyme provided in an embodiment of the present disclosure;

[0020] Figure 2 is a schematic diagram of another method for preparing a carrier for immobilizing an enzyme provided in an embodiment of the present disclosure;

[0021] Figure 3 is a schematic diagram of another method for preparing a carrier for immobilizing an enzyme provided in an embodiment of the present disclosure;

[0022] Figure 4 is a schematic diagram of another method for preparing a carrier for immobilizing an enzyme provided in an embodiment of the present disclosure;

[0023] Figure 5 is a schematic diagram of a method for preparing an immobilized enzyme provided in an embodiment of the present disclosure;

[0024] Figure 6 is a schematic diagram of another method for preparing an immobilized enzyme provided in an embodiment of the present disclosure;

[0025] Figure 7 is a data graph showing the effect of different urea concentrations (first preset concentration) on the relative enzyme activity of immobilized lactase provided by an embodiment of the present disclosure;

[0026] Figure 8 This is a data graph showing the effects of different first preset temperatures on the relative enzyme activity of immobilized lactase provided by an embodiment of the present disclosure;

[0027] Figure 9 This is a data graph showing the effects of different first set time periods on the relative enzyme activity of immobilized lactase provided by an embodiment of the present disclosure;

[0028] Figure 10 is a data graph showing the effects of different second preset temperatures on the relative enzyme activity of immobilized lactase provided by an embodiment of the present disclosure;

[0029] Figure 11 This is a data graph showing the effects of different second set time lengths on the relative enzyme activity of immobilized lactase provided by an embodiment of the present disclosure;

[0030] Figure 12 This is a data graph showing the effect of different cellulose acetate carbamate solution concentrations on the relative enzyme activity of immobilized lactase provided by the embodiments of the present disclosure;

[0031] Figure 13 This is an appearance diagram of a cellulose acetate urethane porous ball provided in an embodiment of the present disclosure;

[0032] Figure 14 is a schematic diagram of a cross section of a cellulose acetate urethane porous ball provided by an embodiment of the present disclosure;

[0033] Figure 15 is a schematic diagram of a cross section of another cellulose acetate urethane porous ball provided by an embodiment of the present disclosure;

[0034] Figure 16 3. It is a data graph showing the enzyme activity, protein yield and relative enzyme activity of different mass ratios of lactase and cellulose acetate carbamate porous balls provided in the embodiments of the present disclosure;

[0035] Figure 17 The relative enzyme activity of the immobilized lactase provided in the embodiments of the present disclosure varies with the reaction batch. DETAILED DESCRIPTION

[0036] In order to be able to understand the features and technical content of the embodiments of the present disclosure in more detail, the implementation of the embodiments of the present disclosure is described in detail below in conjunction with the accompanying drawings. The accompanying drawings are for reference only and are not used to limit the embodiments of the present disclosure. In the following technical description, for the sake of convenience of explanation, a full understanding of the disclosed embodiments is provided through a number of details. However, one or more embodiments can still be implemented without these details. In other cases, to simplify the drawings, well-known structures, steps, and devices can be simplified for display.

[0037] In the description and claims of the embodiments of the present disclosure, as well as in the accompanying drawings, the terms "first," "second," and the like are used to distinguish similar items and are not necessarily used to describe a particular order or precedence. It should be understood that the terms used in this manner are interchangeable where appropriate to describe the embodiments of the present disclosure herein. In addition, the terms "including," "having," and any variations thereof are intended to cover non-exclusive inclusions.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs. The term "plurality" means two or more. In the disclosed embodiment, the character " / " indicates that the objects before and after are in an "or" relationship. For example, A / B means: A or B. The term "and / or" is a description of the association relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or, three relationships of A and B.

[0039] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present disclosure can be combined with each other.

[0040] Those skilled in the art will understand that, in the methods of various embodiments or examples, the order in which the steps are written does not imply a strict order of execution and does not constitute any limitation on the implementation process. The detailed order of execution of each step should be determined by its function and possible internal logic. Unless otherwise specified, all steps of the present application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps A and B, which means that the method may include steps A and B performed sequentially, or steps B and A performed sequentially. For example, it is mentioned that the method may also include step C, which means that step C may be added to the method in any order. For example, the method may include steps A, B and C, or steps A, C and B, or steps C, A and B, etc.

[0041] In this application, open technical features or technical solutions described with words such as "contain," "include," and "includes" do not exclude additional members beyond the listed members unless otherwise specified, and can be regarded as providing both closed features or solutions consisting of the listed members and open features or solutions that include additional members beyond the listed members. For example, if A includes a1, a2, and a3, it may or may not include other members unless otherwise specified. This can be regarded as providing both the feature or solution of "A consists of a1, a2, and a3" and the feature or solution of "A includes not only a1, a2, and a3, but also other members."

[0042] " range " disclosed in the present application can be limited in the form of lower limit and upper limit, and given range is limited by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of special range. The scope limited in this way can be including end value or excluding end value, and any end value can be included or not included independently, and can be arbitrarily combined, that is, any lower limit can form a range with any upper limit combination. For example, if the scope of 60 to 120 and 80 to 110 is listed for a particular parameter, it is understood that the scope of 60 to 110 and 80 to 120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3,4 and 5 are also listed, then the following range can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In the present application, unless otherwise specified, the numerical range " a to b " represents the abbreviation of any real number combination between a and b, wherein a and b are all real numbers. For example, the numerical range "0 to 5" means that all real numbers between "0 to 5" have been listed in this document, and "0 to 5" is just an abbreviation for these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to listing the parameter as, for example, integers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc. For example, when a parameter is expressed as an integer selected from "2 to 10", it is equivalent to listing the integers 2, 3, 4, 5, 6, 7, 8, 9 and 10. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0043] The disclosed embodiments provide a carrier for immobilizing an enzyme, using amino-modified cellulose acetate porous spheres as the carrier.

[0044] Cellulose acetate porous balls are used as carriers to bind the immobilized enzyme. Cellulose acetate porous balls are low-cost and easy to prepare. They also have high strength, high toughness, and are non-flammable. They also degrade in the environment, making them environmentally friendly. Therefore, using cellulose acetate porous balls as carriers for immobilized enzymes is low-cost and biodegradable, allowing the enzyme to be used multiple times. Cellulose acetate porous balls offer advantages such as high porosity, high enzyme loading capacity, and covalent bonding of enzyme molecules to the carrier, making them less susceptible to detachment. This improves enzyme stability.

[0045] Cellulose acetate porous balls are modified with amino groups, and amino groups are introduced into cellulose acetate through chemical reactions to change its surface properties or functions. Cellulose acetate itself carries many unreacted hydroxyl groups. By using amino-modified cellulose acetate porous balls as carriers for immobilized enzymes, porous balls with enzyme-binding active groups (amino groups) on the surface can be prepared. These active groups can covalently bind to chemical groups on the enzyme surface, thereby significantly improving the immobilization efficiency of the enzyme and increasing the enzyme loading capacity. The enzyme is covalently bound to the carrier and is not easy to fall off. In addition, the porous structure of cellulose acetate increases the carrier's internal surface area and enzyme loading capacity, and also makes the carrier have higher mechanical strength, making it suitable for industrial operations. Therefore, amino groups can improve the immobilization efficiency of the enzyme. Cellulose acetate porous balls have stronger adsorption capacity, can more effectively adsorb enzymes, and further improve the stability of the immobilized enzyme.

[0046] Alternatively, the amino-modified cellulose acetate porous balls include cellulose acetate carbamate porous balls, which are formed from cellulose acetate carbamate, which is prepared by modifying cellulose acetate with isocyanic acid. Isocyanic acid modifies cellulose acetate so that cellulose acetate obtains a carbonyl amino group at its hydroxyl vacancy, thereby generating cellulose acetate carbamate. The cellulose acetate carbamate porous balls are prepared by dissolving cellulose acetate carbamate in an organic solvent and then dripping it into water, and the cellulose acetate carbamate porous balls are formed as the organic solvent dissolves. The cellulose acetate carbamate porous balls can be biodegraded, so that the immobilized enzyme can be used multiple times.

[0047] Here, the chemical formula for cellulose acetate carbamate prepared by modifying cellulose acetate with isocyanic acid is: HNCO + CH3COO-Cell-OH → CH3COO-Cell-OCONH2, where HNCO is isocyanic acid, CH3COO-Cell-OH is cellulose acetate, and CH3COO-Cell-OCONH2 is cellulose acetate carbamate. In addition, side reactions will occur during this process, as shown in the following reaction formulas: HNCO + NH2CONH2 → H2NCONHCONH2, where H2NCONHCONH2 is biuret; HNCO + NH3 → NH4 + NCO - , NH4 + NCO - It is ammonium isocyanate.

[0048] Alternatively, the amino group can be provided by isocyanic acid, produced by the decomposition of urea at high temperatures. Urea is inexpensive, and providing the amino group with isocyanic acid further reduces the cost of preparing the immobilized enzyme. The chemical formula for isocyanic acid produced by the decomposition of urea at high temperatures is: NH₂CONH₂ → HNCO + NH₃↑. HNCO is isocyanic acid.

[0049] Optionally, the outer diameter of the amino-modified cellulose acetate porous spheres ranges from 2 mm to 4 mm. Optionally, the outer diameter of the amino-modified cellulose acetate porous spheres ranges from 2.5 mm to 3.5 mm. Optionally, the outer diameter of the amino-modified cellulose acetate porous spheres is 3 mm. By controlling the outer diameter of the amino-modified cellulose acetate porous spheres within the above range, the molding and transfer of the amino-modified cellulose acetate porous spheres are facilitated. And within this range, the number of amino-modified cellulose acetate porous spheres can be increased in the same volume of solution, thereby increasing the specific surface area of ​​the amino-modified cellulose acetate porous spheres, increasing the number of enzymes bound to the carrier, and thereby improving the performance of the immobilized enzyme.

[0050] For example, the outer diameter of the amino-modified cellulose acetate porous spheres can be 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, or any value within the range of 2 mm to 4 mm.

[0051] Optionally, the average pore size of the amino-modified cellulose acetate porous spheres (referring to the average diameter of the multiple small pores inside the amino-modified cellulose acetate porous spheres) ranges from 15nm to 30nm. Optionally, the average pore size of the amino-modified cellulose acetate porous spheres ranges from 15nm to 25nm. Optionally, the average pore size of the amino-modified cellulose acetate porous spheres ranges from 18nm to 22nm. Optionally, the average pore size of the amino-modified cellulose acetate porous spheres is 20nm. By controlling the average pore size of the amino-modified cellulose acetate porous spheres within the above range, the catalytic performance and operational stability of the immobilized enzyme can be improved through nanoscale porous structure and amino functional modification. And within this range of pore size, the number of pores of the amino-modified cellulose acetate porous spheres can be increased, thereby increasing the specific surface area of ​​the porous spheres and increasing the binding amount of the enzyme.

[0052] For example, the average pore size of the amino-modified cellulose acetate porous spheres can be 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 25 nm, 28 nm, 29 nm, 30 nm, or any value within the range of 15 nm to 30 nm.

[0053] Optionally, the specific surface area of ​​the amino-modified cellulose acetate porous spheres ranges from 30 m 2 / g to 50m 2 / g. Optionally, the specific surface area of ​​the amino-modified cellulose acetate porous spheres ranges from 35m 2 / g to 45m 2 / g. Optionally, the specific surface area of ​​the amino-modified cellulose acetate porous spheres ranges from 30m 2 / g to 40m 2 / g. Optionally, the specific surface area of ​​the amino-modified cellulose acetate porous spheres ranges from 40m 2 / g to 50m 2 / g. Optionally, the specific surface area of ​​amino-modified cellulose acetate porous spheres is 40m 2 / g.

[0054] In the embodiment of the present disclosure, the specific surface area of ​​the amino-modified cellulose acetate porous spheres is in the range of 30 m 2 / g to 50m 2 / g, which can increase more enzyme binding sites and increase the binding capacity of the enzyme. It can also avoid the blockage of the pores or mass transfer resistance caused by excessive specific surface area. It can also ensure the mechanical strength of the amino-modified cellulose acetate porous balls and avoid structural collapse. Therefore, through the synergistic effect of moderate pore structure and amino functional modification, an efficient balance is achieved between enzyme loading, mass transfer efficiency and stability. For example, the specific surface area of ​​the amino-modified cellulose acetate porous balls is 30m 2 / g、32m 2 / g、35m 2 / g、38m 2 / g, 40m 2 / g、41m 2 / g、42m 2 / g、45m 2 / g、48m 2 / g, 50m 2 / g, or 30m 2 / g to 50m 2 Any value between / g.

[0055] Combine Figure 1 As shown, the present disclosure provides a method for preparing a carrier for immobilizing an enzyme, comprising:

[0056] S101, modifying cellulose acetate with a modifier to obtain cellulose acetate carbamate.

[0057] S102, dissolving cellulose acetate carbamate in a first organic solvent and then dripping the solvent into water to form cellulose acetate carbamate porous balls.

[0058] In the preparation method of the carrier for immobilized enzyme adopted in the present disclosure, cellulose acetate is used as a substrate, and cellulose acetate is modified into cellulose acetate carbamate, so as to facilitate the preparation of porous cellulose acetate carbamate porous balls, which makes it easy for the cellulose acetate carbamate porous balls to be combined with the enzyme as a carrier. Cellulose acetate is cheap, has good biocompatibility, is easily soluble in organic solvents, and is easy to process. In addition, cellulose acetate tow has the characteristics of high strength, high toughness, and non-flammability, and it can be biodegraded in the environment. Therefore, cellulose acetate is used as a substrate to prepare cellulose acetate carbamate porous balls, and cellulose acetate carbamate porous balls are used as a carrier of the enzyme, thereby achieving enzyme fixation and multiple batches of reuse, high storage stability and use stability, biodegradability, reducing the cost of enzyme use, and having significant industrial application potential and application prospects.

[0059] After obtaining cellulose acetate carbamate, the cellulose acetate carbamate is dissolved in a first organic solvent and then dripped into water to form cellulose acetate carbamate porous balls. Here, the cellulose acetate carbamate porous balls are amino-modified cellulose acetate porous balls. The cellulose acetate carbamate is first dissolved in the first organic solvent and then precipitated in water with the first organic solvent to form a porous spherical structure, which increases the internal surface area, improves the enzyme loading capacity, and ensures the stability of the enzyme during use. The cellulose acetate carbamate porous balls are obtained as carriers to facilitate the binding of the carrier and the enzyme to obtain an immobilized enzyme.

[0060] In some optional embodiments, step S101 modifies cellulose acetate with a modifier to obtain cellulose acetate carbamate, comprising: (a) dispersing cellulose acetate in an aqueous solution of the modifier to obtain a mixed solution; and (b) heating the mixed solution to obtain cellulose acetate carbamate. In this embodiment, cellulose acetate serves as a substrate and is dispersed into the aqueous solution of the modifier to ensure thorough mixing of the modifier and cellulose acetate. The mixed solution is then heated, causing the modifier to decompose at high temperatures to produce amino groups. The amino groups then form carbonyl amino groups at hydroxyl vacancies in the cellulose acetate, thereby producing cellulose acetate carbamate.

[0061] Combine Figure 2 The present disclosure provides another method for preparing a carrier for immobilizing an enzyme, comprising:

[0062] S201, dispersing cellulose acetate into a modifier aqueous solution to obtain a mixed solution.

[0063] S202, heating the mixed solution to obtain cellulose acetate carbamate.

[0064] S203, dissolving cellulose acetate carbamate in a first organic solvent and then dripping the solvent into water to form cellulose acetate carbamate porous balls.

[0065] The preparation method of this embodiment utilizes cellulose acetate as a substrate and an aqueous solution of a modifier as a source of amino groups. The two are first mixed to form a mixed solution, which is then heated to decompose the modifier to produce an intermediate. The intermediate then causes the cellulose acetate to acquire carbonyl amino groups at hydroxyl vacancies, thereby forming cellulose acetate carbamate. The cellulose acetate carbamate is then reacted and processed to produce amino-modified cellulose acetate porous balls, i.e., cellulose acetate carbamate porous balls. The cellulose acetate carbamate porous balls serve as carriers for binding to an enzyme to prepare an immobilized enzyme.

[0066] Optionally, the intermediate substance is isocyanic acid or isocyanate.

[0067] Optionally, the modifier is urea. Here, urea is abundant in source and low in cost. Urea can be decomposed to produce isocyanic acid by high temperature. The reaction is simple and easy to implement, which facilitates the mass production of immobilized enzymes.

[0068] It is understood that the modifier can also be other substances, such as cyanates and isocyanate compounds. However, different modifiers require different processes to produce isocyanic acid or isocyanate. For example, cyanates require acidification, while isocyanate compounds can be added directly. In practical applications, the modifier can be selected based on the application requirements and reaction environment.

[0069] Optionally, in step S201, a modifier aqueous solution with a first preset concentration is prepared, cellulose acetate is dispersed in the modifier aqueous solution at a second preset ratio, and heated at a first preset temperature and stirred for a first set time to obtain a mixed solution.

[0070] In the disclosed embodiments, after mixing cellulose acetate and an aqueous modifier solution, the solution is heated to a first predetermined temperature while being stirred to allow the modifier and cellulose acetate to mix thoroughly, thereby pretreating the cellulose acetate and obtaining a pretreated mixed solution. The aqueous modifier solution has a first predetermined concentration, and a second predetermined ratio of cellulose acetate to the aqueous modifier solution. This ensures that the initial amounts of the cellulose acetate and modifier reactants are sufficient for the modifier to generate sufficient isocyanate to fully modify the cellulose acetate.

[0071] Optionally, the first preset concentration ranges from 15% to 35%. Optionally, the first preset concentration ranges from 20% to 25%. Optionally, the first preset concentration ranges from 15% to 25%. Optionally, the first preset concentration ranges from 20% to 25%.

[0072] For example, the first preset concentration can be 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, or any value in the range of 15% to 35%.

[0073] Optionally, the second preset ratio ranges from 0.5 g to 1.5 g of cellulose acetate to 10 mL of the aqueous modifier solution. Optionally, the second preset ratio ranges from 0.8 g to 1.2 g of cellulose acetate to 10 mL of the aqueous modifier solution. Optionally, the second preset ratio ranges from 0.8 g to 1 g of cellulose acetate to 10 mL of the aqueous modifier solution.

[0074] For example, the second preset ratio can be 0.5 g cellulose acetate: 10 mL modifier aqueous solution, 0.6 g cellulose acetate: 10 mL modifier aqueous solution, 0.8 g cellulose acetate: 10 mL modifier aqueous solution, 1 g cellulose acetate: 10 mL modifier aqueous solution, 1.2 g cellulose acetate: 10 mL modifier aqueous solution, 1.4 g cellulose acetate: 10 mL modifier aqueous solution, 1.5 g cellulose acetate: 10 mL modifier aqueous solution, or any value within the range of 0.5 g to 1.5 g cellulose acetate: 10 mL modifier aqueous solution.

[0075] Optionally, the first preset temperature range is 30℃ to 80℃, optionally, the first preset temperature range is 50℃ to 80℃, optionally, the first preset temperature range is 60℃ to 70℃, optionally, the first preset temperature range is 60℃ to 80℃, optionally, the first preset temperature range is 50℃ to 70℃.

[0076] For example, the first preset temperature can be 30℃, 40℃, 50℃, 52℃, 55℃, 57℃, 59℃, 60℃, 61℃, 63℃, 65℃, 68℃, 70℃, 72℃, 75℃, 80℃, or any value within the range of 30℃ to 80℃.

[0077] Optionally, the first set time length ranges from 1 hour to 6 hours. Optionally, the first set time length ranges from 3 hours to 6 hours. Optionally, the first set time length ranges from 4 hours to 5 hours. Optionally, the first set time length ranges from 3 hours to 5 hours. Optionally, the first set time length ranges from 4 hours to 6 hours.

[0078] For example, the first set duration can be 1 hour, 2 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, or any value within the range of 1 hour to 6 hours.

[0079] Optionally, the mixture is heated at a first preset temperature and stirred at a first speed for a first set time to obtain the mixed solution, wherein the first speed ranges from 150 rpm to 250 rpm, or the first speed ranges from 180 rpm to 220 rpm, or the first speed ranges from 150 rpm to 200 rpm.

[0080] For example, the first rotational speed may be 150 rpm, 160 rpm, 180 rpm, 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, or any value within the range of 150 rpm to 250 rpm.

[0081] Optionally, the first preset temperature is heated in a water bath or an oil bath.

[0082] Optionally, in step S202, the mixed solution is heated to obtain cellulose acetate carbamate, including: (c) heating and stirring the mixed solution and adding a second organic solvent, and heating and evaporating water to obtain a reaction solution; (d) reacting the reaction solution at a second preset temperature for a second set time to obtain cellulose acetate carbamate.

[0083] In the disclosed embodiment, the mixed liquid is mixed in a second organic solvent, and then heated to evaporate the water. The temperature is further increased so that the reaction liquid reacts at a second preset temperature. In this way, the modifier can decompose at the second preset temperature to produce an intermediate substance. The intermediate substance enables cellulose acetate to obtain a carbonyl amino group at its hydroxyl vacancy, thereby generating cellulose acetate carbamate.

[0084] Optionally, the reaction liquid is reacted at a second preset temperature for a second set time to obtain cellulose acetate carbamate, including: after the reaction liquid is reacted at the second preset temperature for the second set time, filtering using a sand core funnel to obtain a modified product, washing the modified product with hot water multiple times, and discharging after drying to obtain cellulose acetate carbamate.

[0085] In this embodiment, after the cellulose acetate and the modifier are evenly mixed, they are transferred to a second organic solvent, and the moisture is removed by heating. Then, the second organic solvent is further heated to increase the temperature. This can increase the reaction temperature of the cellulose acetate and the modifier. In this way, not only can the modifier decompose to produce isocyanic acid, but the isocyanic acid can also modify the cellulose acetate to obtain cellulose acetate carbamate.

[0086] Optionally, the second preset temperature ranges from 120°C to 140°C. Optionally, the second preset temperature ranges from 125°C to 140°C. Optionally, the second preset temperature ranges from 125°C to 130°C. Optionally, the second preset temperature ranges from 130°C to 140°C. Optionally, the second preset temperature ranges from 135°C to 140°C.

[0087] In the embodiment of the present disclosure, the second preset temperature affects the number of amino groups in cellulose acetate carbamate, and thus affects the number of enzymes that can be loaded by the finally formed cellulose acetate carbamate porous balls. Setting the second preset temperature within the above range can ensure that the number of amino groups in cellulose acetate carbamate is maximized, thereby ensuring the loading amount of the enzyme and the activity of the enzyme.

[0088] For example, the second preset temperature can be 120℃, 125℃, 128℃, 130℃, 132℃, 135℃, 136℃, 137℃, 138℃, 140℃, 142℃, 140℃, or any value in the range of 120℃ to 140℃.

[0089] Optionally, the second organic solvent is not miscible with water and has a boiling point above 140°C.

[0090] Optionally, the second organic solvent includes one or more of o-xylene, acetone and dimethyl sulfoxide.

[0091] Optionally, the amount of the second organic solvent is 30 ml to 70 ml, or the amount of the second organic solvent is 40 ml to 60 ml, or the amount of the second organic solvent is 50 ml.

[0092] For example, the second organic solvent may be 30 ml, 35 ml, 40 ml, 45 ml, 50 ml, 55 ml, 60 ml, 65 ml, 70 ml, or any value within the range of 30 ml to 70 ml.

[0093] Optionally, the second set time length ranges from 1 hour to 6 hours, optionally, the second set time length ranges from 2 hours to 5 hours, optionally, the second set time length ranges from 2 hours to 3 hours.

[0094] For example, the second set time length is 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, or any value within the range of 1 hour to 6 hours.

[0095] Combine Figure 3 The present disclosure provides another method for preparing a carrier for immobilizing an enzyme, comprising:

[0096] S301, preparing a modifier aqueous solution with a first preset concentration, dispersing cellulose acetate in the modifier aqueous solution at a second preset ratio, heating at a first preset temperature and stirring for a first preset time to obtain a mixed solution.

[0097] S302, heating and stirring the mixed solution and adding a second organic solvent, and heating to evaporate water to obtain a reaction solution.

[0098] S303 , the reaction liquid is reacted at a second preset temperature for a second preset time to obtain cellulose acetate carbamate.

[0099] S304, dissolving cellulose acetate carbamate in a first organic solvent and then dripping the solvent into water to form cellulose acetate carbamate porous balls.

[0100] In this embodiment, the modifier and cellulose acetate are mixed and pretreated, and then a second organic solvent is added and heated to increase the temperature, so that the modifier and cellulose acetate undergo a modification reaction at a second preset temperature to form cellulose acetate carbamate, and then cellulose acetate carbamate porous balls are prepared. The cellulose acetate carbamate porous balls can be used as a carrier for immobilized enzymes.

[0101] Optionally, in step 102 , the step of dissolving cellulose acetate carbamate in a first organic solvent and then dripping the resulting mixture into water to form cellulose acetate carbamate porous balls includes: (m) dissolving cellulose acetate carbamate in the first organic solvent at a second predetermined concentration until a solution is formed;

[0102] (n) dropping the solution into distilled water to form a cellulose acetate urethane porous ball mixture; (o) filtering, soaking and draining the cellulose acetate urethane porous ball mixture to obtain cellulose acetate urethane porous balls.

[0103] In this embodiment, cellulose acetate carbamate is dissolved in a second organic solvent and then dripped into distilled water, so that the cellulose acetate carbamate is more evenly distributed. Then, it is dripped into distilled water to form porous spherical cellulose acetate carbamate porous balls. The cellulose acetate carbamate porous balls are mixed in water and solvent, and cellulose acetate carbamate porous balls are obtained by filtering, soaking and draining. By selecting a second organic solvent that has good miscibility with cellulose acetate carbamate, the uniform dissolution of cellulose acetate carbamate is achieved to form a homogeneous solution system. When the solution is added dropwise to distilled water, due to the difference in miscibility between the second organic solvent and water, a rapid mass transfer process from solvent to non-solvent is initiated, resulting in phase separation of the polymer chains. In this process, the gradual diffusion of the second organic solvent and the penetration of water molecules work together to promote the formation of a three-dimensional interpenetrating pore structure in the polymer network during the curing process. Subsequent filtration and soaking treatments can effectively remove residual solvents and stabilize the pore structure. The aqueous phase molding process avoids the use of surfactants, and the residual solvent can be completely removed by simple water washing, which is green, energy-saving and pollution-free. Combined Figure 14 and Figure 15 As shown, it can be seen that the cellulose acetate urethane porous ball has a porous structure. Figure 13 As shown, it can be seen that the outer surface of the cellulose acetate urethane porous ball is spherical.

[0104] Optionally, the solubility parameter of the first organic solvent is in the range of 20 to 25 MPa1 / 2 To ensure that the cellulose acetate carbamate and the first organic solvent are uniformly dissolved.

[0105] Optionally, the ratio of the solubility of the first organic solvent to the solubility of water is greater than or equal to 8 MPa1 / 2 , to ensure the driving force for phase separation.

[0106] Optionally, the boiling point of the first organic solvent is in the range of 60 to 150° C., ensuring a moderate volatilization rate to avoid structural collapse due to excessive solidification.

[0107] Optionally, the first organic solvent includes one or more of a ketone solvent, an amide solvent and a chlorinated solvent.

[0108] In the embodiment of the present disclosure, the first organic solvent needs to be able to form a homogeneous solution with cellulose acetate carbamate, and also needs to be partially miscible with water to drive phase separation and form a porous structure, and also needs to have a moderate volatilization rate to avoid structural collapse caused by too rapid solidification.

[0109] Optionally, the first organic solvent includes one or more of N,N'-dimethylformamide, N,N'-dimethylacetamide, N'-methylpyrrolidone, acetone, cyclohexanone, methyl isobutyl ketone, γ-valerolactone, ethyl lactate and propylene carbonate.

[0110] Preferably, the first organic solvent comprises N,N-dimethylformamide.

[0111] Optionally, the second preset concentration ranges from 7.5% to 15%. Optionally, the second preset concentration ranges from 9% to 11%. Here, the second preset concentration regulates key parameters of the microstructure and performance of the cellulose acetate urethane porous spheres. By regulating phase separation kinetics and thermodynamic equilibrium, the second preset concentration achieves synergistic optimization of the cellulose acetate urethane porous spheres from pore size to porosity to strength.

[0112] Optionally, the second preset concentration ranges from 7.5% to 9%. Optionally, the second preset concentration ranges from 10% to 12%. Optionally, the second preset concentration ranges from 12% to 15%. Optionally, the second preset concentration ranges from 10% to 12.5%. Optionally, the second preset concentration ranges from 10% to 15%.

[0113] For example, the second preset concentration can be 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11.5%, 12%, 12.5%, 13%, 14%, 15%, or any value in the range of 7.5% to 15%.

[0114] Combine Figure 4 The present disclosure provides another method for preparing a carrier for immobilizing an enzyme, comprising:

[0115] S401, preparing a modifier aqueous solution with a first preset concentration, dispersing cellulose acetate in the modifier aqueous solution at a second preset ratio, heating at a first preset temperature and stirring for a first preset time to obtain a mixed solution.

[0116] S402, heating and stirring the mixed solution and adding a second organic solvent, and heating to evaporate water to obtain a reaction solution.

[0117] S403 , the reaction liquid is reacted at a second preset temperature for a second preset time to obtain cellulose acetate carbamate.

[0118] S404 , dissolving cellulose acetate carbamate in a first organic solvent to form a solution having a cellulose acetate carbamate concentration of a second preset concentration.

[0119] S405, dropping the solution into distilled water to form a cellulose acetate urethane porous ball mixed solution.

[0120] S406, filtering, soaking and draining the cellulose acetate urethane porous ball mixture to obtain cellulose acetate urethane porous balls.

[0121] In this embodiment, a modifier and cellulose acetate are mixed and pretreated, and then a second organic solvent is added and heated to a predetermined temperature, causing the modifier and cellulose acetate to undergo a modification reaction at a second predetermined temperature to form cellulose acetate carbamate. The prepared cellulose acetate carbamate is dissolved in the second organic solvent and then dripped into distilled water to form a cellulose acetate carbamate porous ball mixture. The cellulose acetate carbamate porous ball mixture is then filtered and drained to prepare the cellulose acetate carbamate porous ball. This completes the carrier preparation.

[0122] The disclosed embodiments provide a method for preparing an immobilized enzyme, wherein a carrier prepared by the method for preparing a carrier for immobilized enzyme described in any one of the above embodiments is combined with an enzyme to obtain an immobilized enzyme.

[0123] The present disclosure provides a method for preparing an immobilized enzyme, comprising: adding cellulose acetate carbamate porous balls to a cross-linker solution and mixing for a third set time to obtain a suspension; repeatedly rinsing the suspension to obtain carrier balls; using an acid solution to dissolve emulsifier to obtain an enzyme solution of a third preset concentration; adding the carrier balls to the enzyme solution, shaking, filtering, and repeatedly rinsing to obtain an initial immobilized enzyme; and rinsing and de-enzyming the initial immobilized enzyme to obtain an immobilized enzyme.

[0124] In the disclosed embodiments, a crosslinking agent is used to bind the cellulose acetate carbamate porous spheres to the enzyme. The carbamate groups of the cellulose acetate carbamate provide reaction sites for crosslinking, while amino groups or other groups on the surface of the enzyme provide another reaction site for crosslinking. The crosslinking agent can connect the cellulose acetate carbamate porous spheres and the enzyme, thereby achieving binding of the cellulose acetate carbamate porous spheres and the enzyme. In addition, the porous spherical structure of the cellulose acetate carbamate porous spheres increases the internal surface area, thereby improving the amount of enzyme binding.

[0125] Optionally, the concentration of the crosslinker solution ranges from 0.5% to 2%, optionally from 0.5% to 1.5%, optionally from 0.5% to 1%, and optionally from 0.75% to 1%. Maintaining the concentration of the crosslinker solution within the above range can protect the enzyme loading and activity and improve curing stability. For example, the concentration of the crosslinker solution can be 0.5%, 0.7%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, or any value within the range of 0.5% to 2%.

[0126] Optionally, the third set time ranges from 3 hours to 5 hours, or the third set time ranges from 4 hours to 5 hours, so as to ensure the binding stability between the cellulose acetate carbamate porous balls and the enzyme.

[0127] For example, the third set duration may be 3 hours, 4 hours, 5 hours, or any value within the range of 3 hours to 5 hours.

[0128] Optionally, the third preset concentration ranges from 0.01 g / ml to 0.1 g / ml, or the third preset concentration ranges from 0.05 g / ml to 0.1 g / ml, so as to ensure the amount of enzyme immobilization.

[0129] For example, the third preset concentration may be 0.01 g / ml, 0.03 g / ml, 0.05 g / ml, 0.07 g / ml, 0.1 g / ml, or any value within the range of 0.01 g / ml to 0.1 g / ml.

[0130] Optionally, the cross-linking agent is a bifunctional cross-linking agent, which can simultaneously connect the amino groups of the cellulose acetate urethane porous spheres and the enzyme for binding.

[0131] Optionally, the cross-linking agent is one or more of glutaraldehyde and genipin.

[0132] Preferably, the cross-linking agent is glutaraldehyde. Glutaraldehyde is selected as the cross-linking agent between the carrier and the enzyme protein. The two aldehyde groups of glutaraldehyde can form Schiff bases (N=C) with the amino groups of the carrier and the enzyme protein, respectively, linking them with a five-carbon bridge to produce an immobilized enzyme. The immobilized enzyme prepared in this way has the advantages of low carrier material cost, biodegradability, high mechanical strength, porous carrier, high enzyme loading capacity, and covalent bonding of the enzyme molecule to the carrier that is not easily detached.

[0133] Optionally, the acid may be an inorganic acid or an organic acid. Optionally, when the acid is an organic acid, the concentration range is 0.1 mol / L to 0.5 mol / L. Optionally, when the acid is an organic acid, the concentration range is 0.1 mol / L to 0.3 mol / L. When the acid is an organic acid, the concentration range is 0.15 mol / L to 0.25 mol / L. For example, the concentration of the organic acid may be 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, or any value between 0.1 mol / L and 0.5 mol / L.

[0134] Combine Figure 5 The present disclosure provides another method for preparing an immobilized enzyme, comprising:

[0135] S501, adding cellulose acetate carbamate porous balls into a cross-linking agent solution and mixing for a third set time to obtain a suspension.

[0136] S502, repeatedly washing the suspension to obtain carrier balls.

[0137] S503: Dissolve the enzyme using an acid solution to obtain an enzyme solution with a third preset concentration.

[0138] S504, adding the carrier balls into the enzyme solution, shaking, filtering and repeatedly washing to obtain the initial immobilized enzyme.

[0139] S505, the initial immobilized enzyme is washed and de-enzymed to obtain the immobilized enzyme.

[0140] In this example, after preparing the cellulose acetate carbamate porous balls, the cellulose acetate carbamate porous balls, which served as carriers, were treated. The cellulose acetate carbamate porous balls and the enzyme were then cross-linked using a cross-linking agent to obtain an immobilized enzyme. The carbamate groups of the cellulose acetate carbamate provide reaction sites for the cross-linking, while amino groups or other groups on the enzyme surface provide another reaction site for the cross-linking. The cross-linking agent can connect the cellulose acetate carbamate porous balls and the enzyme, thereby achieving binding of the cellulose acetate carbamate porous balls and the enzyme.

[0141] Alternatively, the ratio of the mass of the enzyme to the mass of the cellulose acetate carbamate porous spheres is in the range of 0.025 to 1. Alternatively, the ratio of the mass of the enzyme to the mass of the cellulose acetate carbamate porous spheres is in the range of 0.05 to 0.75. Alternatively, the ratio of the mass of the enzyme to the mass of the cellulose acetate carbamate porous spheres is in the range of 0.25 to 0.5. Alternatively, the ratio of the mass of the enzyme to the mass of the cellulose acetate carbamate porous spheres is in the range of 0.05 to 0.5.

[0142] In the disclosed embodiments, the mass ratio of the enzyme to the mass of the cellulose acetate carbamate porous ball is an important parameter that affects the performance of the immobilized enzyme. It not only affects the amount of enzyme bound to the cellulose acetate carbamate porous ball, but also when the mass ratio of the enzyme to the mass of the cellulose acetate carbamate porous ball is greater than 1, the content of the enzyme is too high. Although this will increase the relative enzyme activity, it will cause some enzymes to be unable to be fixed, and there will be too much free enzyme, which will affect subsequent processes. When the mass ratio of the enzyme to the mass of the cellulose acetate carbamate porous ball is less than 0.025, the number of carriers is high, which may result in insufficient utilization of the carrier surface, waste of materials, and will cause the relative enzyme activity of the immobilized enzyme to be lower. Therefore, by keeping the mass ratio of the enzyme to the mass of the cellulose acetate carbamate porous ball within the above-mentioned range, the relative enzyme activity and immobilization efficiency of the prepared immobilized enzyme can be guaranteed, free enzyme can be reduced, and the stability of the immobilized enzyme can be improved.

[0143] For example, the ratio of the mass of the enzyme to the mass of the cellulose acetate urethane porous spheres can be 0.025, 0.03, 0.05, 0.08, 0.1, 0.2, 0.25, 0.3, 0.5, 0.7, 0.75, 0.8, 0.9, 1, or any value within the range of 0.025 to 1.

[0144] Combine Figure 6 The present disclosure provides another method for preparing an immobilized enzyme, comprising:

[0145] S601, preparing a modifier aqueous solution of a first preset concentration, dispersing cellulose acetate therein, wherein the cellulose acetate content is a second preset ratio, heating at a first preset temperature and stirring for a first preset time to obtain a mixed solution.

[0146] S602, heating and stirring the mixed solution and adding a second organic solvent, and heating to evaporate water to obtain a reaction solution.

[0147] S603 , the reaction liquid is reacted at a second preset temperature for a second preset time to obtain cellulose acetate carbamate.

[0148] S604 , dissolving cellulose acetate carbamate in a first organic solvent to form a solution having a cellulose acetate carbamate concentration of a second preset concentration.

[0149] S605, dropping the solution into distilled water to form a cellulose acetate urethane porous ball mixture.

[0150] S606, filtering, soaking and draining the cellulose acetate urethane porous ball mixture to obtain cellulose acetate urethane porous balls.

[0151] S607, adding the cellulose acetate urethane porous balls into the cross-linking agent solution and mixing for a third set time to obtain a suspension.

[0152] S608, repeatedly washing the suspension to obtain carrier balls.

[0153] S609: dissolving the enzyme using an acid solution to obtain an enzyme solution of a third preset concentration.

[0154] S610, adding the carrier balls to the enzyme solution, shaking, filtering and repeatedly washing to obtain the initial immobilized enzyme.

[0155] S611, the initial immobilized enzyme is washed and de-enzymed to obtain the immobilized enzyme.

[0156] In this embodiment, a modifier and cellulose acetate are mixed and pretreated, and then a second organic solvent is added and heated to a predetermined temperature, causing the modifier and cellulose acetate to undergo a modification reaction at a second predetermined temperature to form cellulose acetate carbamate. The prepared cellulose acetate carbamate is dissolved in the second organic solvent and then dripped into distilled water to form a cellulose acetate carbamate porous ball mixture. The cellulose acetate carbamate porous balls are then prepared by filtering and draining. The cellulose acetate carbamate porous balls are then cross-linked with the enzyme using a cross-linking agent to obtain the immobilized enzyme.

[0157] Alternatively, the prepared immobilized enzyme is filtered using a fritted funnel, sealed while keeping it moist, and stored in a refrigerator at 4° C. This ensures the activity of the immobilized enzyme.

[0158] Alternatively, cellulose acetate may be commercially available, or may be formed by esterifying cellulose with acetate.

[0159] The present disclosure also provides an immobilized enzyme, which includes an enzyme and a carrier. The carrier includes the carrier for immobilizing enzyme in any of the above embodiments, or the carrier is prepared using the preparation method of the carrier for immobilizing enzyme in any of the above embodiments.

[0160] The immobilized enzyme provided by the embodiments of the present disclosure includes the carrier for immobilizing the enzyme of any of the above embodiments, or the carrier is prepared using the preparation method of the carrier for immobilizing the enzyme of any of the above embodiments. Therefore, it has the beneficial effects of the carrier for immobilizing the enzyme or the preparation method of the carrier for immobilizing the enzyme of any of the above embodiments, and will not be repeated here.

[0161] The immobilized enzymes provided by the embodiments of the present disclosure have broad applicability and can immobilize virtually any type of enzyme. The specific choice depends on the target application scenario, such as the type of catalytic reaction, substrate properties, and product requirements. Optionally, the enzyme bound to the carrier used for the immobilized enzyme can be a hydrolase, oxidoreductase, transferase, lyase, isomerase, or ligase.

[0162] The embodiments of the present disclosure provide applications of the carrier for immobilizing an enzyme according to any of the aforementioned embodiments in the biological field; or, applications of the carrier prepared by the method for preparing the carrier for immobilizing an enzyme according to any of the aforementioned embodiments in the biological field.

[0163] The biological field encompasses all disciplines and technologies related to life sciences, including but not limited to basic biology (such as molecular biology and cell biology), applied biotechnology (such as enzyme engineering, fermentation engineering, and genetic engineering), and interdisciplinary fields (such as biomedicine, environmental biotechnology, and industrial biotechnology). As a core method of biocatalysis, immobilized enzyme technology is essentially the specific application of biotechnology in engineering and industrial scenarios.

[0164] Furthermore, the carrier for immobilized enzyme and the immobilized enzyme provided by the embodiments of the present disclosure can be applied in the fields of biomedicine, industrial biology, food biology, environmental biology, agricultural biology, molecular biology, and synthetic biology.

[0165] Optionally, when the carrier for immobilizing enzyme is applied to food organisms, the enzyme bound to the carrier for immobilizing enzyme can be lipase, lactase, protease, glucose oxidase, glycosyltransferase or glucose isomerase, etc.

[0166] Optionally, when the carrier for immobilizing enzyme is applied to biomedicine, the enzyme bound to the carrier for immobilizing enzyme can be lipase, penicillin acylase, peroxidase, transaminase, aspartase or DNA ligase.

[0167] Optionally, when the carrier for immobilizing enzymes is applied to industrial organisms, the enzyme bound to the carrier for immobilizing enzymes may be lipase, cellulase or nitrile hydratase.

[0168] Optionally, when the carrier for immobilizing enzymes is applied to environmental organisms, the enzyme bound to the carrier for immobilizing enzymes may be laccase, urease, petroleum degrading enzyme, and the like.

[0169] Optionally, when the carrier for immobilizing enzymes is applied to agricultural organisms, the enzyme bound to the carrier for immobilizing enzymes may be an organophosphorus degrading enzyme, phytase, cellulase, and the like.

[0170] Optionally, when the carrier for immobilizing enzymes is applied to molecular biology and synthetic biology, the enzyme bound to the carrier for immobilizing enzymes may be DNA ligase, penicillin acylase, and the like.

[0171] Specific examples are provided below to specifically illustrate the immobilized enzymes of the disclosed embodiments, their preparation methods, and applications, in order to more clearly illustrate the technical problems, technical solutions, and beneficial effects addressed by the present application. It is apparent that the described embodiments are merely a portion of the present application's embodiments, rather than all of them. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present application and its applications.

[0172] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0173] Cellulose acetate was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., o-xylene, glutaraldehyde, and urea were purchased from Sinopharm Group, and lactase was purchased from Suzhou Fulide Biological Co., Ltd.

[0174] When the immobilized enzyme is immobilized lactase, the immobilized lactase can be used for lactose hydrolysis and galactose production, and can also be used for tagatose production using lactose as raw material.

[0175] Taking lactase as an example, an immobilized enzyme was prepared and the enzyme activity of the immobilized enzyme was tested. The method for testing the enzyme activity of the immobilized enzyme in the embodiment is as follows:

[0176] Enzyme activity definition: Using lactose as substrate, at 50°C and pH 6.0, the amount of enzyme required to hydrolyze 1 μmol of glucose within 1 min is defined as one enzyme activity unit (U).

[0177] Determination method: 2g of immobilized lactase was added to 10mL of a 10% lactose solution (pH 6.0). The reaction was shaken at 50°C, 200rpm, for 30 minutes. The reaction solution was then removed and placed in a boiling water bath for 10 minutes to terminate the reaction. After diluting 50-fold with distilled water, the lactose content in the reaction solution was accurately determined using a high-performance liquid chromatography (HPLC, differential refractive index detector, stationary phase Carbomix Pb to NP column (10:8%, 7.8×300mm, 10μm, Saifen Technology), mobile phase water, and a flow rate of 0.5mL / min. By measuring the lactose content under different conditions, the percentage of lactose converted to products (such as glucose and galactose) relative to the initial lactose content was determined as the conversion rate, which reflects the relative enzymatic activity of the immobilized lactase.

[0178] Immobilized lactase was prepared using the following examples, and the relative enzyme activity of the immobilized lactase prepared in different examples was determined using the above-mentioned measurement method.

[0179] The relative enzyme activities in Examples 1 to 39 were counted with the highest value being 100%, and the relative enzyme activity of the initial batch of Example 40 was recorded as 100%.

[0180] Example 1 Preparation of immobilized lactase:

[0181] Weigh 25g of cellulose acetate and place it in 250ml of 25% (first preset concentration) urea solution, the first preset concentration is 25%, stir and mix, place it on a shaker at 50°C, and oscillate at 200rpm under the first preset temperature of 50°C for the first set time of 5h. Take 100ml of the evenly mixed cellulose acetate urea mixture and place it in a 250ml flat-bottom flask. Place the flask in an oil bath and set the oil temperature to 155°C. Add a large magnet to the flask, turn on the magnetic stirring device of the oil bath, and slowly add 50ml of o-xylene while stirring rapidly. Continue stirring and connect a distillation apparatus to the flask. After a period of time, the water and part of the o-xylene in the flask are evaporated, and the liquid in the bottle begins to heat up. Adjust the oil bath temperature so that the reaction liquid temperature is maintained at 135°C. The second preset temperature is 135°C, the reaction time is 2h, and the second set time is 2h. After the reaction is completed, use a sand core funnel to filter and remove the reaction liquid, and rinse the obtained cellulose acetate carbamate particles with hot water several times.

[0182] 3g of cellulose acetate carbamate particles were placed in a small beaker. 30ml of N,N-dimethylformamide solution was added to a second predetermined concentration of 10%. The solution was dissolved in a 70°C oil bath with magnetic stirring for approximately 1 hour. Once completely dissolved, the solution was aspirated using a 5ml syringe and dripped into water. The droplets formed balls upon entry. The size of the balls could be varied by controlling the dripping speed. The water was magnetically stirred during the dripping process. Filtration was performed to obtain the cellulose acetate carbamate carrier balls.

[0183] 6g of wet cellulose acetate carbamate spheres were placed in a 50ml Erlenmeyer flask, and 30ml of a 1% glutaraldehyde solution was added. The mixture was shaken at 25°C and 150 rpm for 5 hours. After completion, the mixture was filtered and rinsed several times with distilled water. The cross-linked glutaraldehyde support was placed in a 50ml Erlenmeyer flask, and an enzyme solution consisting of 3g of lactase and 30ml of 0.2M pH 6.0 phosphate buffer was added. Here, the mass ratio of lactase to wet cellulose acetate carbamate spheres was 0.5. The mixture was shaken at 25°C and 150 rpm for 12 hours. After completion, the mixture was filtered and rinsed several times with distilled water. The immobilized enzyme was added to the buffer solution and shaken at 25°C and 150 rpm for 1 to 2 hours to wash out any unimmobilized enzyme within the spheres. Filtering was performed to obtain immobilized lactase.

[0184] Example 2

[0185] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the concentration of urea (the first preset concentration) is 15%. The remaining steps and parameters are the same as those in Example 1.

[0186] Example 3

[0187] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the concentration of urea (the first preset concentration) is 20%. The remaining steps and parameters are the same as those in Example 1.

[0188] Example 4

[0189] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the concentration of urea (the first preset concentration) is 30%. The remaining steps and parameters are the same as those in Example 1.

[0190] Example 5

[0191] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the concentration of urea (the first preset concentration) is 35%. The remaining steps and parameters are the same as those in Example 1.

[0192] Example 6

[0193] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the concentration of urea (the first preset concentration) is 40%. The remaining steps and parameters are the same as those in Example 1.

[0194] The immobilized lactase prepared in Examples 1 to 6 was used to catalyze the hydrolysis of lactose, and the enzyme activity of the immobilized lactase was measured. The relative enzyme activities corresponding to different Examples are shown in FIG. Figure 7 As shown, it can be seen that the urea concentration (first preset concentration) has a certain effect on the enzymatic activity of the immobilized lactase, but the effect is not significant. The enzymatic activity of the immobilized lactase prepared at different urea concentrations can all reach above 80%. In addition, the enzymatic activity of the immobilized lactase is not linearly related to the urea concentration. When the urea concentration ranges from 20% to 25%, the enzymatic activity of the immobilized lactase prepared is the highest.

[0195] Example 7

[0196] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the first preset temperature is 30° C. The remaining steps and parameters are the same as those in Example 1.

[0197] Example 8

[0198] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the first preset temperature is 40° C. The remaining steps and parameters are the same as those in Example 1.

[0199] Example 9

[0200] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the first preset temperature is 60° C. The remaining steps and parameters are the same as those in Example 1.

[0201] Example 10

[0202] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the first preset temperature is 70° C. The remaining steps and parameters are the same as those in Example 1.

[0203] Example 11

[0204] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the first preset temperature is 80° C. The remaining steps and parameters are the same as those in Example 1.

[0205] The immobilized lactase prepared in Example 1, Example 7 to Example 11 was tested for its enzyme activity. The relative enzyme activities of different examples were as follows: Figure 8As shown, the first preset temperature has a certain impact on the enzymatic activity of the immobilized lactase, but the impact is not significant. The enzymatic activity of the immobilized lactase in different embodiments can reach above 80%. The enzymatic activity of the immobilized lactase first increases with the increase of the first preset temperature. After reaching 50°C, the increase in the enzymatic activity of the immobilized lactase is not obvious. The treatment temperature range for the highest enzymatic activity of the immobilized lactase is 50°C to 80°C.

[0206] Example 12

[0207] The preparation of immobilized lactase in this example differs from that in Example 1 in that the first set time is 1 hour. The remaining steps and parameters are the same as those in Example 1.

[0208] Example 13

[0209] The preparation of immobilized lactase in this example differs from that in Example 1 in that the first set time is 2 hours. The remaining steps and parameters are the same as those in Example 1.

[0210] Example 14

[0211] The preparation of immobilized lactase in this example differs from that in Example 1 in that the first set time is 3 hours. The remaining steps and parameters are the same as those in Example 1.

[0212] Example 15

[0213] The preparation of immobilized lactase in this example differs from that in Example 1 in that the first set time is 4 hours. The remaining steps and parameters are the same as those in Example 1.

[0214] Example 16

[0215] The preparation of immobilized lactase in this example differs from that in Example 1 in that the first set time is 6 hours. The remaining steps and parameters are the same as those in Example 1.

[0216] Example 17

[0217] The preparation of immobilized lactase in this example differs from that in Example 1 in that the first set time is 7 hours. The remaining steps and parameters are the same as those in Example 1.

[0218] The relative enzyme activities of the immobilized lactase prepared in Example 1, Example 12 to Example 17 were measured. The relative enzyme activities of the immobilized lactase in different examples are shown in FIG. Figure 9As shown, it can be seen that the first set time has a certain effect on the enzymatic activity of the immobilized lactase, but the effect is not significant. The enzymatic activity of the immobilized lactase in different embodiments can reach more than 90%. When the first set time is within 2 hours to 5 hours, the enzymatic activity of the immobilized lactase is higher. Furthermore, when the first set time is 5 hours, the enzymatic activity of the immobilized lactase is the highest.

[0219] Example 18

[0220] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the second preset temperature is 120° C. The remaining steps and parameters are the same as those in Example 1.

[0221] Example 19

[0222] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the second preset temperature is 125° C. The remaining steps and parameters are the same as those in Example 1.

[0223] Example 20

[0224] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the second preset temperature is 130° C. The remaining steps and parameters are the same as those in Example 1.

[0225] Example 21

[0226] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the second preset temperature is 140° C. The remaining steps and parameters are the same as those in Example 1.

[0227] Example 22

[0228] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the second preset temperature is 145° C. The remaining steps and parameters are the same as those in Example 1.

[0229] The relative enzyme activities of the immobilized lactase prepared in Example 1 and Example 18 to Example 22 were measured. The relative enzyme activities of the immobilized lactase in different examples are shown in FIG. Figure 10 As shown, it can be seen that the second preset temperature has a greater impact on the enzymatic activity of the immobilized lactase. When the temperature is high, the enzymatic activity of the immobilized lactase will drop rapidly. When the second preset temperature is 125°C, the enzymatic activity of the immobilized lactase is the highest. When the second preset temperature is between 130°C and 140°C, the enzymatic activity of the immobilized lactase is also high.

[0230] Example 23

[0231] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the second set time is 1 hour. The remaining steps and parameters are the same as those in Example 1.

[0232] Example 24

[0233] The preparation of immobilized lactase in this example differs from that in Example 1 in that the second set time is 3 hours. The remaining steps and parameters are the same as those in Example 1.

[0234] Example 25

[0235] The preparation of immobilized lactase in this example differs from that in Example 1 in that the second set time is 4 hours. The remaining steps and parameters are the same as those in Example 1.

[0236] Example 26

[0237] The preparation of immobilized lactase in this example differs from that in Example 1 in that the second set time is 5 hours. The remaining steps and parameters are the same as those in Example 1.

[0238] Example 27

[0239] The preparation of immobilized lactase in this example differs from that in Example 1 in that the second set time is 7 hours. The remaining steps and parameters are the same as those in Example 1.

[0240] The relative enzyme activities of the immobilized lactase prepared in Example 1 and Example 23 to Example 27 were measured. The relative enzyme activities of the immobilized lactase in different examples are shown in FIG. Figure 11 As shown, it can be seen that the second set time has a certain effect on the enzyme activity of the immobilized lactase. When the second set time reaches 7h, the enzyme activity of the immobilized lactase will drop rapidly. The enzyme activity of the immobilized enzyme with the second set time of 2h to 5h is the highest.

[0241] Example 28

[0242] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the second preset concentration is 5%. The remaining steps and parameters are the same as those in Example 1.

[0243] Example 29

[0244] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the second preset concentration is 7.5%. The remaining steps and parameters are the same as those in Example 1.

[0245] Example 30

[0246] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the second preset concentration is 12.5%. The remaining steps and parameters are the same as those in Example 1.

[0247] Example 31

[0248] The preparation of immobilized lactase in this embodiment differs from that in Example 1 in that the second preset concentration is 15%. The remaining steps and parameters are the same as those in Example 1.

[0249] The relative enzyme activities of the immobilized lactase prepared in Example 1, Example 28 to Example 31 were measured. The relative enzyme activities of the immobilized lactase in different examples are shown in FIG. Figure 12 As shown, it can be seen that the second preset concentration has a greater impact on the enzymatic activity of the immobilized lactase. When the second preset concentration is less than or equal to 7.5%, the enzymatic activity of the immobilized lactase is low. When the second preset concentration is between 10% and 15%, the enzymatic activity of the immobilized enzyme is higher. More specifically, when the second preset concentration is between 10% and 12.5%, the enzymatic activity of the immobilized enzyme is the highest.

[0250] Example 32

[0251] The preparation of immobilized lactase in this example is different from that in Example 1 in that the mass ratio of lactase to wet cellulose acetate carbamate beads is 0.025. The remaining steps and parameters are the same as those in Example 1.

[0252] Example 33

[0253] The preparation of immobilized lactase in this example differs from that in Example 1 in that the mass ratio of lactase to wet cellulose acetate carbamate beads is 0.05. The remaining steps and parameters are the same as those in Example 1.

[0254] Example 34

[0255] The preparation of immobilized lactase in this example differs from that in Example 1 in that the mass ratio of lactase to wet cellulose acetate carbamate beads is 0.25. The remaining steps and parameters are the same as those in Example 1.

[0256] Example 35

[0257] The preparation of immobilized lactase in this example differs from that in Example 1 in that the mass ratio of lactase to wet cellulose acetate carbamate beads is 0.75. The remaining steps and parameters are the same as those in Example 1.

[0258] Example 36

[0259] The preparation of immobilized lactase in this example differs from that in Example 1 in that the mass ratio of lactase to wet cellulose acetate carbamate beads is 1. The remaining steps and parameters are the same as those in Example 1.

[0260] The relative enzyme activities of the immobilized lactase prepared in Examples 32 to 36 were measured. The relative enzyme activities of different examples were as follows: Figure 16As shown, it can be seen that the higher the lactase content, the higher the relative enzyme activity of the immobilized lactase. However, with the increase of lactase, the protein yield and enzyme activity yield gradually decrease. Here, the protein yield and enzyme activity yield reflect the protein recovery efficiency and the retention degree of enzyme activity. It can be seen that the more lactase in the immobilized lactase, the better. When the mass ratio of lactase to cellulose acetate carbamate balls is in the range of 0.25 to 0.5, the prepared immobilized lactase has a higher relative enzyme activity and can also ensure the protein yield and enzyme activity yield.

[0261] The immobilized lactase prepared in this example was enzymatically hydrolyzed using the following examples, and the enzyme content after enzymatic hydrolysis in different examples was measured.

[0262] Example 37

[0263] The immobilized lactase prepared in this example is different from that in Example 1 in that the enzyme activity test parameters for the immobilized enzyme are different. When the immobilized lactase in this example is tested for enzyme activity, the reaction time is 2 hours at 200 rpm.

[0264] After testing, the relative enzyme activity of Example 37 was 83%.

[0265] This indicates that the immobilized lactase prepared in this example has a high relative enzyme activity even when the enzymatic hydrolysis time is increased, and the immobilized lactase has a high stability.

[0266] Example 38

[0267] The immobilized lactase prepared in this example is different from that in Example 1 in that the enzyme activity test parameters for the immobilized enzyme are different. When the immobilized lactase in this example is tested for enzyme activity, the amount of immobilized lactase used is 0.5 g, the concentration of the lactose solution is 30%, and the reaction is shaken at 200 rpm for 6 h.

[0268] After testing, the relative enzyme activity of Example 38 was 63%.

[0269] Here, the amount of immobilized enzyme is small and the lactose content is high, but it can still convert 63% of the relative enzyme activity, which shows that the immobilized enzyme lactase prepared in this example has high enzyme activity and good stability.

[0270] Example 39

[0271] The immobilized lactase prepared in this example differs from that in Example 1 in that the enzyme activity assay parameters for the immobilized lactase are different. In the enzyme activity assay of the immobilized lactase in this example, an oscillation reaction at 200 rpm for 2 hours constitutes one batch, with the initial enzyme activity recorded as 100%. The immobilized lactase was stored in a refrigerator at 4°C between assays, and the relative enzyme activity after different uses of the immobilized lactase was calculated.

[0272] like Figure 17 As shown, the relative enzyme activity of the immobilized lactase in the first batch is defined as 100%. The immobilized enzyme prepared in Example 39 showed no significant decrease in activity after 20 days of use and storage after batch 40. This indicates that the immobilized lactase prepared in this example has good stability. Furthermore, it can be seen that the relative enzyme activity of the immobilized lactase in the first batch is not the highest; it increases with each batch and fluctuates around 100%.

[0273] Example 40

[0274] The preparation of the immobilized enzyme in this example differs from that in Example 1 in that lactase is replaced with glucose oxidase, resulting in an immobilized glucose oxidase. The remaining steps and parameters are the same as those in Example 1.

[0275] Test method: 5 g of immobilized glucose oxidase was placed in a conical flask containing 20 ml of 10% glucose, 0.55 g of calcium carbonate was added as a buffer, and the reaction was carried out at 55°C and 200 rpm for a period of time to measure the glucose content.

[0276] Comparative Example 1

[0277] A certain amount of cellulose acetate was weighed and dissolved in a 1:2 mixture of DMSO (dimethyl sulfoxide) and ethanol. The mixture was added dropwise to distilled water through a No. 6 syringe needle to form spheres. After filtration and washing, the spheres were sealed and stored to prevent drying, yielding porous cellulose acetate spheres. The cellulose acetate spheres were then mixed with twice the volume of a 0.05 mol / L NaIO4 solution and shaken overnight at 4°C. The activated carrier was then washed thoroughly with distilled water, dried, and stored sealed at 4°C.

[0278] A solution containing 6 g of activated carrier was mixed with a solution containing 3 g of glucose oxidase and shaken at 4°C overnight. After filtration and washing, 200 ml of 0.5 mol / L NaCl solution was added and shaken for 2 h and dried. The mixture was then thoroughly washed with distilled water and dried to prepare immobilized glucose oxidase.

[0279] In Comparative Example 1, the vicinal diol structure in the glycosyl compound molecule was oxidized by NaIO4 to form an aldehyde group, which then formed a Schiff's base with the amino group in the protein molecule.

[0280] Test method: 5 g of immobilized glucose oxidase was placed in a conical flask containing 20 ml of 10% glucose, 0.55 g of calcium carbonate was added as a buffer, and the reaction was carried out at 55°C and 200 rpm for a period of time to measure the glucose content.

[0281] The relative enzyme activities of the immobilized glucose oxidase prepared in Example 40 and Comparative Example 1 are shown in Table 1. It can be seen that the relative enzyme activity of the immobilized glucose oxidase prepared using the carrier prepared in this example is higher.

[0282] Table 1

[0283] Reaction time The relative enzyme activity of comparative example 1 Relative enzyme activity of Example 40 7h 39.4％ 42.5％ 18h 56.4％ 62.8％

[0284] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0285] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A carrier for immobilizing an enzyme, characterized in that: Amino-modified cellulose acetate porous spheres were used as carriers.

2. The carrier for immobilizing an enzyme according to claim 1, characterized in that The amino-modified cellulose acetate porous spheres are formed from cellulose acetate carbamate, which is prepared by modifying cellulose acetate with isocyanic acid.

3. The carrier for immobilizing an enzyme according to claim 2, characterized in that Isocyanic acid is produced by the decomposition of urea at high temperatures.

4. The carrier for immobilizing an enzyme according to any one of claims 1 to 3, characterized in that The outer diameter of the amino-modified cellulose acetate porous spheres is in the range of 2 mm to 4 mm, and / or the average pore size of the amino-modified cellulose acetate porous spheres is in the range of 15 nm to 30 nm, and / or the specific surface area of ​​the amino-modified cellulose acetate porous spheres is in the range of 30 m 2 / g to 50m 2 / g.

5. The method for preparing a carrier for immobilized enzyme according to any one of claims 1 to 4, characterized in that: include: Modifying cellulose acetate with a modifier to obtain cellulose acetate carbamate; Cellulose acetate carbamate is dissolved in a first organic solvent and then dropped into water to form cellulose acetate carbamate porous balls.

6. The preparation method according to claim 5, characterized in that The steps of modifying cellulose acetate with a modifier to obtain cellulose acetate carbamate include: dispersing cellulose acetate in an aqueous solution of a modifier to obtain a mixed solution; The mixed solution is heated to obtain cellulose acetate carbamate.

7. The preparation method according to claim 6, characterized in that The steps of dispersing cellulose acetate into a modifier aqueous solution to obtain a mixed solution include: preparing a modifier aqueous solution having a first preset concentration, dispersing cellulose acetate in the modifier aqueous solution at a second preset ratio, heating at a first preset temperature and stirring for a first preset time to obtain a mixed solution; Preferably, the first preset concentration ranges from 15% to 35%, or the first preset concentration ranges from 20% to 25%; Preferably, the second preset ratio is 0.5 g to 1.5 g of cellulose acetate: 10 mL of the modifier aqueous solution; or, the second preset ratio is 0.8 g to 1.2 g of cellulose acetate: 10 mL of the modifier aqueous solution; Preferably, the first preset temperature range is 30°C to 80°C, or the first preset temperature range is 50°C to 80°C, or the first preset temperature range is 60°C to 70°C, or the first preset temperature range is 60°C to 80°C, or the first preset temperature range is 50°C to 70°C, or the first preset temperature range is 70°C to 80°C; Preferably, the first set time ranges from 1 hour to 6 hours, or the first set time ranges from 3 hours to 6 hours, or the first set time ranges from 4 hours to 5 hours, or the first set time ranges from 2 hours to 5 hours; Preferably, the modifying agent comprises urea.

8. The preparation method according to claim 6, characterized in that The step of heating the mixed solution to obtain cellulose acetate carbamate comprises: The mixed solution is heated and stirred, and a second organic solvent is added, and the temperature is increased to evaporate water to obtain a reaction solution; The reaction liquid is reacted at a second preset temperature for a second set time to obtain cellulose acetate carbamate; Preferably, the second preset temperature ranges from 120° C. to 140° C., or the second preset temperature ranges from 125° C. to 140° C., or the second preset temperature ranges from 125° C. to 130° C., or the second preset temperature ranges from 130° C. to 140° C., or the second preset temperature ranges from 135° C. to 140° C.; Preferably, the amount of the second organic solvent is 30 ml to 70 ml, or the amount of the second organic solvent is 40 ml to 60 ml, or the amount of the second organic solvent is 50 ml; Preferably, the second organic solvent comprises one or more of o-xylene, acetone and dimethyl sulfoxide; Preferably, the second set time length ranges from 1 hour to 6 hours, or the second set time length ranges from 2 hours to 5 hours, or the second set time length ranges from 2 hours to 3 hours.

9. The preparation method according to claim 5, characterized in that The step of dissolving cellulose acetate carbamate in a first organic solvent and then dripping the solvent into water to form cellulose acetate carbamate porous balls comprises: dissolving cellulose acetate carbamate in a first organic solvent to form a solution of a second predetermined concentration; The solution is dropped into distilled water, and the solution forms a cellulose acetate urethane porous ball mixture in the distilled water; Filtering, soaking and draining the cellulose acetate urethane porous ball mixture to obtain cellulose acetate urethane porous balls; Preferably, the first organic solvent comprises one or more of a ketone solvent, an amide solvent and a chlorinated solvent; Preferably, the second preset concentration range is 7.5% to 15%, or the second preset concentration range is 9% to 11%, or the second preset concentration range is 10% to 12.5%, or the second preset concentration range is 10% to 15%.

10. A method for preparing an immobilized enzyme, characterized in that: The carrier is combined with the enzyme to obtain an immobilized enzyme, wherein the carrier is prepared by the method for preparing a carrier for immobilized enzyme according to any one of claims 5 to 9.

11. The preparation method according to claim 10, characterized in that: The immobilized enzyme is obtained by combining the carrier with the enzyme, including: adding cellulose acetate urethane porous balls to the crosslinker solution and mixing for a third set time to obtain a suspension; The suspension is repeatedly washed to obtain carrier balls; dissolving the enzyme using an acid solution to obtain an enzyme solution having a third predetermined concentration; Add the carrier balls into the enzyme solution, shake well, filter and rinse repeatedly to obtain the initial immobilized enzyme; washing and de-enzyming the initial immobilized enzyme to obtain the immobilized enzyme; Preferably, the ratio of the mass of the enzyme to the mass of the cellulose acetate carbamate porous ball is in the range of 0.025 to 1, or the ratio of the mass of the enzyme to the mass of the cellulose acetate carbamate porous ball is in the range of 0.05 to 0.75, or the ratio of the mass of the enzyme to the mass of the cellulose acetate carbamate porous ball is in the range of 0.25 to 0.5; Preferably, the concentration of the crosslinking agent solution is in the range of 0.5% to 2%, or the concentration of the crosslinking agent solution is in the range of 0.5% to 1.5%, or the concentration of the crosslinking agent solution is in the range of 0.5% to 1%, or the concentration of the crosslinking agent solution is in the range of 0.75% to 1%; Preferably, the third set time ranges from 3 hours to 5 hours, or the third set time ranges from 4 hours to 5 hours; Preferably, the third preset concentration ranges from 0.01 g / ml to 0.1 g / ml, or the third preset concentration ranges from 0.05 g / ml to 0.1 g / ml.

12. An immobilized enzyme, characterized in that The invention comprises an enzyme and a carrier, wherein the carrier comprises the carrier for immobilizing an enzyme according to any one of claims 1 to 4, or the carrier is prepared by the method for preparing the carrier for immobilizing an enzyme according to any one of claims 5 to 9.

13. Use of the carrier for immobilizing an enzyme according to any one of claims 1 to 4 in the biological field; or use of the carrier prepared by the method for preparing the carrier for immobilizing an enzyme according to any one of claims 5 to 9 in the biological field.

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