Reusable enzyme-loaded compound as well as preparation method and application thereof
By using silicon-based nanocarrier loading enzymes, the enzyme-carrying complex is formed, which solves the problem that enzymes are prone to inactivate and cannot be reused in harsh environments, and achieves efficient, stable and economical application of enzymes.
Patent Information
- Application Number
- CN202311827289.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-27
- Publication Date
- 2025-06-27
AI Technical Summary
Existing enzymes are prone to inactivate and cannot be reused in harsh environments, limiting their efficiency and economicality in multi-field applications.
Silicon-based nanocarrier is used as a carrier, and the enzyme is loaded on the surface of the silicon-based nanocarrier through a surface modification agent to form a reusable enzyme carrier complex. The support has a solid structure and a patterned structure, which improves the loading and catalytic efficiency of the enzyme.
The stability, activity and dispersion of the enzyme are improved, and the enzyme-carrying complex has good thermal stability and pH adaptability, which realizes the reusable use of the enzyme, reduces production costs and improves catalytic efficiency.
Smart Images
Figure CN120210176A_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the technical field of multifunctional nanocarriers, and particularly relates to a reusable enzyme-loaded complex and its preparation method and application. Background Art
[0002] Enzymes are the chips of green biological manufacturing in nature. They can catalyze reaction substrates to synthesize chemicals. Compared with chemical catalysts, they have better selectivity and specificity and bind to substrates under mild conditions, using the active center to catalyze complex chemical reactions. Enzymes are also indispensable in our daily life and have broad application prospects in many fields such as food chemistry, fine chemistry, medical chemistry, drug synthesis, fuel industry, and control of environmental pollution.
[0003] However, most enzymes are relatively fragile and are easily inactivated by harsh environments. In addition, many enzymes are water-soluble and cannot be separated from the system after the catalytic reaction is completed and cannot be reused. Summary of the Invention
[0004] Embodiments of this application provide a reusable enzyme-loaded complex and its preparation method and application.
[0005] In a first aspect, embodiments of this application provide a reusable enzyme-loaded complex, which includes a silicon-based nanocarrier and an enzyme loaded on the silicon-based nanocarrier. The silicon-based nanocarrier is a solid structure, and the surface of the solid structure has a patterned structure formed by recessing from the surface to the center. The enzyme is loaded on the surface of the solid structure through a surface modifier.
[0006] According to the embodiments of the first aspect of this application, the silicon-based nanocarrier satisfies at least one of the following conditions:
[0007] (1) The depth of the patterned structure recessing from the surface to the center is 5 nm to 15 nm;
[0008] (2) The particle size of the silicon-based nanocarrier is 10 nm - 5000 nm;
[0009] (3) The channel width of the patterned structure is 1 nm - 100 nm.
[0010] According to the embodiments of the first aspect of this application, the raw material components of the silicon-based nanocarrier include 0.1 wt% - 50 wt% silicon source, 0.1 wt% - 20 wt% surfactant, and 0.1 wt% - 20 wt% dispersant. The silicon-based nanocarrier is formed by self-assembly of the silicon source, surfactant, and dispersant.
[0011] According to an embodiment of the first aspect of the present application, the silicon source includes one or more of dimethylsilanol, methyl orthosilicate, ethyl orthosilicate, Prussian blue containing a siloxane bond, tetraethyl orthosilicate, alkoxysilane, silane coupling agent, silicon tetrachloride, and sodium silicate; and / or, the surfactant includes one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate, cetyltrimethylammonium bromide, sodium octyl sulfonate, polyvinylpyrrolidone, and polyvinyl alcohol; and / or, the dispersant includes one or more of sodium carboxymethyl cellulose, polymer dispersant, n-butanol, and cyclohexane.
[0012] According to an embodiment of the first aspect of the present application, the raw material components of the silicon-based nanocarrier further include 0.1 wt% - 30 wt% metal ions, and the silicon-based nanocarrier is formed by self-assembly of a silicon source, a surfactant, metal ions, and a dispersant.
[0013] According to an embodiment of the first aspect of the present application, the metal ions include one or more of manganese ions, zinc ions, zirconium ions, calcium ions, copper ions, ferrous ions, cerium ions, cobalt ions, and palladium ions;
[0014] According to an embodiment of the first aspect of the present application, the molar ratio of the metal ions to the silicon source is 1:0.01 - 100.
[0015] According to an embodiment of the first aspect of the present application, the silicon-based nano-mesoporous carrier includes one or more of a copper-silicon-based nanocarrier, an iron-silicon-based nanocarrier, a calcium-silicon-based nanocarrier, a cerium-silicon-based nanocarrier, a cobalt-silicon-based nanocarrier, a manganese-silicon-based nanocarrier, a zirconium-silicon-based nanocarrier, a lead-silicon-based nanocarrier, a copper-zirconium-silicon-based nanocarrier, a copper-iron-silicon-based nanocarrier, and a manganese-copper-silicon-based nanocarrier; and / or,
[0016] The surface modifier includes one or more of polyethyleneimine, branched polyethyleneimine, Pluronic F-127, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide, sodium carboxymethyl cellulose, Tween-80, polyglutamic acid, polyaspartic acid, collagen, glutaraldehyde, calcium lignosulfonate, polylysine, cysteine, and dopamine; and / or,
[0017] The enzyme includes any one or more of laccase, protease, peroxidase, lipase, dehydrogenase, oxidoreductase, oxygenase, and hydroxylase.
[0018] According to an embodiment of the first aspect of the present application, the enzyme-loaded complex further includes an activator, and the activator is loaded on the silicon-based nanocarrier;
[0019] The activator includes one or more of coenzyme A, coenzyme B12, adenosine triphosphate, coenzyme NAD + 、coenzyme NADP + 、coenzyme FAD, coenzyme FMN, hydrogen ions, chloride ions, and bromide ions.
[0020] According to an embodiment of the first aspect of the present application, the mass ratio of the enzyme to the silicon-based nanocarrier is 50:1 to 1:50.
[0021] According to an embodiment of the first aspect of the present application, the mass ratio of the enzyme to the surface modifier is 60:1 to 2:1; preferably 20:1 to 10:1.
[0022] In a second aspect, an embodiment of the present application provides a method for preparing a reusable enzyme-loaded complex, including the following steps: stirring and mixing a silicon source, a surfactant, and a dispersant, and obtaining a silicon-based nanocarrier after the reaction; stirring and mixing a surface modifier, an enzyme, and the silicon-based nanocarrier in an aqueous solution, and obtaining a reusable enzyme-loaded complex after centrifugation and washing.
[0023] According to an embodiment of the second aspect of the present application, the step of stirring and mixing a silicon source, a surfactant, and a dispersant and obtaining a silicon-based nanocarrier after the reaction satisfies at least one of the following conditions:
[0024] (1) The concentration of the silicon source is 1 - 1000 mg / mL, preferably 5 - 200 mg / mL, more preferably 5 - 100 mg / mL, and further preferably 10 - 50 mg / mL;
[0025] The concentration of the surfactant is 0.1 - 100 mg / mL, preferably 0.5 - 50 mg / mL, more preferably 0.5 - 10 mg / mL, and further preferably 1 - 5 mg / mL;
[0026] The concentration of the dispersant is 1 - 1000 mg / mL, preferably 5 - 200 mg / mL, more preferably 5 - 100 mg / mL, and further preferably 10 - 50 mg / mL;
[0027] (2) The stirring and mixing is carried out at 50°C - 100°C for reflux operation;
[0028] (3) After the reaction, the silicon-based nanocarrier to be dried is obtained by centrifugation, and the silicon-based nanocarrier is obtained after the silicon-based nanocarrier to be dried is dried, and the drying method includes at least one of natural air drying, freeze drying, and spray drying.
[0029] According to an embodiment of the first aspect of the present application, the step of stirring and mixing a surface modifier, an enzyme, and the silicon-based nanocarrier in an aqueous solution, and obtaining a reusable enzyme-loaded complex after centrifugation and washing satisfies at least one of the following conditions:
[0030] The concentration of the surface modifier is 10 - 100 mg / mL, preferably 10 - 50 mg / mL;
[0031] The concentration of the silicon-based nanocarrier is 1 - 80 mg / mL, preferably 5 - 20 mg / mL;
[0032] The concentration of the enzyme is 0.1 - 50 mg / mL, preferably 0.1 - 10 mg / mL.
[0033] In a third aspect, the embodiments of the present application provide the above-mentioned reusable enzyme-loaded complex for use in the fields of catalysis, drug delivery, wastewater treatment, medicine, and sterilization.
[0034] For the enzyme-loaded complex according to the embodiments of the present application, the silicon-based nanocarrier is a solid structure, and the surface of the solid structure has a patterned structure that is recessed from the surface towards the core. The recessed patterned structure can provide a higher specific surface area, which helps to increase the enzyme loading amount; the solid structure can enable the enzyme to be loaded on the surface of the silicon-based nanocarrier, increasing the contact area with the substrate and improving the catalytic efficiency. The inventors of the present application noticed in experiments that the enzyme-loaded complex has good thermal stability and pH adaptability, and thus has good reusability. This may be because the solid structure and the patterned structure of the silicon-based nanocarrier cause the silicon-based nanocarrier to be a pressure for enzyme dispersion.
[0035] The embodiments of the present application can effectively improve the stability, activity, and dispersibility of the enzyme by assembling the above-mentioned silicon-based nanocarrier with the enzyme. The surface modifier can enable the silicon-based nanocarrier to better bind to the enzyme, achieving a uniform and efficient catalytic effect for all the loaded enzymes. Since the particle size of the silicon-based nanocarrier is small, the enzyme-loaded complex has good dispersibility in an aqueous solution. The enzyme-loaded complex not only has better reusability itself, but also since the enzyme is loaded on the surface of the silicon-based nanocarrier, the enzyme can be simply and conveniently replaced and self-assembled. When replacing different enzymes, the discarded enzyme can be effectively peeled off and a new enzyme can be quickly assembled to form a new-functional enzyme-loaded complex, achieving the efficient utilization of the nanocarrier, improving the operability of the enzyme-loaded complex in the catalytic process, saving production costs, and providing catalytic efficiency. Description of the Drawings
[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings required to be used in the embodiments of the present application. Obviously, the following described drawings are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the drawings.
[0037] Figure 1 SEM image of the reusable enzyme-loaded complex prepared according to Example 1 of the present application;
[0038] Figure 2Reusability relative enzyme activity graph of the reusable enzyme-loaded complex prepared according to Example 1 of the present application.
[0039] Figure 3 Schematic process diagram for preparing the reusable enzyme-loaded complex according to the examples of the present application. Detailed implementation manners
[0040] Hereinafter, embodiments of the reusable enzyme-loaded complex and its preparation method of the present application are specifically disclosed with appropriate reference to the accompanying drawings, but there may be cases where unnecessary detailed descriptions are omitted. For example, there are cases where detailed descriptions of well-known matters and repeated descriptions of actually identical structures are omitted. This is to prevent the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the accompanying drawings and the following description are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter recited in the claims.
[0041] Reusable enzyme-loaded complex
[0042] In a first aspect, embodiments of the present application provide a reusable enzyme-loaded complex, which includes a silicon-based nanocarrier and an enzyme loaded on the silicon-based nanocarrier. The silicon-based nanocarrier is a solid structure, and the surface of the solid structure has a patterned structure formed by depressions from the surface towards the core. The enzyme is loaded on the surface of the solid structure through a surface modifier.
[0043] For the enzyme-loaded complex according to the embodiments of the present application, the silicon-based nanocarrier is a solid structure, and the surface of the solid structure has a patterned structure with depressions from the surface towards the core. The depressions in the patterned structure can provide a relatively large specific surface area, which helps to increase the enzyme loading amount; the solid structure can load the enzyme on the surface of the silicon-based nanocarrier, increasing the contact area with the substrate and improving the catalytic efficiency. The inventors of the present application noticed in experiments that the enzyme-loaded complex has good thermal stability and pH adaptability, and thus has good reusability. This may be because the solid structure and the patterned structure of the silicon-based nanocarrier relieve the dispersion pressure on the enzyme.
[0044] The embodiments of the present application can be prepared by assembling the above-mentioned silicon-based nanocarriers with enzymes, which can effectively improve the stability, activity and dispersibility of the enzymes. The surface modifier can enable better binding between the silicon-based nanocarrier and the enzyme, so that all the loaded enzymes achieve the effect of uniform and efficient catalysis. Due to the small particle size of the silicon-based nanocarrier, the enzyme-loaded complex has good dispersibility in an aqueous solution. The enzyme-loaded complex not only has better reusability itself, but also since the enzyme is loaded on the surface of the silicon-based nanocarrier, the enzyme can be simply and conveniently replaced and self-assembled. When different enzymes are replaced, the discarded enzyme can be effectively peeled off and a new enzyme can be quickly assembled to form an enzyme-loaded complex with new functions, achieving the efficient utilization of the nanocarrier, improving the operability of the enzyme-loaded complex in the catalytic process, saving production costs and providing catalytic efficiency.
[0045] In some embodiments, the surface modifier includes one or more of polyethyleneimine, branched polyethyleneimine, Pluronic F-127, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide, sodium carboxymethylcellulose, Tween-80, polyglutamic acid, polyaspartic acid, collagen, glutaraldehyde, calcium lignosulfonate, polylysine, cysteine, dopamine. The surface modifier can enable better binding between the silicon-based nanocarrier and the enzyme, so that all the loaded enzymes achieve the effect of uniform and efficient catalysis.
[0046] In some embodiments, the enzyme includes various functional natural enzymes, including any one or more of laccase, protease, peroxidase, lipase, dehydrogenase, oxidoreductase, oxygenase and hydroxylase. The enzyme can also include various functional artificially synthesized enzymes, and the embodiments of the present application are not limited thereto.
[0047] Exemplarily, the enzyme-loaded complexes in the embodiments of the present application include, but are not limited to, laccase@SiO2, protease@SiO2, peroxidase@SiO2, lipase@SiO2, dehydrogenase@SiO2, oxidoreductase@SiO2, oxygenase and hydroxylase@SiO2.
[0048] In some embodiments, the depth of the patterned structure recessed from the surface towards the center is 5 nm to 15 nm. The depth of the recess in the above range can increase the specific surface area and at the same time facilitate sufficient contact between the loaded enzyme and the substrate.
[0049] Exemplarily, the depth of the patterned structure recessed from the surface towards the center is 5 nm, 5.5 nm, 6 nm, 6.5 nm, 7 nm, 7.5 nm, 8 nm, 8.5 nm, 8.9 nm, 9 nm, 9.5 nm, 9.7 nm, 10 nm, 10.5 nm, 11 nm, 11.5 nm, 12 nm, 12.5 nm, 12.6 nm, 13 nm, 13.5 nm, 14 nm, 14.1 nm, 14.5 nm, 15 nm or a range composed of any two of the above values.
[0050] In some embodiments, the particle size of the silicon-based nanocarrier is 10 nm - 5000 nm. The relatively small particle size of the silicon-based nanocarrier enables good dispersion of the enzyme-loaded complex in an aqueous solution.
[0051] Exemplarily, the depth of the patterned structure recessed from the surface towards the center is 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 220 nm, 240 nm, 260 nm, 280 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm or a range composed of any two of the above values.
[0052] In some embodiments, as Figure 1 shown, the patterned structure includes irregular channels distributed on the surface of the silicon-based nanocarrier, and the channel width of the patterned structure is 1 nm - 100 nm. The channel width of the patterned structure within the above range facilitates sufficient contact between the loaded enzyme and the substrate, and also facilitates the replacement and self-assembly of the enzyme and the carrier.
[0053] Exemplarily, the channel width of the patterned structure is 1 nm, 1.5 nm, 2 nm, 2.5 nm, 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 6.5 nm, 7 nm, 7.5 nm, 8 nm, 8.5 nm, 8.9 nm, 9 nm, 9.5 nm, 9.7 nm, 10 nm, 10.5 nm, 11 nm, 11.5 nm, 12 nm, 12.5 nm, 12.6 nm, 13 nm, 13.5 nm, 14 nm, 14.1 nm, 14.5 nm, 15 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm or a range composed of any two of the above values.
[0054] In some embodiments, the raw material components of the silicon-based nanocarrier include 0.1 wt% - 50 wt% silicon source, 0.1 wt% - 20 wt% surfactant, and 0.1 wt% - 20 wt% dispersant. The silicon-based nanocarrier is formed by self-assembly of the silicon source, surfactant, and dispersant.
[0055] The silicon-based nanocarrier in the embodiments of the present application includes the above raw material components. The raw material components are simple and are formed by self-assembly. The preparation method is simple and is conducive to mass production.
[0056] In some embodiments, the silicon source includes one or more of dimethylsilanol, methyl orthosilicate, ethyl orthosilicate, Prussian blue containing siloxane bonds, tetraethyl orthosilicate, alkoxysilane, silane coupling agent, silicon tetrachloride, and sodium silicate.
[0057] In some embodiments, the surfactant includes one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate, cetyltrimethylammonium bromide, sodium octyl sulfonate, polyvinylpyrrolidone, and polyvinyl alcohol.
[0058] In some embodiments, the dispersant includes one or more of sodium carboxymethyl cellulose, polymer dispersant, n-butanol, and cyclohexane.
[0059] In some embodiments, the raw material components of the silicon-based nanocarrier further include 0.1 wt% - 30 wt% metal ions. The silicon-based nanocarrier is formed by self-assembly of the silicon source, surfactant, metal ions, and dispersant.
[0060] Exemplarily, the mass content of the metal ions can be 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.88 wt%, 0.9 wt%, 0.95 wt%, 1.0 wt%, 2.0 wt%, 3.0 wt%, 4.0 wt%, 5.0 wt%, 6.0 wt%, 7.0 wt%, 8.0 wt%, 9.0 wt%, 10 wt%, 12 wt%, 14 wt%, 16 wt%, 18 wt%, 20 wt%, 22 wt%, 24 wt%, 26 wt%, 28 wt%, 30 wt% or a range composed of any two of the above values.
[0061] In some embodiments, the metal ions include one or more of manganese ions, zinc ions, zirconium ions, calcium ions, copper ions, ferrous ions, cerium ions, cobalt ions, and palladium ions. The metal ions can promote the catalytic reaction, and the presence of the metal ions can make the catalytic effect of the enzyme better.
[0062] In some embodiments, the molar ratio of the metal ions to the silicon source is 1:50 - 1000.
[0063] Exemplarily, the molar ratio of metal ions to silicon source is 1:0.03, 1:0.04, 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09, 1:0.10, 1:0.11, 1:0.12, 1:0.15, 1:0.16, 1:0.17, 1:0.18, 1:0.20, 1:0.22, 1:0.25, 1:0.30, 1:0.35, 1:0.40, 1:0.45, 1:0.50, 1:0.55, 1:0.60, 1:0.65, 1:0.70, 1:0.75, 1:0.80, 1:0.85, 1:0.90, 1:0.95, 1:1.00, 1:
[0064] 1.01, 1:1.02, 1:2, 1:3, 1:5, 1:10, 1:20, 1:30 or a range composed of any two of the above values.
[0065] In some embodiments, the silicon-based nano-mesoporous carrier includes one or more of a copper-silicon-based nano-carrier, an iron-silicon-based nano-carrier, a calcium-silicon-based nano-carrier, a cerium-silicon-based nano-carrier, a cobalt-silicon-based nano-carrier, a manganese-silicon-based nano-carrier, a zirconium-silicon-based nano-carrier, a lead-silicon-based nano-carrier, a copper-zirconium-silicon-based nano-carrier, a copper-iron-silicon-based nano-carrier, and a manganese-copper-silicon-based nano-carrier.
[0066] In some embodiments, the enzyme-loaded complex further includes an activator, and the activator is loaded on the silicon-based nano-carrier;
[0067] The activator includes one or more of coenzyme A, coenzyme B12, adenosine triphosphate, coenzyme NAD + , coenzyme NADP + , coenzyme FAD, coenzyme FMN, hydrogen ions, chloride ions, and bromide ions. The activator can activate the action of the enzyme, and some activators can cooperate with the enzyme to catalyze different reactions to form a cascade reaction.
[0068] In some embodiments, the mass ratio of the enzyme to the silicon-based nano-carrier is 50:1 to 1:50.
[0069] Exemplarily, the mass ratio of the enzyme to the silicon-based nano-carrier is 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50 or a range composed of any two of the above values.
[0070] In some embodiments, the mass ratio of the enzyme to the surface modifier is 60:1 to 2:1; illustratively, the mass ratio of the enzyme to the silicon-based nanocarrier is 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 8:1, 6:1, 4:1, 2:1. Preferably, it is 20:1 to 10:1.
[0071] By regulating the mass ratio of enzyme to silicon-based nanocarrier and the mass ratio of enzyme to surface modifier, the loading amount of enzyme on the surface of silicon-based nanocarrier can be adjusted as needed, which is beneficial to expand the application range of enzyme-loaded complexes.
[0072] Method for preparing reusable enzyme-loaded complex
[0073] In a second aspect, an embodiment of the present application provides a method for preparing a reusable enzyme-loaded complex.
[0074] like Figure 3 As shown, the preparation method comprises the following steps:
[0075] Step S100, stirring and mixing a silicon source, a surfactant and a dispersant to obtain a silicon-based nanocarrier after reaction;
[0076] Step S200, the surface modifier, enzyme and silicon-based nanocarrier are stirred and mixed in an aqueous solution, and a reusable enzyme-loaded complex is obtained after centrifugation and washing.
[0077] The method for preparing a reusable enzyme-loaded complex according to the embodiment of the present application does not require grinding equipment, high-pressure homogenization equipment, high-temperature calcining furnaces or microfluidic equipment, etc., has low requirements for equipment, reduces energy consumption in production, and has a certain degree of universality, being applicable to almost all enzymes, having low production costs, and being capable of continuous online operation.
[0078] [Step S100]
[0079] In some embodiments, in the step of mixing the silicon source, the surfactant and the dispersant by stirring and reacting to obtain the silicon-based nanocarrier, the concentration of the silicon source is 1-1000 mg / mL, preferably 5-200 mg / mL, more preferably 5-100 mg / mL, and further preferably 10-50 mg / mL.
[0080] The silicon-based nanocarrier prepared by the preparation method of the embodiment of the present application is a solid structure. The surface of the solid structure has a patterned structure that depresses from the surface to the core. The depressed patterned structure can provide a higher specific surface area, which helps to increase the enzyme loading amount. The solid structure can load the enzyme on the surface of the silicon-based nanocarrier, increase the contact area with the substrate, and improve the catalytic efficiency. The inventors of the present application noticed in the experiment that the enzyme-loaded complex has good thermal stability and pH adaptability, and thus has good reusability.
[0081] In some embodiments, the concentration of the surfactant is 0.1-100 mg / mL, preferably 0.5-50 mg / mL, more preferably 0.5-10 mg / mL, and further preferably 1-5 mg / mL.
[0082] In some embodiments, the concentration of the dispersant is 1-1000 mg / mL, preferably 5-200 mg / mL, more preferably 5-100 mg / mL, and further preferably 10-50 mg / mL.
[0083] In some embodiments, the stirring and mixing is carried out at 50°C-100°C for reflux operation. The reflux operation can remove the surfactant in the system.
[0084] Exemplarily, the stirring and mixing is carried out at 50°C, 55°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 95°C, 100°C for reflux operation, or the reflux operation is carried out within the range composed of any two of the above values.
[0085] By regulating the stirring conditions and the reactant concentration, the reaction rate and reaction time can be adjusted, which is beneficial to obtaining the silicon-based nanocarrier with the required content, and can reduce the risk of agglomeration of solid substances in the silicon-based nanocarrier, which is beneficial to the subsequent operation of loading the enzyme.
[0086] In some embodiments, after the reaction, the silicon-based nanocarrier to be dried is obtained by centrifugation. The silicon-based nanocarrier to be dried is dried to obtain the silicon-based nanocarrier. The drying methods include at least one of natural air drying, freeze drying, and spray drying.
[0087] [Step S200]
[0088] In some embodiments, in the step of stirring and mixing a surface modifier, an enzyme and a silicon-based nanocarrier in an aqueous solution, centrifuging and washing to obtain a reusable enzyme-loaded complex, the concentration of the surface modifier is 10-100 mg / mL, preferably 10-50 mg / mL;
[0089] In some embodiments, the concentration of the silicon-based nanocarrier is 1-80 mg / mL.
[0090] Exemplarily, the concentration of the silicon-based nanocarrier is 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL or a range composed of any two of the above values. Preferably, the concentration of the silicon-based nanocarrier is 5-20 mg / mL.
[0091] In some embodiments, the concentration of the enzyme is 0.1-50 mg / mL. Exemplarily, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL or a range composed of any two of the above values. Preferably, it is 0.1-10 mg / mL.
[0092] By regulating the stirring conditions and the concentrations of the reactants, the reaction rate and reaction time can be adjusted, which is beneficial to obtaining the enzyme-loaded complex with the required content, and can reduce the risk of aggregation of solid substances in the enzyme-loaded complex, which is beneficial to the subsequent action of the enzyme.
[0093] In a third aspect, the embodiments of the present application provide an application of the above-mentioned reusable enzyme-loaded complex product in the fields of catalysis, drug delivery, wastewater treatment, medicine, and sterilization.
[0094] The reusable enzyme-loaded complex in this application can be applied in the fields of catalysis, drug delivery, wastewater treatment, medicine, sterilization, and other fields that may be involved.
[0095] The present application will be described below in conjunction with specific embodiments.
[0096] Example 1
[0097] The reusable enzyme-loaded complex prepared in an aqueous solution in this example includes the following steps:
[0098] (1) Prepare 30 ml of a urea solution with a concentration of 0.4 mol / L. Add 3.0 g of cetyltrimethylammonium bromide (CTAB), 1.5 ml of n-butanol, and 16.38 ml of cyclohexane to the urea solution. Then add a 0.1 mol / L copper and zirconium metal ion solution to the system and stir at a speed of 550 rpm at room temperature for 2 h.
[0099] (2) Add 3.14 ml of a silicon source solution to the solution in step (1). Stir the mixture at 550 rr pm at room temperature for 30 min, and then incubate at 70 °C for 20 h.
[0100] (3) Centrifuge the solution in step (2) at 10000 rpm for 10 min to obtain a solid product. Disperse the solid product in ethanol and isopropanol respectively, centrifuge for 10 min for one washing, then discard the supernatant, and dry the washed solid product. Then reflux in an HCl ethanol solution at 82 °C for 6 h, wash twice with ethanol, and dry again to obtain a silicon-based nanocarrier with a particle size of about 500 nm and a pore size of about 30 nm.
[0101] (4) Disperse 20 mg of the silicon-based nanocarrier in step (3) and 5 mg of polyethyleneimine in 1 ml of an ethanol solution. Mix at 37 °C and 200 rpm for 12 h, then wash 5 times with deionized water. Incubate with 2 mg of laccase in a PBS solution with a pH of 7.0 at 4 °C for 12 h, and centrifuge at 8000 rpm for 15 min to obtain a reusable enzyme-loaded complex. The enzyme-loaded complex is used for catalytic reactions. After repeating the catalysis ten times, it can still retain about 70% of the enzyme activity.
[0102] (5) When the enzyme needs to be replaced, add an enzyme eluent to the enzyme-loaded complex to restore it to the state of the silicon-based nanocarrier, and repeat steps (1)-(4) for the immobilization of a new enzyme. After the enzyme-loaded complex is dried, Figure 1 The scanning electron microscope test chart shows that its microstructure is uniformly distributed flower-shaped or spherical nanoparticles; Figure 2 This is the performance of the repeated use of the enzyme-loaded complex.
[0103] Example 2
[0104] The reusable enzyme-loaded complex prepared in the aqueous solution in this embodiment comprises the following steps:
[0105] (1) Prepare 30 ml of a urea solution with a concentration of 0.4 mol / L. Add 3.0 g of cetyltrimethylammonium bromide (CTAB), 1.23 ml of n-butanol, and 13 ml of cyclohexane to the urea solution. Then add a 0.1 mol / L copper and zirconium metal ion solution to the system and stir at a speed of 550 rpm at room temperature for 2 h.
[0106] (2) Add 3.14 ml of a silicon source solution to the solution in step (1). Stir the mixture at 550 rpm at room temperature for 30 min, and then incubate it at 70 °C for 20 h.
[0107] (3) Centrifuge the solution in step (2) at 10000 rpm for 10 min to obtain a solid product. Disperse the solid product in ethanol and isopropanol respectively, centrifuge for 10 min for one washing, then discard the supernatant, and dry the washed solid product. Then reflux it in an HCl ethanol solution at 82 °C for 6 h, wash it twice with ethanol, and dry it again to obtain a silicon-based nanocarrier with a particle size of about 400 nm and a pore size of about 25 nm.
[0108] (4) Disperse 20 mg of the silicon-based nanocarrier in step (3), 5 mg of polyethyleneimine, and 10 mg of glutaraldehyde in 1 ml of an ethanol solution. Mix them at 37 °C and 200 rpm for 12 h, then wash them 5 times with deionized water. Incubate them with 2 mg of an enzyme in a PBS solution with a pH of 7.0 at 4 °C for 12 h, and centrifuge at 8000 rpm for 15 min to obtain a reusable enzyme-loaded complex. Use the enzyme-loaded complex for catalytic reactions. After repeating the catalysis ten times, it can still retain about 75% of the enzyme activity.
[0109] (5) When the enzyme needs to be replaced, add an enzyme eluent to the enzyme-loaded complex to restore the state of the silicon-based nanocarrier, and repeat steps (1)-(4) for the immobilization of a new enzyme.
[0110] Example 3
[0111] The reusable enzyme-loaded complex prepared in the aqueous solution in this embodiment comprises the following steps:
[0112] (1) Prepare 30 ml of a urea solution with a concentration of 0.4 mol / L. Add 3.0 g of cetyltrimethylammonium bromide (CTAB), 1.23 ml of n-butanol, and 13 ml of cyclohexane to the urea solution. Then add a 0.1 mol / L copper and zirconium metal ion solution to the system and stir at a speed of 550 rpm at room temperature for 2 h.
[0113] (2) Add 3.64 ml of the silicon source solution to the solution in step (1), stir the mixture at 550 rpm at room temperature for 30 min, and then incubate it at 70 °C for 20 h.
[0114] (3) Centrifuge the solution in step (2) at 10000 rpm for 10 min to obtain a solid product. Disperse the solid product in ethanol and isopropanol respectively, centrifuge for 10 min for one - time washing, then discard the supernatant, and dry the washed solid product. Then reflux it in HCl ethanol solution at 82 °C for 6 h, wash it twice with ethanol, and dry it again to obtain a silicon - based nanocarrier with a particle size of about 700 nm and a pore size of about 50 nm.
[0115] (4) Disperse 20 mg of the silicon - based nanocarrier in step (3) and 10 mg of glutaraldehyde in 1 ml of ethanol solution, mix them at 37 °C and 200 rpm for 12 h, then wash them 5 times with deionized water, incubate them with 2 mg of enzyme in PBS solution with a pH of 7.0 at 4 °C for 12 h, and centrifuge at 8000 rpm for 15 min to obtain a reusable enzyme - loaded complex. Use the enzyme - loaded complex for catalytic reactions. After repeating the catalysis ten times, it can still retain about 70% of the enzyme activity.
[0116] (5) When the enzyme needs to be replaced, add an enzyme eluent to the enzyme - loaded complex to restore the state of the silicon - based nanocarrier, and repeat steps (1) - (4) for the immobilization of a new enzyme.
[0117] Example 4
[0118] The steps for preparing the reusable enzyme - loaded complex in an aqueous solution in this example are as follows:
[0119] (1) Prepare 30 ml of a urea solution with a concentration of 0.4 mol / L, and add 3.0 g of cetyltrimethylammonium bromide (CTAB), 1.93 ml of n - butanol, and 15.38 ml of cyclohexane to the urea solution. Then add a 0.1 mol / L copper and zirconium metal ion solution to the system and stir it at a speed of 550 rpm at room temperature for 2 h.
[0120] (2) Add 3.14 ml of the silicon source solution to the solution in step (1), stir the mixture at 550 rpm at room temperature for 30 min, and then incubate it at 70 °C for 20 h.
[0121] (3) Centrifuge the solution in step (2) at 10000 rpm for 10 min to obtain a solid product. Disperse the solid product in ethanol and isopropanol respectively, centrifuge for 10 min for one - time washing, then discard the supernatant, and dry the washed solid product. Then reflux it in HCl ethanol solution at 82 °C for 6 h, wash it twice with ethanol, and dry it again to obtain a silicon - based nanocarrier with a particle size of about 600 nm and a pore size of about 40 nm.
[0122] (4) Incubate 20 mg of the silicon-based nanocarrier from step (3) with 2 mg of the enzyme in PBS solution at pH 7.0 for 12 h at 4 °C, and centrifuge at 8000 rpm for 15 min to obtain a reusable enzyme-loaded complex. The enzyme-loaded complex can be used for catalytic reactions and still retain approximately 80% of its enzyme activity after being reused ten times.
[0123] (5) When the enzyme needs to be replaced, add an enzyme eluent to the enzyme-loaded complex to restore the state of the silicon-based nanocarrier, and repeat steps (1)-(4) for the immobilization of a new enzyme.
[0124] Example 5
[0125] The steps for preparing the reusable enzyme-loaded complex in an aqueous solution in this example are as follows:
[0126] (1) Prepare 30 ml of a urea solution with a concentration of 0.4 mol / L, and add 3.0 g of cetyltrimethylammonium bromide (CTAB), 2 g of sodium dodecyl sulfate, 1.93 ml of n-butanol, and 15.38 ml to the urea solution. Then add a 0.1 mol / L copper and zirconium metal ion solution to the system and stir at a speed of 550 rpm at room temperature for 2 h.
[0127] (2) Add 3.14 ml of the silicon source solution to the solution from step (1), stir the mixture at 550 rpm at room temperature for 30 min, and then incubate at 70 °C for 20 h.
[0128] (3) Centrifuge the solution from step (2) at 10000 rpm for 10 min to obtain a solid product. Disperse the solid product in ethanol and isopropanol respectively, centrifuge for 10 min for one washing, discard the supernatant, and dry the washed solid product. Then reflux in an HCl ethanol solution at 82 °C for 6 h, wash twice with ethanol, and dry again to obtain a silicon-based nanocarrier with a particle size of approximately 200 nm and a pore size of approximately 20 nm.
[0129] (4) Disperse 20 mg of the silicon-based nanocarrier from step (3), 5 mg of polyethyleneimine, and 10 mg of glutaraldehyde in 1 ml of ethanol solution, mix at 37 °C and 200 rpm for 12 h, wash 5 times with deionized water, incubate with 2 mg of the enzyme in PBS solution at pH 7.0 for 12 h at 4 °C, and centrifuge at 8000 rpm for 15 min to obtain a reusable enzyme-loaded complex. The enzyme-loaded complex can be used for catalytic reactions and still retain approximately 70% of its enzyme activity after being reused ten times.
[0130] (5) When the enzyme needs to be replaced, add an enzyme eluent to the enzyme-loaded complex to restore the state of the silicon-based nanocarrier, and repeat steps (1)-(4) for the immobilization of a new enzyme.
[0131] Example 6
[0132] The reusable enzyme - loaded complex prepared in an aqueous solution in this example comprises the following steps:
[0133] (1) Prepare 30 ml of a urea solution with a concentration of 0.4 mol / L. Add 3.0 g of cetyltrimethylammonium bromide (CTAB), 2.2 g of sodium octylsulfonate, 2.5 g of polyvinylpyrrolidone, 1.23 ml of n - butanol, and 15.38 ml of cyclohexane to the urea solution. Then add a 0.1 mol / L copper and zirconium metal ion solution to the system and stir at a speed of 550 rpm at room temperature for 2 h.
[0134] (2) Add 3.14 ml of a silicon source solution to the solution in step (1). Stir the mixture at 550 rr pm at room temperature for 30 min, and then incubate at 70 °C for 20 h.
[0135] (3) Centrifuge the solution in step (2) at 10000 rpm for 10 min to obtain a solid product. Disperse the solid product in ethanol and isopropanol respectively, centrifuge for 10 min for one - time washing, then discard the supernatant, and dry the washed solid product. Then reflux in an HCl ethanol solution at 82 °C for 6 h, wash twice with ethanol, and dry again to obtain a silicon - based nanocarrier with a particle size of about 800 nm and a pore size of about 50 nm.
[0136] (4) Disperse 20 mg of the silicon - based nanocarrier in step (3), 5 mg of polyethyleneimine, 10 mg of glutaraldehyde, and 12 mg of calcium lignosulfonate in 1 ml of an ethanol solution. Mix at 37 °C and 200 rpm for 12 h, then wash 5 times with deionized water. Incubate with 2 mg of enzyme in a PBS solution with a pH of 7.0 at 4 °C for 12 h, and centrifuge at 8000 rpm for 15 min to obtain a reusable enzyme - loaded complex. The enzyme - loaded complex is used for catalytic reactions, and after repeating the catalysis ten times, it can still retain about 75% of the enzyme activity.
[0137] (5) When the enzyme needs to be replaced, add an enzyme eluent to the enzyme - loaded complex to restore the state of the silicon - based nanocarrier, and repeat steps (1) - (4) for the immobilization of a new enzyme.
[0138] Comparative Example 1
[0139] The enzyme - loaded complex prepared in an aqueous solution in this comparative example comprises the following steps:
[0140] (1) Prepare 30 ml of a urea solution with a concentration of 0.01 mol / L. Add 3.0 g of cetyltrimethylammonium bromide (CTAB), 1.7 ml of n-butanol, and 13.38 ml of cyclohexane to the urea solution. Then add a 0.1 mol / L copper and zirconium metal ion solution to the system and stir at a speed of 550 rpm for 2 h at room temperature.
[0141] (2) Add 0.15 ml of a silicon source solution to the solution in step (1). Stir the mixture at 550 rr pm at room temperature for 30 min, and then incubate at 70 °C for 2 h.
[0142] (3) Centrifuge the solution in step (2) at 10000 rpm for 10 min to obtain a solid product. Disperse the solid product in ethanol and isopropanol respectively, centrifuge for 10 min for one washing, then discard the supernatant, and dry the washed solid product. After refluxing in an HCl ethanol solution at 82 °C for 6 h, wash it twice with ethanol and then dry it. A silicon-based nanocarrier with a regular morphology and a pore structure on the surface cannot be obtained.
[0143] (4) Disperse 20 mg of the silicon-based nanocarrier in step (3) and 0.1 mg of polyethyleneimine in 1 ml of an ethanol solution. Mix at 37 °C and 200 rpm for 12 h, then wash 5 times with deionized water. Incubate with 2 mg of laccase in a PBS solution with a pH of 7.0 at 4 °C for 12 h, and centrifuge at 8000 rpm for 15 min. A reusable enzyme-loaded complex cannot be obtained and it does not have the performance of being reusable.
[0144] Comparative Example 2
[0145] The enzyme-loaded complex prepared in this comparative example in an aqueous solution includes the following steps:
[0146] (1) Prepare 30 ml of a urea solution with a concentration of 0.01 mol / L. Add 3.0 g of cetyltrimethylammonium bromide (CTAB), 1.7 ml of n-butanol, and 13.38 ml of cyclohexane to the urea solution and stir at a speed of 550 rpm for 2 h at room temperature.
[0147] (2) Add 0.15 ml of a silicon source solution to the solution in step (1). Stir the mixture at 550 rr pm at room temperature for 30 min, and then incubate at 70 °C for 2 h.
[0148] (3) Centrifuge the solution from step (2) at 10,000 rpm for 10 min to obtain a solid product. Disperse the solid product in ethanol and isopropanol respectively, centrifuge for 10 min for one washing, discard the supernatant, and dry the washed solid product. Then reflux it in an HCl ethanol solution at 82 °C for 6 h, wash it twice with ethanol, and dry it again, but a silicon-based nanocarrier with a regular morphology and a pore structure on the surface cannot be obtained.
[0149] (4) Disperse 20 mg of the silicon-based nanocarrier from step (3) and 0.1 mg of polyethyleneimine in 1 ml of an ethanol solution, mix them at 37 °C and 200 rpm for 12 h, then wash them 5 times with deionized water, incubate them with 2 mg of laccase in a PBS solution with a pH of 7.0 at 4 °C for 12 h, and centrifuge at 8,000 rpm for 15 min. A reusable enzyme-loaded complex cannot be obtained and it does not have the performance of being reusable.
[0150] The above examples and comparative examples illustrate and describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above examples. What is described in the above examples and the specification only explains the principle of the present invention, rather than limiting the scope of the present invention in any way. Without departing from the scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the claimed protection. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of various equivalent modifications or substitutions, and these modifications or substitutions should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims
1. A reusable enzyme-loaded complex, characterized in that, It includes a silicon-based nanocarrier and an enzyme loaded on the silicon-based nanocarrier. The silicon-based nanocarrier is a solid structure, and the surface of the solid structure has a patterned structure formed by recessing from the surface to the centroid. The enzyme is loaded on the surface of the solid structure through a surface modifier.
2. The reusable enzyme-loaded complex according to claim 1, wherein The silicon-based nanocarrier satisfies at least one of the following conditions: (1) The depth of the recess from the surface to the centroid of the patterned structure is 5 nm to 100 nm; (2) The particle size of the silicon-based nanocarrier is 10 nm - 5000 nm; (3) The channel width of the patterned structure is 1 nm - 100 nm.
3. The reusable enzyme-loaded complex according to claim 1, characterized in that, The raw material components of the silicon-based nanocarrier include 0.1 wt% - 50 wt% of a silicon source, 0.1 wt% - 20 wt% of a surfactant, and 0.1 wt% - 20 wt% of a dispersant. The silicon-based nanocarrier is formed by self-assembly of the silicon source, the surfactant, and the dispersant.
4. The reusable enzyme-loaded complex according to claim 3, wherein The silicon source includes one or more of dimethylsilanol, methyl orthosilicate, ethyl orthosilicate, Prussian blue containing a siloxane bond, tetraethyl orthosilicate, alkoxysilane, silane coupling agent, silicon tetrachloride, sodium silicate; and / or, The surfactant includes one or more of sodium dodecyl sulfate, sodium dodecyl sulfonate, cetyltrimethylammonium bromide, sodium octyl sulfonate, polyvinylpyrrolidone, polyvinyl alcohol; and / or, The dispersant includes one or more of sodium carboxymethyl cellulose, polymer dispersant, n-butanol, cyclohexane.
5. The reusable enzyme-carrying complex according to claim 3, wherein The raw material components of the silicon-based nanocarrier further include 0.1 wt% - 30 wt% of metal ions, and the silicon-based nanocarrier is formed by self-assembly of the silicon source, the surfactant, the metal ions, and the dispersant; Optionally, the metal ions include one or more of manganese ions, zinc ions, zirconium ions, calcium ions, copper ions, ferrous ions, cerium ions, cobalt ions, palladium ions; Optionally, the molar ratio of metal ions to the silicon source is 1:0.01 - 100.
6. The reusable enzyme-loaded complex according to claim 1, wherein The silicon-based nano-mesoporous carrier includes one or more of copper-silicon-based nanocarrier, iron-silicon-based nanocarrier, calcium-silicon-based nanocarrier, cerium-silicon-based nanocarrier, cobalt-silicon-based nanocarrier, manganese-silicon-based nanocarrier, zirconium-silicon-based nanocarrier, lead-silicon-based nanocarrier, copper-zirconium-silicon-based nanocarrier, copper-iron-silicon-based nanocarrier, manganese-copper-silicon-based nanocarrier; and / or, The surface modifier includes one or more of polyethyleneimine, branched polyethyleneimine, Pluronic F-127, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide, sodium carboxymethyl cellulose, Tween-80, polyglutamic acid, polyaspartic acid, collagen, glutaraldehyde, calcium lignosulfonate, polylysine, cysteine, dopamine; and / or, The enzyme includes any one or more of laccase, protease, peroxidase, lipase, dehydrogenase, oxidoreductase, oxygenase, and hydroxylase.
7. The reusable enzyme-carrying complex according to any one of claims 1-6, characterized in that, The enzyme-loaded complex further includes an activator, and the activator is loaded on the silicon-based nanocarrier; The activator includes one or more of coenzyme A, coenzyme B12, adenosine triphosphate, coenzyme NAD + , coenzyme NADP + , coenzyme FAD, coenzyme FMN, hydrogen ions, chloride ions, bromide ions.
8. The reusable enzyme-loaded complex according to claim 1, wherein The mass ratio of the enzyme to the silicon-based nanocarrier is 50:1 to 1:
50.
9. The reusable enzyme-loaded complex according to claim 1, characterized in that, The mass ratio of the enzyme to the surface modifier is 60:1 to 2:1; preferably 20:1 to 10:
1.
10. A method for preparing a reusable enzyme-loaded complex, characterized in that, It includes the following steps: Stir and mix a silicon source, a surfactant and a dispersant, and obtain a silicon-based nanocarrier after reaction; Stir and mix a surface modifier, an enzyme and the silicon-based nanocarrier in an aqueous solution, and obtain a reusable enzyme-loaded complex after centrifugation and washing.
11. The preparation method according to claim 10, characterized in that, The step of stirring and mixing the silicon source, the surfactant and the dispersant to obtain a silicon-based nanocarrier after reaction satisfies at least one of the following conditions: (1) The concentration of the silicon source is 1 - 1000 mg / mL, preferably 5 - 200 mg / mL, more preferably 5 - 100 mg / mL, and further preferably 10 - 50 mg / mL; The concentration of the surfactant is 0.1 - 100 mg / mL, preferably 0.5 - 50 mg / mL, more preferably 0.5 - 10 mg / mL, and further preferably 1 - 5 mg / mL; The concentration of the dispersant is 1 - 1000 mg / mL, preferably 5 - 200 mg / mL, more preferably 5 - 100 mg / mL, and further preferably 10 - 50 mg / mL; (2) The stirring and mixing is carried out with reflux operation at 50°C - 100°C; (3) After reaction, a silicon-based nanocarrier to be dried is obtained by centrifugation, and the silicon-based nanocarrier is obtained after the silicon-based nanocarrier to be dried is dried. The drying method includes at least one of natural air drying, freeze drying, and spray drying.
12. The preparation method according to claim 10, characterized in that, The step of stirring and mixing the surface modifier, the enzyme and the silicon-based nanocarrier in an aqueous solution, and obtaining a reusable enzyme-loaded complex after centrifugation and washing satisfies at least one of the following conditions: The concentration of the surface modifier is 10 - 100 mg / mL, preferably 10 - 50 mg / mL; The concentration of the silicon-based nanocarrier is 1 - 80 mg / mL, preferably 5 - 20 mg / mL; The concentration of the enzyme is 0.1 - 50 mg / mL, preferably 0.1 - 10 mg / mL.
13. Application of the reusable enzyme-loaded complex according to any one of claims 1 - 12 in the fields of catalysis, drug delivery, wastewater treatment, medicine, and sterilization.