Preparation method and application of a bionic enzyme aerogel reactor

Abstract

The present invention relates to the technical field of preparation methods of bionic enzyme reactors, and specifically discloses a preparation method and application of a bionic enzyme aerogel reactor, which specifically includes the following steps: Step S1, first prepare titanium dioxide nanofibers (TiNFs) through solvothermal treatment; Step S2, subject the aqueous dispersion containing titanium dioxide nanofibers (TiNFs), chitosan methacrylate (CSMA) and a crosslinking agent to freeze casting and vacuum drying to obtain a bionic C / TiNFs aerogel reactor with vertically arranged channels; Step S3, polymerize and modify glycidyl methacrylate (GMA), methacryloylethyl sulfobetaine (SBMA) and N,N'-methylenebisacrylamide (MBA) crosslinking agent on the skeleton of the bionic aerogel reactor; Step S4, covalently immobilize tannase in the bionic aerogel reactor under mild conditions to obtain a bionic tannase-pGSC / TiNFs enzyme reactor. The bionic tannase-pGSC / TiNFs enzyme reactor in the present invention is composed of TiNFs, CSMA, polyglycidyl methacrylate (PGMA), poly(methacryloylethyl sulfobetaine) (PSBMA) and immobilized tannase, has vertically arranged channels, and can act as a tissue cell reactor.

Description

Technical Field

[0001] The present invention relates to the technical field of the preparation method of bionic enzyme reactors, in particular to a preparation method and application of a bionic enzyme aerogel reactor. Background Art

[0002] Enzymes have characteristics such as high specificity, good biocompatibility, and high catalytic activity, and are widely used in fields such as food production, biochemical detection, and pharmaceuticals. However, obstacles such as insufficient stability, enzyme denaturation, and difficulties in separation and reuse limit the efforts to continuously convert reactants into products and reduce production costs. In this regard, enzyme immobilization technology is considered an effective way to overcome the above challenges. Currently, a variety of enzyme immobilization methods have been developed, including physical adsorption, encapsulation, and chemical bonding. Although the stability of the immobilized enzyme has been improved to a certain extent, it is still insufficient overall. Summary of the Invention

[0003] Aiming at the technical problems mentioned in the background art, the present invention provides a preparation method and application of a bionic enzyme aerogel reactor.

[0004] The technical solution adopted by the present invention is as follows: A preparation method of a bionic enzyme aerogel reactor specifically includes the following steps:

[0005] Step S1, first prepare titanium dioxide nanofibers (TiNFs) through solvothermal treatment;

[0006] Step S2, subject a water dispersion containing titanium dioxide nanofibers (TiNFs), chitosan methylacrylate (CSMA), and a crosslinking agent to freeze casting and vacuum drying to obtain a bionic C / TiNFs aerogel reactor with vertically arranged channels;

[0007] Step S3, polymerize and modify glycidyl methacrylate (GMA) monomer, sulfobetaine methacrylate (SBMA) monomer, and N, N'-methylenebisacrylamide (MBA) crosslinking agent on the framework of the bionic aerogel reactor;

[0008] Step S4, covalently immobilize tannase in the bionic aerogel reactor under mild conditions to obtain a bionic tannase-pGSC / TiNFs enzyme reactor.

[0009] The present invention is further provided such that the bionic tannase-pGSC / TiNFs enzyme reactor prepared in step S4 is cylindrical.

[0010] The present invention is further provided such that the bionic tannase-pGSC / TiNFs enzyme reactor prepared in step S4 exhibits a 3D hierarchical cell-like structure with honeycomb pores in its structure.

[0011] The present invention is further configured such that the size of the honeycomb pores is 20 - 100 μm, the reticular pore walls are composed of nanofibers and polymers, and the thickness is 0.2 - 2 μm.

[0012] The present invention is further configured such that the bionic tannase - pGSC / TiNFs enzyme reactor also exhibits unidirectional, vertically arranged, and interconnected channels longitudinally.

[0013] The present invention is further configured as an application of a bionic enzyme aerogel reactor, applying the bionic tannase - pGSC / TiNFs enzyme reactor to biocatalytic engineering.

[0014] The beneficial effects of the present invention are as follows: Compared with the prior art, the bionic tannase - pGSC / TiNFs enzyme reactor in the present invention is composed of TiNFs, CSMA, PGMA, PSBMA, and immobilized tannase, and has vertically arranged channels. Due to the characteristics of the reactor having a honeycomb structure, rapid mass transfer, a comfortable microenvironment, and a substrate enrichment effect modified by PSBMA, compared with an enzyme reactor without PSBMA and free tannase, the bionic tannase - pGSC / TiNFs enzyme reactor exhibits enhanced pH value and thermal tolerance, thermal stability, storage stability, and reusability. The bionic tannase - pGSC / TiNFs enzyme reactor can act as a tissue cell reactor and can continuously and effectively convert tannin into gallic acid and glucose under continuous flow conditions, demonstrating the application of biomass conversion. Description of the Drawings

[0015] Figure 1 It is a schematic diagram of the preparation method of the bionic tannase - pGSC / TiNFs enzyme reactor with vertically arranged channels in the present invention and its application in the continuous - flow catalytic conversion of tannin.

[0016] Figure 2 It is the SEM photos of (a) the bionic enzyme reactor, (b - e) the transverse (b - d) and vertical cross - section (e) in the present invention, and (f) the water contact angle diagram of the bionic tannase - pGSC / TiNFs enzyme reactor.

[0017] Figure 3 It is the (a) Zeta potential values of different samples in water after grinding, (b) water flux of different samples under the action of gravity without additional auxiliary pressure, (c) compressive stress - strain curves of different samples, and (d) diagram of the immobilized amount of tannase in different reactors in the present invention.

[0018] Figure 4 It is the diagram of the effects of (a) immobilization time, (b) tannase concentration, (c) pH value, and (d) reaction temperature on the immobilized amount and activity of the bionic tannase - pGSC / TiNFs enzyme reactor in the present invention.

[0019] Figure 5 This is a graph showing the effects of different conditions on the activities of immobilized and free tannase in the present invention; (a) pH value, (b) temperature, (c) storage time, and (d) multiple cycles.

[0020] Figure 6 This is a graph showing the continuous flow catalytic conversion of tannins using the biomimetic tannase-pGSC / TiNFs enzyme reactor in the present invention; (a) photograph of the test equipment, (b) conversion efficiency at different flow rates, (c) conversion efficiency for 10 consecutive cycles using 100 mL of solution per cycle, and (d) conversion efficiency for continuous use for 15 days. Detailed implementation manners

[0021] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0022] To solve the problems existing in the background art, the present application proposes the following technical solutions: A preparation method and application of a biomimetic enzyme aerogel reactor, which specifically includes the following steps:

[0023] The manufacturing process is as Figure 1 shown in a.

[0024] Step S1: First, titanium dioxide nanofibers (TiNFs) are prepared by solvothermal treatment.

[0025] Step S2: A water dispersion containing titanium dioxide nanofibers (TiNFs), chitosan methacrylate (CSMA), and a crosslinking agent is freeze-cast and vacuum-dried to obtain a biomimetic C / TiNFs aerogel reactor with vertically arranged channels.

[0026] Step S3: Glycidyl methacrylate (GMA) monomer, 2-(Methacryloyloxy)ethyl dimethylammonium betaine (SBMA) monomer, and N,N'-methylenebisacrylamide (MBA) crosslinking agent are polymerized and modified on the framework of the biomimetic aerogel reactor.

[0027] Step S4, covalently immobilize tannase in the biomimetic aerogel reactor under mild conditions to obtain the biomimetic tannase-pGSC / TiNFs enzyme reactor (Tannase-pCSC / TiNFs). The biomimetic tannase-pGSC / TiNFs enzyme reactor has high porosity and water flux. When the solution flows through the reactor, the reactant tannin can be effectively converted into gallic acid and glucose (Figure 1b).

[0028] Among them, the biomimetic tannase-pGSC / TiNFs enzyme reactor prepared in step S4 is cylindrical. The biomimetic tannase-pGSC / TiNFs enzyme reactor exhibits a 3D hierarchical cell-like structure with the characteristics of honeycomb pores. The size of the honeycomb pores is 20 - 100 μm. The reticular pore walls are composed of nanofibers and polymers with a thickness of 0.2 - 2 μm. The biomimetic tannase-pGSC / TiNFs enzyme reactor also shows unidirectional, vertically arranged and interconnected channels longitudinally.

[0029] The further explanations are as follows:

[0030] The preparation method of the biomimetic CSMA / TiNFs (C / TiNFs) aerogel is as follows:

[0031] Mix 20.0 g of titanium dioxide nanofiber (TiNFs) (20 mg g –1 ), 10.0 g of chitosan methacrylate (CSMA) (20 mg g –1 ), and 100 μL of polyethylene glycol diglycidyl ether and stir for 20 min. Add the mixture to a cylindrical mold, and then perform a directional freezing process using liquid nitrogen. Lyophilize the frozen sample and heat-treat it at 60 o °C for 4 h to obtain the biomimetic C / TiNFs aerogel.

[0032] The synthesis method of the biomimetic tannase-pGMA / SBMA / CSMA / TiNFs (Tannase-pGSCS / TiNFs) enzyme reactor is as follows:

[0033] Dissolve 400 mg of glycidyl methacrylate, 12 mg of K 2 S 2 O 8 , 2 mL of methacryloylethyl sulfobetaine, and 12 mg of N,N'-methylenebisacrylamide in 4 mL of deionized water. Drop the resulting solution onto the above-mentioned biomimetic C / TiNFs aerogel. The sample is at 80 oHeat-treat at C for 1 h to obtain a bionic pGMA / SBMA / CSMA / TiNFs (pGSC / TiNFs) reactor. Wash the sample with deionized water to remove residual chemicals. Subsequently, add the pGSC / TiNFs reactor to a phosphate buffer solution (10 mL, pH 7.0) containing tannase at a concentration of 0.5 mg mL –1 −1. o Incubate the mixture at 4

[0034] C for 12 h. Wash the obtained tannase-immobilized enzyme reactor (Tannase-pGSCS / TiNFs) several times with phosphate buffer (pH 7.0) and store it in the refrigerator.

[0035] In addition, the methods for measuring the enzyme activity, enzyme immobilization amount, and conversion efficiency of the bionic tannase-pGSCS / TiNFs enzyme reactor in continuous flow mode are as follows: –1 −1

[0036] Study the tannase activity by measuring the amount of gallic acid produced from methyl gallate. The amount of immobilized enzyme in the bionic tannase-pGSCS / TiNFs enzyme reactor is measured using the Bradford method. The continuous flow catalytic conversion test is carried out in a common chromatographic column. Place the bionic tannase-pGSCS / TiNFs enzyme reactor with a diameter of 26 mm and a height of 26 mm at the bottom of the column. Inject an aqueous solution containing tannin (1 mg mL Figure 2 −1) into the top surface of the bionic tannase-pGSCS / TiNFs enzyme reactor using a peristaltic pump. Collect the initial solution and the filtered solution after catalytic treatment, and analyze them by ultraviolet-visible absorption spectroscopy.

[0037] Figure 2 In summary, observe the surface and internal morphology of the bionic tannase-pGSC / TiNFs enzyme reactor. The bionic tannase-pGSC / TiNFs enzyme reactor is cylindrical and can be easily prepared in a cylindrical mold (

[0038] a); Figure 2 b-d show the scanning electron microscope (SEM) images in the transverse direction. The bionic tannase-pGSCS / TiNFs enzyme reactor exhibits a 3D hierarchical cell-like structure, and its structure is similar to the honeycomb pores in natural trees;

[0039] In addition, the bionic tannase-pGSC / TiNFs enzyme reactor is hydrophilic, and water droplets can be completely absorbed within 1 second ( Figure 2 as shown in f).

[0040] In further designs, the surface ζ potential of the prepared samples was studied. As Figure 3 shown in a, the C / TiNFs sample prepared from titanium dioxide nanofibers (TiNFs) and chitosan methacrylate (CSMA) has a positive potential of +10.5 mV. Subsequently, after modification with PGMA and PSBMA, the surface potential decreased to approximately +5.60 mV. In addition, the tannase-pGSC / TiNFs enzyme reactor has a negative potential of -5.31 mV, indicating that tannase was successfully immobilized. Thanks to the excellent water wettability and high porosity (99.2%), the aqueous solution can flow through the reactor rapidly under the action of gravity without additional auxiliary pressure.

[0041] Among them, the water fluxes of the bionic C / TiNFs, pGSC / TiNFs, and tannase-pGSC / TiNFs enzyme reactors with a thickness of 15 mm were measured to be 2023, 2454, and 2432 Lm–2h–1 respectively ( Figure 3 b). The high water flux and low physical flow resistance contribute to the rapid reactant-product exchange in continuous flow catalytic reactions.

[0042] In addition, Figure 3 c shows the compressive stress-strain curves of the three samples. Among them, the compressive stress of the bionic tannase-pGSC / TiNFs enzyme reactor (83.3 kPa) at 70% strain is higher than that of pGSC / TiNFs (60.4 kPa) and C / TiNFs (24.9 kPa). The compressive stress of the bionic tannase-pGSC / TiNFs enzyme reactor at 70% stress is still higher than that of the bionic porous materials with similar structures reported in the literature.

[0043] Among them, the amount of enzyme immobilized on the bionic reactor modified with PSBMA was measured. As Figure 3 shown in d, the amount of tannase immobilized on tannase-pGSC / TiNFs (98.4 mg g -1 ) is slightly lower than that of tannase-pGC / TiNFs (104.5 mg g -1 ).

[0044] Among them, the present invention studied the effects of tannase concentration, reaction time, pH value, and temperature on the amount of immobilized enzyme binding and enzyme activity. The results showed that when the amount of tannase used was 0.5 mgmL -1 , the pH value was 7.0, and the reaction temperature was 4 oUnder the conditions of C and a reaction time of 12 h, the best immobilization efficiency can be achieved ( Figure 4 ). The data shows that the enzyme binding amount, immobilization efficiency and enzyme activity are 98.4 mg / g -1 , 84.2% and 50.5 U / g -1 .

[0045] In addition, the effects of pH value, temperature, storage time and cycle number on the activities of Tannase-pGC / TiNFs, Tannase-pGSC / TiNFs and free tannase were also studied. As Figure 5 shown in a, the optimal pH value of free tannase is 7.0, while Tannase-pGC / TiNFs and Tannase-pGSC / TiNFs show the maximum activity at pH 6.5 and 6.0 respectively. In addition, in the pH range of 5.0 to 6.0, Tannase-pGC / TiNFs and Tannase-pGSC / TiNFs maintain 81.6–94.3% and 91.3–100% of their activities respectively; in contrast, free tannase maintains a lower range of 56.7-81.2%. At pH 8.0, the retained activities of Tannase-pGC / TiNFs (62.5%) and Tannase-pGSC / TiNFs (81.2%) are higher than that of free tannase (50.8%).

[0046] The results show that immobilized tannase has higher stability than free tannase in a wide pH range. This phenomenon is attributed to the modified polymers PGMA and PSBMA, which endow Tannase-pGC / TiNFs and Tannase-pGSC / TiNFs with a buffered microenvironment and excellent protective effects, resulting in a shielding effect.

[0047] Among them, the stabilities of immobilized tannase and free tannase at different temperatures were also studied. As Figure 5 shown in b, free tannase shows the maximum activity at 45 o °C, while Tannase-pGC / TiNF and Tannase-pGSC / TiNF reach the optimal activity at 50 o °C. In addition, when the temperature rises from 45 o °C to 65 o °C, the activities of Tannase-pGC / TiNFs and free tannase decrease sharply to 71.2% and 48.1% respectively, while Tannase-pGSC / TiNFs maintains a relatively high activity of 80.6%. Even at 70 oAt high temperatures of C, the residual activity of Tannase-pGSC / TiNFs is still superior to that of Tannase-pGC / TiNFs and free tannase. These results are due to the covalent bond between tannase and the solid support in pGC / TiNFs and pGSC / TiNFs, which improves the molecular rigidity and thermal stability of the enzyme protein.

[0048] The storage stability of free and immobilized tannase was determined by incubating free tannase in a buffer solution at pH = 7.0 and immobilized enzyme in a buffer solution at pH = 6.5 for 63 days. Free tannase retained 17.8% of its initial activity, while tannase-pGC / TiNFs and tannase-pGSC / TiNFs retained 64.6% and 70.5% of the enzyme activity respectively ( Figure 5 c). In addition, after 10 consecutive cycles, Tannase-pGC / TiNFs and Tannase-pGSC / TiNFs showed high activity retention rates of 96.3% and 96.5%, but the relative activity of free tannase was 66.5% ( Figure 5 d). These results indicate that the protein structure of tannase is stable after covalent immobilization on the designed support.

[0049] Among them, in addition, the continuous flow catalytic conversion of tannin to gallic acid and glucose was also evaluated. As Figure 6 shown in a, the biomimetic tannase-pGSC / TiNFs enzyme reactor was fixed in the device, and the aqueous solution containing the reactants was pumped to the top surface of the biomimetic tannase-pGSC / TiNFs enzyme reactor. The catalytic reaction could be completed when the aqueous solution flowed through the Tannase-pGSC / TiNFs enzyme reactor. The effect of flow rate on the conversion efficiency was also studied. As Figure 6 shown in b, when the flow rate was lower than 1.0 mg mL –1 -1, the conversion efficiency exceeded 99.6%. When the flow rate was further increased, the efficiency would gradually decrease. Too high a flow rate might lead to insufficient contact between the immobilized tannase and the reactants.

[0050] The recyclability and long-term stability of the biomimetic tannase-pGSC / TiNFs enzyme reactor were studied. In the presence of the biomimetic tannase-pGSC / TiNFs enzyme reactor, the flow catalytic conversion of tannin was continuously repeated for 10 cycles, and the conversion efficiency decreased slightly with the increase in the number of cycles ( Figure 6 c). The biomimetic tannase-pGSC / TiNFs enzyme reactor could still maintain 98.1% efficiency after 10 cycles, showing good recyclability. In addition, when the biomimetic tannase-pGSC / TiNFs enzyme reactor was continuously operated for 15 days, the conversion efficiency could still be maintained at 82.4% ( Figure 6d), Therefore, the prepared biomimetic tannase-pGSC / TiNFs enzyme reactor has high conversion efficiency, good recyclability and long-term stability in the continuous bioconversion of tannins.

[0051] In summary, the biomimetic tannase-pGSC / TiNFs enzyme reactor in the present invention is composed of TiNFs, CSMA, PGMA, PSBMA and immobilized tannase, and has vertically arranged channels. Due to the characteristics of the reactor such as honeycomb structure, rapid mass transfer, comfortable microenvironment and substrate enrichment effect modified by PSBMA, compared with the enzyme reactor without PSBMA and free tannase, the biomimetic tannase-pGSC / TiNFs enzyme reactor exhibits enhanced pH and heat tolerance, thermal stability, storage stability and reusability. The modified PSBMA provides a buffered microenvironment and excellent protection, which helps to achieve high enzyme catalytic stability. Importantly, the biomimetic tannase-pGSC / TiNFs enzyme reactor can act as a tissue cell reactor to continuously and effectively convert tannins into gallic acid and glucose under continuous flow conditions, demonstrating the application potential of biomass conversion.

[0052] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a bionic enzyme aerogel reactor, characterized in that: The specific steps include: Step S1, first preparing titanium dioxide nanofibers by solvent thermal treatment; Step S2, freeze-casting and vacuum drying an aqueous dispersion containing titanium dioxide nanofibers, chitosan methacrylate (CSMA) and a cross-linking agent to obtain a biomimetic C / TiNFs aerogel reactor with vertically arranged channels; Step S3, polymerizing and modifying glycidyl methacrylate (GMA) monomer, methacryloylethyl sulfobetaine (SBMA) monomer and N,N'-methylenebisacrylamide (MBA) crosslinker on the skeleton of the biomimetic aerogel reactor; Step S4, covalently immobilizing tannase in a biomimetic aerogel reactor under mild conditions, thereby obtaining a biomimetic tannase-pGSC / TiNFs enzyme reactor.

2. The method for preparing a biomimetic enzyme aerogel reactor according to claim 1, characterized in that: The biomimetic tannase-pGSC / TiNFs enzyme reactor prepared in step S4 is cylindrical.

3. The method for preparing a biomimetic enzyme aerogel reactor according to claim 1, characterized in that: The biomimetic tannase-pGSC / TiNFs enzyme reactor prepared in step S4 exhibits a 3D hierarchical cell-like structure with honeycomb pores in the structure.

4. The method for preparing a biomimetic enzyme aerogel reactor according to claim 3, characterized in that: The size of the honeycomb holes is 20-100 μm, and the mesh hole walls are composed of nanofibers and polymers, with a thickness of 0.2-2 μm.

5. The method for preparing a biomimetic enzyme aerogel reactor according to claim 1, characterized in that: The biomimetic tannase-pGSC / TiNFs enzyme reactor also exhibits unidirectional, vertically arranged and interconnected channels in the longitudinal direction.

6. An application of a biomimetic enzyme aerogel reactor as claimed in any one of claims 1 to 5, characterized in that: The bionic tannase-pGSC / TiNFs enzyme reactor is applied in biocatalysis engineering.

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