Preparation method and application of hollow hierarchical porous MOF material

Abstract

The application discloses a preparation method and application of a hollow hierarchical porous MOF material, and successfully generates the hollow hierarchical porous MOF material with a hollow hierarchical porous core-shell structure by adopting a combined strategy of PVP surface modification and Tris-HCl buffer etching, and the structural integrity of the hollow hierarchical porous MOF material is maximally maintained in a water-involved condition as a carrier for an enzyme molecule encapsulation process. In addition, the hollow hierarchical porous metal organic framework is modified by 3-aminopropyltrimethoxysilane, and higher enzyme loading efficiency is achieved. The experimental results show that the maximum enzyme loading capacity is 216.3 mg / mL, the relative activity of the immobilized enzyme is increased by 15 times compared with the CRL@ZIF-8 activity, and the immobilized enzyme has good thermal, chemical and operation stability. The immobilized enzyme is used for catalyzing synthesis of phytosterol ester, and the maximum conversion rate is 88.8%, and the immobilized enzyme shows excellent catalytic performance. The application provides a new idea for developing the MOF immobilized enzyme for biotechnological applications.

Description

Technical Field

[0001] This invention belongs to the field of immobilized enzyme synthesis, and specifically relates to a method for preparing hollow hierarchical porous MOF materials and their applications. Background Technology

[0002] Enzymes, due to their high catalytic activity, high selectivity, and favorable reaction conditions, have become competitive tools in biosynthesis and are widely used in industries such as food, textiles, environment, and medicine. Enzyme immobilization is an effective method to improve enzyme stability. Compared with free enzymes, enzyme immobilization endows enzymes with reusability and ease of isolation, protects enzymes from direct exposure to harsh environments, and creates a special microenvironment that stabilizes enzyme conformation and enhances enzyme activity, thereby positively impacting the final catalytic yield and product benefits. However, developing structurally controllable and high-performance carriers to improve the stability and activity of immobilized enzymes remains an urgent and challenging task.

[0003] Metal-organic frameworks (MOFs) are a class of porous materials formed by the self-assembly of metal ions or metal clusters with organic ligands. Due to their diverse topologies and properties, ultra-high specific surface area, abundant and easily tunable pore structures, and good stability, MOFs have attracted widespread attention in gas adsorption, biomedicine, biosensing, and enzyme immobilization catalysis. As a subfamily of MOFs, zeolite imidazole frameworks (ZIFs) are porous crystalline materials with a zeolite framework structure formed by the coordination of transition metal ions with imidazole derivative ligands. They have been widely reported as ideal enzyme immobilization supports due to their mild synthesis conditions and biocompatibility.

[0004] Currently, there are three main strategies for preparing immobilized enzymes on / within MOFs: in-situ encapsulation, surface linking, and pore trapping using pre-synthesized MOFs. For in-situ encapsulation, the enzyme is typically encapsulated within the MOF during its synthesis. Due to the shielding effect of the MOF, the resulting immobilized enzyme exhibits excellent stability under certain harsh conditions. However, the shell surrounding the immobilized enzyme increases mass transfer limitations between the enzyme and substrate, and some organic precursors in MOFs (e.g., 2-methylimidazole (2-MIM) in ZIF-8) can directly interact with the enzyme, thereby reducing its catalytic activity. Surface linking, on the other hand, immobilizes the enzyme on the MOF surface through covalent bonds or physical adsorption. This immobilization method is independent of the pore structure of MOFs and is simple to design and highly operable. However, its immobilization sites are mainly located in limited surface areas, making it easy for the enzyme to detach from the MOF surface. Furthermore, since most synthesized MOFs are micropores or small mesopores, they significantly hinder enzyme passage and the diffusion efficiency of substrates and products. Therefore, the immobilization efficiency of enzymes immobilized on MOF surfaces is quite poor. For pore trapping in pre-synthesized MOFs, enzyme diffusion into the MOF pores is beneficial for increasing enzyme loading and reducing enzyme leaching. However, the enzyme immobilization efficiency depends on the strict design of the MOF pores to match the enzyme molecule size. Therefore, the development of novel hollow hierarchical porous MOFs offers a novel and effective way to overcome these limitations. The MOF shell has abundant porous structure and internal hollow cavities, providing sufficient space for the enzyme catalytic system and offering shorter diffusion paths for reactant and product transport. Furthermore, the well-organized framework protects the enzyme molecules, resulting in excellent catalytic efficiency. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a method for preparing hollow hierarchical porous MOF materials and their applications.

[0006] The first aspect of the present invention provides a method for preparing a hollow hierarchical porous MOF material, comprising the following steps:

[0007] S1. Preparation of spherical ZIF-8: The ZIF-8 precursors, sulfuric acid heptahydrate ZnSO4·7H2O and 2-methylimidazolium 2-MIM, were dissolved in anhydrous methanol to obtain reaction solution A and reaction solution B. Then, reaction solution B was added to reaction solution A and mixed. The mixture was stirred and reacted, then centrifuged, washed with anhydrous methanol, and dried under vacuum to obtain spherical ZIF-8.

[0008] S2. Polyvinylpyrrolidone was added during the stirring growth of spherical ZIF-8. After co-incubation, the precipitate was centrifuged, the supernatant was removed, and weakly basic cationic tris(hydroxymethyl)aminomethane hydrochloride buffer was added. The mixture was soaked and stirred to dissolve and eliminate the spherical colloidal soft template. The mixture was centrifuged again at high speed, the etching solution was extracted with a syringe, washed with anhydrous methanol, and vacuum dried to obtain PVP / HZIF-8.

[0009] S3. PVP / HZIF-8 was added to an anhydrous ethanol solution of 3-aminopropyltrimethoxysilane, mixed and stirred, centrifuged to precipitate, then washed several times with anhydrous ethanol and dried under vacuum to obtain N-PVP / HZIF-8.

[0010] Preferably, in step S1, the stirring reaction temperature is 15–35°C and the stirring time is 0.5–2 h.

[0011] Preferably, in step S2, the stirring reaction temperature is 15–35°C and the stirring time is 0.5–2 h.

[0012] Preferably, in step S3, the stirring reaction temperature is 55–65°C, and the stirring time is 6–8 hours.

[0013] Preferably, in step S3, the mass ratio of the spherical ZIF-8 to PVP is 1:3 to 1:5;

[0014] Preferably, the mass-to-volume ratio (mg / μL) of PVP / HZIF-8 to 3-aminopropyltrimethoxysilane is 0.5:10 to 1.5:10.

[0015] A second aspect of the present invention provides a hollow hierarchical porous MOF material prepared by the above-described preparation method.

[0016] A third aspect of the present invention provides a method for preparing immobilized enzymes using the above-mentioned hollow hierarchical porous MOF material as a carrier, comprising the following steps:

[0017] A1. Add Candida rugosa lipase to Tris-HCl buffer, shake to dissolve, and then centrifuge at 4°C and 8000r / min in a refrigerated centrifuge for 10 min to obtain the supernatant.

[0018] A2. Transfer the enzyme supernatant to a stoppered Erlenmeyer flask, add N-PVP / HZIF-8, and place it in a magnetic stirrer at room temperature for enzyme immobilization. During enzyme loading, add GA (0.5%, v / v) in two batches to activate the vector and cross-link it with the enzyme protein.

[0019] A3. After enzyme immobilization, the product was separated by freeze centrifugation, washed three times with Tris-HCl buffer to remove excess lipase, and then freeze-dried to obtain the immobilized enzyme CRL-N-PVP / HZIF-8.

[0020] A fourth aspect of the present invention provides an immobilized enzyme prepared by the above method.

[0021] The fifth aspect of the present invention provides the application of the above-mentioned immobilized enzyme as a biocatalyst in the catalytic synthesis of phytosterol esters.

[0022] The present invention has the following beneficial effects

[0023] (1) This invention innovatively, conveniently, and efficiently solves the problem of ZIF-8's collapse and dissociation during soft template removal due to its water intolerance by employing a combined strategy of PVP surface modification and Tris-HCl buffer etching. This successfully generates a near-hollow hierarchical porous core-shell structure, and under aqueous conditions, the structural integrity is maintained to the maximum extent during enzyme immobilization and encapsulation. Furthermore, to improve the immobilization effect (enzyme loading and catalytic activity), the hollow hierarchical porous metal-organic framework was modified with 3-aminopropyltrimethoxysilane, thereby achieving higher enzyme loading efficiency. This method avoids the severe structural collapse of PVP / ZIF-8 in aqueous systems due to water attack, the excessive release of the precursor imidazole, and enzyme leaching inactivation. This is of great significance for improving the stability of MOFs and expanding their application in aqueous environments.

[0024] (2) The results of the N-PVP / HZIF-8 immobilization effect study in this invention showed that the maximum enzyme loading was 216.3 mg / mL. Compared with the activity of in-situ immobilized CRL@ZIF-8, the relative activity of CRL-N-PVP / HZIF-8 was increased by 15 times, and it also exhibited good thermal, chemical, and operational stability. Furthermore, the prepared CRL-N-PVP / HZIF-8 was used to catalyze the synthesis of phytosterol esters, achieving a maximum conversion rate of 88.8%, demonstrating excellent catalytic performance. This provides a new multi-faceted approach for developing MOF-immobilized enzymes for biotechnological applications. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1Morphology of spherical ZIF-8 and N-PVP / HZIF-8: (a, c) SEM of spherical ZIF-8; (b) TEM of spherical ZIF-8; (d) SEM of N-PVP / HZIF-8;

[0027] Figure 2 SEM and TEM images of the substances in Example 1: (a, b) PVP / ZIF-8; (c, d) PVP / HZIF-8; (e, f) N-PVP / HZIF-8; (g, h) CRL-N-PVP / HZIF-8;

[0028] Figure 3 XRD patterns of each substance in Example 1 (a); FTIR patterns of each substance and free CRL in Example 1 (b);

[0029] Figure 4 XPS spectra of each substance in Example 1: (a, b, c, d and e represent ZIF-8, PVP / ZIF-8, PVP / HZIF-8, N-PVP / HZIF-8 and CRL-N-PVP / HZIF-8 respectively, and from left to right are C1s, N1s and O1s spectra).

[0030] Figure 5 a) N2 adsorption-desorption isotherms; b) pore size distribution of ZIF-8 and its composites based on DFT; c) TEM and EDS images of N-PVP / HZIF-8 (top) and CRL-N-PVP / HZIF-8 (bottom) (elements involved: C, N, Zn, P, S from left to right; scale bar is 100 nm); d) CLSM image of CRL-N-PVP / HZIF-8 at an excitation wavelength of 488 nm;

[0031] Figure 6 Thermogravimetric analysis (a) and static water contact angle (b) of ZIF-8 and its composites;

[0032] Figure 7 SEM image of ZIF-8 dissociating and collapsing in a pure water system (a); TEM image of CRL-ZIF-8 synthesized in a Tris-HCl buffer system (b);

[0033] Figure 8 (a) shows the potential reaction process for the structural stability mechanism of N-PVP / HZIF-8 under immersion conditions; (b) and (c) are SEM images of CRL-ZIF-8 and CRL-N-PVP / HZIF-8 synthesized in the Tris-HCl buffer system.

[0034] Figure 9The effects of initial enzyme concentration (a) and immobilization time (b) on the protein loading and relative activity of CRL-N-PVP / HZIF-8 were investigated; the enzyme immobilization effects of ZIF-8 and the composite material were compared (c); the activities of CRL@ZIF-8 and CRL-N-PVP / HZIF-8 were compared (d); and the relative activities of free CRL and CRL-N-PVP / HZIF-8 before and after heat and trypsin treatment were compared. Optimal activity before treatment was taken as 100% for each group (e); the reusability of the immobilized lipase was assessed (f) (optimal activity of the free enzyme in a, b, c, and d was defined as 100%).

[0035] Figure 10 This is a schematic diagram of the esterification reaction of α-linolenic acid and phytosterol;

[0036] Figure 11 Temperature (a), substrate molar ratio (b), MS The effect of addition amount (c) and catalyst addition amount (d) on the conversion rate of phytosterols is shown in the figure.

[0037] Figure 12 This is a schematic diagram of the preparation process of the hollow hierarchical porous MOF material obtained by the present invention and the enzyme immobilization and encapsulation process. Detailed Implementation

[0038] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will understand that the invention can be implemented in other embodiments without these specific details. In other instances, detailed descriptions of well-known systems, apparatuses, circuits, and methods are omitted so as not to obscure the description of the invention with unnecessary detail.

[0039] In this invention, the precursors ZIF-8, sulfuric acid heptahydrate ZnSO4·7H2O and 2-methylimidazolium 2-MIM, are first dissolved in anhydrous methanol solution. The mixture is stirred to allow 2-MIM to directly assemble and coordinate on the surface of ZnSO4·7H2O colloidal spheres, thereby synthesizing the most basic novel spherical ZIF-8, rather than the most common dodecahedral form of ZIF-8. The specific preparation of spherical ZIF-8 includes: dissolving the ZIF-8 precursor ZnSO4·7H2O and 2-MIM in anhydrous methanol, respectively. ZnSO4·7H2O dissolves in anhydrous methanol, utilizing its solubility in methanol to form stable spherical colloidal aggregates of zinc sulfate heptahydrate, which serve as soft templates for hollow hierarchical porous ZIF-8. Then, an anhydrous methanol solution of 2-MIM is added, and the mixture is stirred to allow 2-MIM to directly assemble and coordinate on the surface of the zinc sulfate heptahydrate colloidal aggregates. A white precipitate is obtained by centrifugation, followed by washing several times with anhydrous methanol and vacuum drying overnight to obtain spherical ZIF-8.

[0040] Under normal circumstances, each Zn in ZIF-8 material 2+ All four 2-methylimidazolium linkages are well coordinated. Due to the bonding between the central metal ion and the nitrogen atoms in the ligands, a key characteristic of ZIF-8 is its strong chemical and thermal stability. However, recent research has revealed that for the previously assumed robust zeolite imidazolium framework ZIF-8 (2-methylimidazolium 2-MIM and Zn(II)), the metal-coordination bonds are highly sensitive to the presence of water molecules and more readily lose N-Zn coordination linkages at acidic pH values. This allows the hydroxyl groups (OH) generated by water to hydroxylate the central metal ion, forming a Zn-O bond; the hydrogen ions generated by water can also hydroxylate the Zn-O bond. 2+ The linker that detaches from the coordination junction is protonated, forming an NH bond at the 2-methylimidazolium terminus. Furthermore, in biocatalysis, the "interfering" anion in the buffer component (PO4 in phosphate buffer solution) 3- Other components (such as 2-MIM) may also replace 2-MIM to form zinc complexes with an amorphous phase, leading to irreversible structural degradation and leaching and inactivation of the immobilized enzyme molecules. Therefore, modification of ZIF-8 and careful analysis and selection of buffer components are essential to develop strategies for improving its tolerance to water "attacks" and resistance to interfering coordinating ions. This is a crucial prerequisite for the application of ZIF-8 in post-permeation immobilization methods in enzyme immobilization, thereby overcoming the limitations imposed by the structural stability of MOFs on the practical development of enzyme immobilization and industrial biocatalysis.

[0041] Secondly, the PVP / HZIF-8 composite material was produced by adding PVP during the mixing and stirring assembly growth process of ZIF-8. During this process, the PVP polymer is adsorbed into the particles through weak coordination interactions between its pyrrolidone ring (C=O) and zinc atoms in ZIF-8, or through hydrophobic interactions between its nonpolar groups and organic linkers in ZIF-8. After incubation, the precipitate was centrifuged, and the supernatant was removed. Immediately afterward, a weakly basic cationic tris(hydroxymethyl)aminomethane hydrochloride buffer (Tris-HCl buffer, 0.05 mol·L⁻¹) was added. -1 (pH=8.0), soak for 20 minutes with intermittent stirring to dissolve and eliminate the spherical colloidal soft template. Centrifuge again at high speed, extract the etching solution with a syringe, wash three times with anhydrous methanol, and vacuum dry to obtain PVP / HZIF-8;

[0042] Here, PVP is a widely used surfactant containing a hydrophilic component (pyrrolidone moiety) and a relatively large hydrophobic group (alkyl group). It not only acts as a capping agent, assisting in controlling the size and shape of certain nanoparticles during synthesis, but also commonly serves as an excellent stabilizer in material synthesis, exhibiting significant resistance to ZIF-8 hydrolytic damage. Furthermore, a weakly basic cationic Tris-HCl buffer is used as an etchant to remove the template, and simultaneously as a buffer component to maintain enzyme activity during enzyme loading. This minimizes forward contact with the ZIF-8 surface by promoting the reverse reaction during ZIF-8 hydrolysis. The above synthetic strategy avoids severe structural collapse and the large-scale release of the precursor imidazole caused by PVP / ZIF-8 etching in an aqueous system.

[0043] Then, in order to improve the effect of immobilized enzyme, PVP / HZIF-8 was modified with 3-aminopropyltrimethoxysilane to obtain amino-functionalized N-PVP / HZIF-8.

[0044] Finally, the *Candida rugosa* lipase (CRL) molecule was immobilized onto the carrier to obtain the immobilized enzyme CRL-N-PVP / HZIF-8. Notably, *Candida rugosa* lipase (CRL) inevitably contains phosphate, a buffer component for maintaining enzyme activity. Based on N-PVP / HZIF-8, this is the first time that phosphate has been used during enzyme loading to achieve a certain degree of ordered pore expansion and enhanced stability of the carrier, rather than the disordered disintegration and destruction widely reported.

[0045] Example 1

[0046] Reference Figure 12 A method for preparing a hollow hierarchical porous MOF material includes the following steps:

[0047] S1. Preparation of spherical ZIF-8: 0.144 g ZnSO4·7H2O and 0.535 g 2-MIM were dissolved in 15 mL of anhydrous methanol to obtain solution A and solution B. The reaction solution B was added to the reaction solution A at room temperature using the in-situ synthesis method. The mixture was stirred and reacted for 1 h. After centrifugation, a white precipitate was obtained. The precipitate was then washed three times with anhydrous methanol and dried overnight in a vacuum drying oven at 60 °C to obtain the most basic spherical ZIF-8.

[0048] S2 and PVP / HZIF-8 composite materials are produced by surface modification through the addition of 0.15 g of PVP during the mixing and stirring assembly growth process of ZIF-8. During this process, the PVP polymer is adsorbed into the particles through weak coordination interactions between its pyrrolidone ring (C=O) and the zinc atoms in ZIF-8, or through hydrophobic interactions between its nonpolar groups and the organic linkers in ZIF-8. Polyvinylpyrrolidone (PVP) is added during the stirring growth of spherical ZIF-8 particles. After co-incubation, the precipitate is centrifuged, the supernatant is removed, and immediately 15 mL of weakly basic cationic tris(hydroxymethyl)aminomethane hydrochloride buffer (Tris-HCl buffer, 0.05 mol·L⁻¹) is added. -1 (pH=8.0), the mixture was soaked at room temperature with intermittent stirring for 20 minutes to dissolve and eliminate the spherical colloidal soft template. After high-speed centrifugation, the etching solution was extracted with a syringe, washed three times with anhydrous methanol, and vacuum dried to obtain PVP / HZIF-8.

[0049] S3. Add 50 mg PVP / HZIF-8 to 10 mL of ethanol solution containing 800 μL of 3-aminopropyltrimethoxysilane (APTS), stir at 60 °C for 7 h, centrifuge to precipitate, wash several times with ethanol and dry under vacuum to obtain N-PVP / HZIF-8.

[0050] Furthermore, the immobilized enzyme, namely the biocatalyst CRL-N-PVP / HZIF-8, was prepared. The specific enzyme immobilization steps are as follows:

[0051] A1. Add 1g of Candida rugosa lipase (CRL) lyophilized powder to 50mL of Tris-HCl buffer (0.05M, pH 7.5), shake to dissolve for 30 minutes, and then centrifuge at 4℃ and 8000r / min in a refrigerated centrifuge for 10 minutes to obtain the supernatant for later use.

[0052] A2. Transfer a certain amount of enzyme supernatant into a stoppered Erlenmeyer flask, add 50 mg of dried N-PVP / HZIF-8 prepared in Example 1, and place it in a magnetic stirrer at 500 rpm at room temperature for enzyme immobilization. During enzyme loading, add 10 mL of GA (0.5%, v / v) in two batches to activate the carrier and cross-link it with the enzyme protein.

[0053] A3. After enzyme immobilization, the product was separated by refrigerated centrifugation (4℃, 7000rpm, 10min), washed three times with Tris-HCl buffer to remove excess lipase, and then freeze-dried for 24h to obtain immobilized enzyme CRL-N-PVP / HZIF-8, which was stored at 4℃ for later use.

[0054] Here, a weakly alkaline cationic Tris-HCl buffer is used as a buffer component to maintain enzyme activity. The Candida rhombifolia lipase CRL molecules are immobilized into the cavity and pores of the carrier through osmosis. At the same time, there are physical adsorption and glutaraldehyde (GA)-assisted covalent cross-linking interactions on the outer surface of the carrier to form the immobilized enzyme, i.e., the biocatalyst CRL-N-PVP / HZIF-8.

[0055] Structural Study of CRL-N-PVP / HZIF-8

[0056] (a) Morphological characterization of the samples produced at each stage of the preparation of CRL-N-PVP / HZIF-8 in Example 1. Results are shown in […]. Figure 1 and Figure 2 .

[0057] Depend on Figure 1 (a) and Figure 1 (c) The results show that the ZIF-8 prepared in Example 1 of this invention consists of relatively uniform, regular, and well-defined spherical particles with a size of approximately 800 nm. The surface of the spheres is composed of tiny and dense ZIF-8 nanocrystals. Figure 2 (a) Figure 2 (c) and Figure 2 (e) The results show that the surface morphology of PVP / ZIF-8, PVP / HZIF-8, and N-PVP / HZIF-8 is basically the same as that of ZIF-8, indicating that surface modification, etching, and amino functionalization of spherical ZIF-8 did not destroy the original spherical structure. (Comparison) Figure 1 (d) and Figure 2 (g) The results show that after N-PVP / HZIF-8 fixation of CRL, no significant morphological changes were observed in N-PVP / HZIF-8. However, it is noteworthy that the core-shell surface changed from dense to rough, with obvious gaps. Figure 1(b) TEM results show that ZIF-8 is almost a solid sphere, with extremely subtle apparent voids in the central structure. Figure 2 (b) TEM results show that the ZIF-8 solid spheres remained essentially unchanged after PVP surface modification (i.e., PVP / ZIF-8), with only a slight increase in the ZIF-8 shell thickness. This is likely due to the interaction between PVP and ZIF-8, attracting more ZIF-8 to stack in the shell. Figure 2 (d) TEM results show that after etching, PVP / ZIF-8 forms a nearly hollow core-shell structure with a shell thickness ranging from 30 to 40 nm. However, the cavity also contains a very small number of negligible bulk ZIF-8 nanocrystals. Figure 2 (f) TEM results showed that the functionalization of APTS (i.e., N-PVP / HZIF-8) did not significantly alter the hollow core-shell structure of PVP / HZIF-8, while after enzyme loading, the structure of the hollow core-shell of PVP / HZIF-8 changed significantly. Figure 2 (h)TEM showed that a large amount of solid filling shadow appeared in the original hollow cavity, and the shell wall produced obvious large mesoporous channels. This indicates that during the enzyme immobilization process, substances in the enzyme solution entered the carrier cavity and interacted with it. At the same time, the highly ordered shell pores provided the possibility for CRL molecules in the solution to enter and be immobilized into the spherical hollow cavity.

[0058] (b) The crystallographic changes of samples at different stages of the preparation process of CRL-N-PVP / HZIF-8 were studied using X-ray diffraction (XRD). The results are shown in [Figure number missing]. Figure 3 .

[0059] Depend on Figure 3 (a) The results show that the characteristic peaks of the XRD pattern of ZIF-8 prepared in Example 1 at 2θ = 7.4°, 10.4°, 12.8°, 14.7°, 16.6°, and 18.0° are consistent with those widely reported in previous studies and can be attributed to the (011), (002), (112), (022), (013), and (222) crystal planes of ZIF-8 nanomaterials. Compared with ZIF-8, the characteristic peaks of the products obtained after each step of PVP modification, etching, and amino functionalization modification remained intact, but the peak intensity gradually decreased. This indicates that the crystal structure of ZIF-8 did not change during the synthesis process, but its lattice constant increased. This may be because each step of the treatment increased the coordination defects of the composite material to varying degrees. It is worth noting that the XRD data of CRL-N-PVP / HZIF-8 showed no diffraction peaks, indicating that it has an amorphous structure. This may be because the enzyme-carrying system contains "interfering components" and Zn. 2+Irregular coordination results in molecular coordination collapse, leading to the emergence of amorphous CRL-N-PVP / HZIF-8 with a random network topological framework, instead of retaining the sodalite topological structure of crystalline ZIF-8.

[0060] (c) To further clarify the changes in chemical bonds and functional groups of the support before and after immobilization, Fourier transform infrared spectroscopy (FTIR) was used to characterize the products involved in different stages of the CRL-N-PVP / HZIF-8 synthesis process. The results are shown in […]. Figure 3 .

[0061] Depend on Figure 3 (b) The results show that at 421cm -1 The peak observed at 900-1350 cm⁻¹ is attributed to the stretching vibration of the Zn-N bond. -1 The peaks within this range correspond to the in-plane bending of the imidazole ring, followed by the peaks at 1350-1500 cm⁻¹. -1 The peaks within this range are related to the stretching vibrations of the imidazole ring, and are at 1582 cm⁻¹. -1 The strong peak at the location is attributed to the C=N stretching mode in the imidazole ring, further confirming the integrity of the covalent bonds in the immobilized enzyme carrier at different stages. The gradual decrease in intensity may be due to the generation of unsaturated coordination metal sites after different stages of carrier treatment, which is consistent with the aforementioned XRD results. PVP modification or APTS modification in the preparation process may have resulted in non-covalent interactions with the carrier, making the characteristic peaks of the relevant chemical substances undetectable, or unable to be clearly identified due to absorption band overlap or peak shift. Furthermore, no absorption peaks specific to ZIF-8 were observed in CRL-N-PVP / HZIF-8, further confirming that the enzyme carrier system contains Zn similar to that in ZIF-8. 2+ The irregular coordination of the material disrupted the normal FTIR imaging of ZIF-8. Notably, Free CRL and CRL-N-PVP / HZIF-8 at 1657 cm⁻¹... -1 and 1537cm -1 The two peaks at these locations show consistent characteristic peaks, which represent the amide I (stretching vibration of the CO bond) and II (combination of NH bending mode and CH stretching mode) bands in the enzyme protein, respectively, revealing the successful loading of CRL in PVP / HZIF-8.

[0062] (d) The surface elemental composition and chemical state of the products synthesized at different stages during the CRL-N-PVP / HZIF-8 synthesis process were investigated using X-ray photoelectron spectroscopy (XPS). This further determined the changes in the elemental composition of the CRL-N-PVP / HZIF-8 material structure during the preparation process. The results are shown in [see attached table]. Figure 4 .

[0063] Depend on Figure 4 The results showed that the high-resolution XPS spectra of C1s for each sample (ZIF-8, PVP / ZIF-8, PVP / HZIF-8, N-PVP / HZIF-8, and CRL-N-PVP / HZIF-8) could be divided into three sub-peaks. The strong peak at the lower binding energy of 284.8 eV can be attributed to the C=O bond, while the smaller peak at the higher binding energy of ~286 eV indicates 2-MeIM or CN introduced from other substances. Another small characteristic peak at 288 eV can be assigned to C=O bonds related to carbonates or enzyme protein molecules. The increased peak area of ​​CN and C=O in PVP / ZIF-8 compared to ZIF-8 can be attributed to the presence of the hydrophilic component (pyrrolidone) in PVP, demonstrating the successful introduction of PVP. The peak areas of CC, CN, and C=O in CRL-N-PVP / HZIF-8 were significantly increased compared to N-PVP / HZIF-8. This can be attributed to the presence of the characteristic amide bond (HN-C=O) of the protein, indicating that CRL was successfully immobilized in the carrier material.

[0064] The high-resolution XPS spectra of O1s for each sample (ZIF-8, PVP / ZIF-8, PVP / HZIF-8, N-PVP / HZIF-8, and CRL-N-PVP / HZIF-8) can be divided into three sub-peaks, representing Zn-O, carbonate O=C, and H2O, which is physically and chemically bonded to the material surface, with binding energies of 530.7, 531.7, and 533 eV, respectively. Notably, among the samples from different stages of immobilized enzyme preparation, the H2O peak area in PVP / HZIF-8 is the largest. This may be due to the etching process requiring a Tris-HCl buffer system, and the relatively abundant presence of N-Zn bonds on the reverse side, which readily adsorb water and form bonds.

[0065] The high-resolution XPS spectra of N1s for each sample (ZIF-8, PVP / ZIF-8, PVP / HZIF-8, N-PVP / HZIF-8, and CRL-N-PVP / HZIF-8) all showed three distinct nitrogen atoms. The signal around 398.8 eV was mainly attributed to the (CN) in the imidazole group. The peak with an intermediate binding energy of ~399.4 eV indicated the presence of an NH bond, while the peak with a higher binding energy of ~400 eV indicated the presence of an N-Zn bond. The increased NH peak area in N-PVP / HZIF-8 compared to PVP / HZIF-8 confirmed the successful modification of PVP / HZIF-8 by APTS for amino functionalization. The N-Zn peak area of ​​the samples at different stages of immobilized enzyme carrier synthesis showed an orderly decrease, indicating that the bonding strength of ZIF-8 was gradually perturbed during carrier synthesis. After immobilization of the enzyme protein, the NH peak area increased significantly, while the abnormal increase in the N-Zn peak was due to the interaction between the NH2 side chain group in the protein molecule and the unsaturated zinc atom of the ligand. Furthermore, the CN peak area decreased significantly due to the removal of the imidazole ring. The reduction in the number of organic linkers facilitates the formation of amorphous MOFs, which typically significantly enhances the activity of the enzyme encapsulated in the MOF. These results confirm the efficient immobilization of lipase on N-PVP / HZIF-8 and validate the findings obtained from the XRD and FTIR studies described above.

[0066] (e) Since a large Brunauer-Emmett-Teller (BET) specific surface area is an important characteristic of an excellent support, nitrogen adsorption / desorption analysis was performed on the obtained CRL-N-PVP / HZIF-8 and the products (ZIF-8, PVP / ZIF-8, PVP / HZIF-8, N-PVP / HZIF-8, and CRL-N-PVP / HZIF-8) at different stages of the immobilized support synthesis process to characterize the surface area and porosity of the samples. The results are as follows: Figure 5 As shown in Table 1.

[0067] Depend on Figure 5(a) The results show that ZIF-8 and PVP / ZIF-8 exhibit Type I isotherms, indicating that micropores are a major component of the nanostructure. Inconspicuous desorption hysteresis loops were observed in the pressure range of 0.5–0.85, indicating the presence of mesoporous micropores within the dense core-shell structures of ZIF-8 and PVP / ZIF-8. This is due to the inevitable generation of minute self-assembly dislocation defects during the one-pot synthesis of ZIF-8 using colloidal aggregates as soft templates. In contrast, both the etched PVP / HZIF-8 and the amino-functionalized N-PVP / HZIF-8 exhibited Type IV isotherms with large and obvious hysteresis loops, indicating the presence of mesopores. This can be attributed to the modification treatment continuously attacking and stretching the originally dense shell walls, increasing the degree of defects.

[0068] Depend on Figure 5 (b) Pore size distribution results show that the main mesopores in PVP / HZIF-8 and N-PVP / HZIF-8 are concentrated around 7.5 nm, while those in ZIF-8 and PVP / ZIF-8 are concentrated around 3.5 nm. Clearly, the proportion of mesopores increases significantly in this order. Generally, the presence of mesopores can provide a larger pore volume and may reduce the surface area of ​​microporous materials. Higher specific surface area and pore volume are beneficial for enzyme loading.

[0069] Table 1

[0070]

[0071] As shown in Table 1, after immobilization of CRL, the BET specific surface area decreased from 1,405.53 m² to 74.26 m². 2 ·g -1 The mesopore volume decreased from 0.46 cm³ / g to 0.23 cm³ / g, likely due to the large amount of CRL filling the hollow cavity. Notably, the micropore volume of CRL-N-PVP / HZIF-8 almost disappeared. This can be attributed to the repeated use of the same buffer solution as during enzyme loading and the further action of phosphate doped in the CRL solution, which orderly expands the pores between the shell walls, resulting in an average pore size of 13.03 nm for the immobilized enzyme. This facilitates more efficient penetration of free CRL (5.0 nm × 4.2 nm × 3 nm) into the shell channels and hollow spheres, and reduces diffusion resistance in the subsequent catalytic reaction system. These results are highly consistent with the aforementioned morphological and structural characterization analysis. This also indicates that the N-PVP / HZIF-8 with a hollow hierarchical porous core-shell structure prepared in this invention can serve as a suitable and excellent platform for immobilized enzymes and subsequent biocatalysis.

[0072] (f) Energy-dispersive spectroscopy (EDS) was used to visually analyze N-PVP / HZIF-8 and CRL-N-PVP / HZIF-8 to further confirm the presence of CRL molecules in the immobilized support. Results are as follows: Figure 5 As shown in (c).

[0073] Depend on Figure 5 (c) The results show that C, N, Zn, P, and S elements are uniformly distributed in N-PVP / HZIF-8 and CRL-N-PVP / HZIF-8, exhibiting a uniform spherical shape. Among them, the distribution density of the characteristic protein elements P and S differs significantly between the two. Compared with the sparse distribution of P and S in N-PVP / HZIF-8, the distribution of P and S in CRL-N-PVP / HZIF-8 is particularly dense after CRL fixation. At the same time, other elements also show varying degrees of density differences, indicating that the lipase CRL has been successfully fixed on or in N-PVP / HZIF-8.

[0074] (g) To further confirm that the enzyme protein was indeed immobilized in the spherical chambers and shell channels of N-PVP / HZIF-8, confocal imaging visualization analysis was performed on the fluorescent CRL-N-PVP / HZIF-8. The results are as follows: Figure 5 As shown in (d).

[0075] Figure 5 (d) The results show that no green rings appeared in the CLSM image at an excitation wavelength of 488 nm for CRL-N-PVP / HZIF-8, clearly demonstrating that the protein was uniformly immobilized in the core-shell structure, with partial aggregation of nanoparticles. This is likely due to the high concentration of the nanoparticle suspension and the low surface zeta potential of the immobilized enzyme. These results collectively confirm that N-PVP / HZIF-8 is entirely feasible as an immobilization carrier for encapsulating the model enzyme CRL.

[0076] (h) Thermogravimetric analysis (TGA) curves and water contact angle measurements of CRL-N-PVP / HZIF-8 and the carrier at different stages of the preparation process are shown in the figure. Figure 6 .

[0077] Depend on Figure 6(a) The results show that the initial phase occurs in the range of approximately 200 °C, which may be attributed to the decomposition of methanol and water trapped in the ZIF-8 pores and the carbonization of excess guest molecules (2-MIM) in the ZIF-8 framework. In the phase of approximately 200–550 °C, the addition of PVP significantly enhances the thermal stability of ZIF-8. A second weight loss of 14.5% was observed in CRL-N-PVP / HZIF-8, primarily due to the thermal decomposition of CRL protein within this temperature range. The weight loss of the enzyme protein in the TGA was approximately consistent with the enzyme load determined by the Protein Quantification Assay Kit (BCA). However, the thermogravimetric analysis curve of free CRL relative to CRL-N-PVP / HZIF-8 showed a significant difference, exhibiting a sharp decrease in weight loss, indicating improved thermal stability of the immobilized enzyme. It is also worth noting that the final weight loss of the carrier at different stages all occurred around 550℃, corresponding to the decomposition of organic ligands caused by MOF framework collapse. The lower decomposition rate of CRL-N-PVP / HZIF-8 indicates that CRL (containing PO4) 3- The incorporation of ) gives this composite material an exciting and unique property of thermal stability.

[0078] Figure 6 (b) Water contact angle results showed that ZIF-8, PVP / ZIF-8, PVP / HZIF-8, N-PVP / HZIF-8, and CRL-N-PVP / HZIF-8 had water contact angles of 107.3°, 86.3°, 65.0°, 50.4°, and 29.8°, respectively. The slight decrease in water contact angle of PVP / ZIF-8 compared to the highly hydrophobic ZIF-8 is attributed to the presence of a strongly hydrophilic pyrrolidone ring in PVP. The decrease in water contact angle of PVP / HZIF-8 is due to the etching process being performed in a Tris-HCl buffer solution, where adsorbed H₂O can further dissociate and bind to Zn sites, thus weakening the original hydrophobicity. The low hydrophobicity of N-PVP / HZIF-8 may be due to the presence of hydrophilic APTS on its surface. The presence of hydrophilic carrier properties will facilitate enzyme binding. The hydrophilic layer formed by the aggregation of adsorbed enzyme molecules is the reason for the continued decrease in the water contact angle of CRL-N-PVP / HZIF-8. At the same time, the multi-point covalent cross-linking between enzyme molecules and between the enzyme and the carrier increases the rigidity of the enzyme and the stability of the framework, thereby improving the tolerance of ZIF-8 in the aqueous environment.

[0079] Study on the preparation strategy of CRL-N-PVP / HZIF-8

[0080] (i) Experiments of this invention have also revealed that when the white product ZIF-8 is transferred to deionized water to eliminate the soft template of the spherical colloidal aggregates, the solution becomes clear and transparent after standing for 20 minutes. The original white product cannot be collected after high-speed centrifugation; it can only be collected after centrifugation with methanol added as a "precipitant" for ZIF-8. Furthermore, this invention utilizes scanning electron microscopy images... Figure 7 (a) confirms that removing the colloidal soft template with deionized water causes ZIF-8 to collapse and dissociate into fragments, and the original regular spherical morphology cannot be maintained. Here, a weakly alkaline cationic Tris-HCl buffer is used as an etching agent to remove the template, and also as a buffer component to maintain enzyme activity during enzyme loading. In addition, the interaction of the relatively large hydrophobic carbon chains (alkyl groups in PVP) in the N-PVP / HZIF-8 structure may create a microenvironment that is resistant to water "attack" to a certain extent. Figure 8 (a) describes the potential mechanism for the structural stability of N-PVP / HZIF-8 under aqueous conditions. SEM images confirm that CRL-ZIF-8 without PVP surface modification exhibits large-scale shell shedding and fragmentation of some particles. Figure 8 b and Figure 7 b). The regular spherical morphology of the CRL-N-PVP / HZIF-8 nanocomposite material was largely maintained, and the shell surface structure was relatively intact. Figure 8 c). The above synthetic strategy avoids severe structural collapse and excessive release of the precursor imidazole caused by PVP / ZIF-8 etching in aqueous systems, while minimizing carrier fragmentation and enzyme leaching inactivation due to water and phosphate "attack" during CRL immobilization in N-PVP / HZIF-8. These results demonstrate the feasibility of the CRL-N-PVP / HZIF-8 strategy synthesized in this paper, which is of great significance for improving the stability of MOFs and expanding their application in aquatic environments.

[0081] Study on the Immobilization Effect of N-PVP / HZIF-8

[0082] (j) The initial enzyme concentration and immobilization time are important factors affecting the protein load and relative activity of the immobilized enzyme during the preparation of CRL-N-PVP / HZIF-8. Figure 9(a) It can be seen that with the increase of the initial enzyme concentration, the relative activity of the immobilized enzyme shows a trend of first steadily increasing and then decreasing, reaching a maximum of 68.6% at an initial enzyme concentration of 20 mg / mL. Simultaneously, the protein loading also shows a change of first increasing and then gradually decreasing, reaching a maximum of 194.6 mg / g at an initial enzyme concentration of 20 mg / mL. This is because a lower initial enzyme concentration leads to a lower enzyme loading on the carrier, resulting in insufficient utilization of the cavity and mesopore spaces of the carrier, leading to lower catalytic efficiency. Furthermore, after the protein loading and relative activity of the immobilized enzyme reach their peak, increasing the initial enzyme concentration does not further improve the immobilization effect of N-PVP / HZIF-8. This phenomenon can be explained by the fact that the higher concentration of CRL solution contains excessive phosphate, which reacts with the Zn in the carrier. 2+ While the combination of complexes positively influences the formation of large mesopores in the shell, excess complexes compete with enzyme proteins for entry into the core chamber. This not only supports the structure but also occupies space for the enzyme load, reducing mass transfer efficiency and decreasing catalytic activity. Furthermore, higher concentrations of enzyme solutions can generate lipase dimers, covering the catalytic sites of the enzyme molecule. Simultaneously, enzyme protein molecules aggregate to form large clusters that are difficult to accommodate within the core-shell structure through mesoporous channels. Therefore, for subsequent experiments, an initial enzyme concentration of 20 mg / mL was chosen.

[0083] (k) Regarding the effect of immobilization time on the protein load and relative activity of immobilized enzymes, as follows: Figure 9 As shown in (b), with increasing immobilization time, the relative activity of the immobilized enzyme initially increased and then gradually decreased, reaching a maximum of nearly 80% at 2.5 h. The enzyme loading initially increased slowly and then stabilized over time, with a maximum loading of 216.3 mg / mL. Since enzyme molecules undergo physical adsorption followed by covalent binding during immobilization, and then enter the pores before reaching the chamber, the increase in relative activity and enzyme loading in the curves follows the explanation that the carrier space was not fully occupied by the enzyme protein. With prolonged immobilization time, enzyme immobilization reached equilibrium. As is known in this invention, glutaraldehyde is also an enzyme inactivator, capable of penetrating protein active sites and reacting with catalytically essential amino acid residues, ultimately leading to a decrease in enzyme activity. Furthermore, excessive enzyme loading increases steric hindrance of the enzyme molecule, hindering the binding of the substrate to the active site, and also slightly reducing the activity of the immobilized enzyme. To maximize the effect of N-PVP / HZIF-8 immobilization and prevent partial inactivation of the enzyme protein during a longer immobilization time, the optimal immobilization time for the immobilized enzyme was selected as 2.5 h.

[0084] (l) To gain a deeper understanding of the impact of different steps in the preparation of the N-PVP / HZIF-8 immobilization carrier on the immobilization effect, various samples generated during the N-PVP / HZIF-8 synthesis process were immobilized with CRL according to the immobilization procedure of the target immobilized enzyme. The results are as follows: Figure 9 As shown in (c), compared to CRL-ZIF-8, CRL-PVP / ZIF-8, CRL-PVP / HZIF-8, and CRL-N-PVP / ZIF-8, it is evident that CRL-N-PVP / HZIF-8 has the highest relative activity and enzyme loading. Comparing the loading of CRL-N-PVP / HZIF-8 and CRL-PVP / HZIF-8 demonstrates that the appropriate use of cross-linking agents in enzyme immobilization strategies contributes to higher enzyme loading. Generally, increased enzyme loading naturally corresponds to higher enzyme activity within a certain range. However, comparing the relative activity and loading of CRL-N-PVP / HZIF-8 and CRL-N-PVP / ZIF-8 highlights the importance of etching to create hollow structures, which can generate more space for enzyme loading. Although the H2O in the enzyme solution and the small amount of phosphate from the lyophilized enzyme powder can contribute to further etching and pore expansion during immobilization, pre-etching reduces the risk of further protonation of imidazole released during carrier decomposition, which could inactivate the enzyme protein. Meanwhile, the highly hydrophobic ZIF-8 and PVP / ZIF-8 are difficult to disperse well, existing to some extent in a floating and aggregated state. This makes it difficult for CRL molecules to diffuse and bind to the carrier in aqueous solution, resulting in low protein loading and relative activity. This indicates that the N-PVP / HZIF-8 immobilization carrier prepared in this invention is well-designed for each stage of the CRL immobilization process.

[0085] (m) To further explore the advantages of this immobilization carrier, an in-situ immobilized enzyme CRL@ZIF-8 was prepared using a one-pot method as a control, while the optimal activity of the free enzyme was defined as 100%. Results are as follows: Figure 9 As shown in (d), the figure clearly shows that the enzyme activity of CRL-N-PVP / HZIF-8 is more than 15-fold higher than that of CRL@ZIF-8. This result may be due to two reasons: firstly, the unfolding effect and competitive coordination caused by the organic ligand 2-methylimidazole in the reaction system lead to the inactivity of the resulting enzyme @ZIF-8. Secondly, the guest enzyme has difficulty diffusing through the dense microporous framework of the in-situ encapsulated ZIF-8, thus significantly limiting the catalytic rate of the encapsulated enzyme.

[0086] (n) To further explore the possibility of the N-PVP / HZIF-8 core-shell structure protecting fragile biomacromolecules from harsh environments, since one advantage of embedded enzymes is enhanced stability due to their shielding effect. In this invention, free CRL and CRL-N-PVP / HZIF-8 were exposed to trypsin (6 mg / mL) respectively. -1 The harsh conditions of exposure to heat (80°C) for 20 minutes can jeopardize the maintenance of enzyme activity through unfolding. The activity of each sample under optimal conditions was used as a control (100%) before treatment. Results are as follows: Figure 9 As shown in (e), after trypsin and heat treatment, the relative activities of the free enzyme were only 70.5% and 76.3%, respectively, while the enzyme protein in CRL-N-PVP / HZIF-8 was protected by the "armor," and its biological activity was preserved at 83.7% and 92.6% of the original. The above data indicate that CRL-N-PVP / HZIF-8 has good stability, which further shows that most of the CRL molecules are encapsulated in the hollow structure of N-PVP / HZIF-8.

[0087] (o) The reusability of this immobilized enzyme was evaluated by using a CRL-N-PVP / HZIF-8 cycle to hydrolyze p-nitrophenyl palmitate (p-NPP). Results are as follows: Figure 9 As shown in (f), the relative activity decreased significantly after the first reaction cycle, which may be due to leakage of CRL weakly adsorbed on the support surface during the separation and washing steps. Notably, the relative activity of the immobilized enzyme decreased slowly in subsequent cycles. This may be because, on the one hand, the hollow mesoporous core-shell structure protects the CRL from direct exposure to the reaction environment, preserving the structural integrity of the enzyme's active site; on the other hand, the improved pore structure optimizes the substrate transport channels, improving mass transfer efficiency. Of course, it is also possible that products from the previous cycle were not completely removed (this is too revealing and analytical, so it can be omitted). CRL-N-PVP / HZIF-8 retained over 70% activity after eight consecutive cycles, indicating that the developed biocatalyst has operational stability and can be used repeatedly and continuously.

[0088] CRL-N-PVP / HZIF-8 Catalyzed Synthesis of Phytosterol Esters

[0089] Candida rugosa lipase (CRL) is a commercially available lipase widely used in various fields. It catalyzes the esterification reaction of phytosterols with linolenic acid to produce phytosterol esters. As an important class of plant derivatives, phytosterol esters play a significant role in improving phytosterol absorption, protecting cardiovascular health, exhibiting antioxidant activity, and preventing cancer.

[0090] To evaluate the ability of CRL-N-PVP / HZIF-8 to produce phytosterol esters, CRL-N-PVP / HZIF-8 was applied to catalyze the esterification of phytosterols and α-linolenic acid (see [link to study]). Figure 10 The production conditions of phytosterol esters were optimized, including catalytic reaction temperature, substrate molar ratio, and molecular sieve MS. The results of adding CRL-N-PVP / HZIF-8 are as follows: Figure 11 As shown in (ad).

[0091] Reaction temperature: Generally, increasing the temperature can increase the solubility of the substrate and decrease the viscosity of the reaction system, intensifying the interaction between the enzyme and the substrate, thereby increasing the reaction rate. While high temperatures enhance the mixing of reactants, the enzyme structure may be damaged, and its activity may decrease. Figure 11 As shown in (a), within the test temperature range of 35-55℃, the esterification rate was lowest at 35℃ (47.9%). As the temperature increased, the esterification rate rose, reaching a maximum phytosterol conversion rate of 75.4% at 45℃. However, further increases in temperature naturally led to a gradual decrease in the phytosterol conversion rate.

[0092] Substrate molar ratio: Theoretically, using phytosterols equal in number to fatty acid residues is sufficient to completely convert fatty acid residues into the corresponding phytosterol esters. Since esterification is a reversible reaction, the reaction can be directed towards the formation of phytosterol esters by removing products or adding excess fatty acids, thereby promoting the conversion rate of phytosterols and obtaining more of the target product. However, the presence of excess fatty acids in the reaction system can severely inhibit lipase activity and may also damage the immobilized enzyme structure; furthermore, it reduces the fluidity of the reaction system, hindering substrate access to the active site and thus impeding the esterification reaction. Figure 11 As shown in (b), the molar ratio of phytosterols to fatty acids in the experiment ranged from 1:1 to 1:5. The substrate conversion rate was 74.7% when the alcohol-acid molar ratio was 1:1, and the esterification rate reached its maximum of 82.4% when the ratio increased to 1:2. However, the esterification rate gradually decreased with increasing alcohol-acid molar ratio; therefore, this experimental result is highly consistent with the theoretical analysis described above.

[0093] MS (molecular sieve) addition amount: Water generated during esterification can inhibit the reaction. Removing the generated water can disrupt the chemical equilibrium and promote the esterification reaction in the forward direction. However, a certain amount of water is usually required to maintain the active conformation of the lipase and obtain the best catalytic effect. Figure 11As shown in (c), the conversion rate of phytosterols increased with MS from 50 mg to 125 mg. However, with further addition of MS, the conversion rate decreased. This is because the water in the system and the trace amounts of water produced by the esterification reaction may be excessively removed by MS, leading to a change in the active conformation of lipase and thus inhibiting the activity of lipase.

[0094] Catalyst dosage: Generally, when the substrate concentration is sufficiently high, the enzyme concentration, i.e., the amount of enzyme added, is positively correlated with the enzyme-catalyzed reaction rate. While enzymes, as biocatalysts, do not affect the equilibrium of the esterification reaction, they can significantly shorten the time required to reach equilibrium. For example... Figure 11 As shown in (d), when the amount of immobilized enzyme added exceeds a certain range, further increases may not yield higher benefits. This is because while increasing the contact between the substrate and the active site of the catalyst, an excessively high concentration of catalyst can also affect the effective mixing between substrates. Furthermore, due to the spatial constraints, competition for substrates can inhibit the esterification reaction.

[0095] This invention is not limited to the specific embodiments described above. Any modifications made by those skilled in the art based on the above concept without creative effort are within the scope of protection of this invention.

Claims

1. A method for preparing a hollow hierarchical porous MOF material, characterized in that, Includes the following steps: S1. Preparation of spherical ZIF-8: The ZIF-8 precursors zinc sulfate heptahydrate (ZnSO4·7H2O) and 2-methylimidazolium (2-MIM) were dissolved in anhydrous methanol to obtain reaction solution A and reaction solution B. Then, reaction solution B was added to reaction solution A and mixed. The mixture was stirred and reacted, then centrifuged, washed with anhydrous methanol, and dried under vacuum to obtain spherical ZIF-8. S2. Polyvinylpyrrolidone was added during the stirring growth of spherical ZIF-8. After co-incubation, the precipitate was centrifuged, the supernatant was removed, and weakly basic cationic tris(hydroxymethyl)aminomethane hydrochloride buffer was added. The mixture was soaked and stirred to dissolve and eliminate the spherical colloidal soft template. The mixture was centrifuged again at high speed, the etching solution was extracted with a syringe, washed with anhydrous methanol, and vacuum dried to obtain PVP / HZIF-8. S3. PVP / HZIF-8 was added to an anhydrous ethanol solution of 3-aminopropyltrimethoxysilane, mixed, stirred and reacted, centrifuged to precipitate, then washed with anhydrous ethanol and dried under vacuum to obtain N-PVP / HZIF-8.

2. The method for preparing hollow hierarchical porous MOF material according to claim 1, characterized in that, In step S1, the stirring reaction temperature is 15-35°C and the stirring time is 0.5-2 hours.

3. The method for preparing hollow hierarchical porous MOF material according to claim 1, characterized in that, In step S2, the stirring reaction temperature is 15–35°C, and the stirring time is 0.5–2 h.

4. The method for preparing hollow hierarchical porous MOF material according to claim 1, characterized in that, In step S3, the temperature of the stirring reaction is 55-65°C, and the stirring time is 6-8 hours.

5. The method for preparing hollow hierarchical porous MOF material according to claim 1, characterized in that, The mass ratio of the spherical ZIF-8 to PVP is 1:3 to 1:

5.

6. The method for preparing hollow hierarchical porous MOF material according to claim 1, characterized in that, The mass-to-volume ratio of PVP / HZIF-8 to 3-aminopropyltrimethoxysilane is 0.5 mg:10 μL to 1.5 mg:10 μL.

7. A hollow hierarchical porous MOF material prepared by the preparation method according to any one of claims 1-6.

8. A method for preparing immobilized enzymes using the hollow hierarchical porous MOF material of claim 7 as an immobilization carrier, characterized in that, Includes the following steps: A1, will Candida rugosa Lipase was added to Tris-HCl buffer, shaken to dissolve, and then centrifuged in a refrigerated centrifuge at 4°C and 8000 rpm for 10 min to obtain the supernatant. A2. Transfer the enzyme supernatant to a stoppered Erlenmeyer flask, add N-PVP / HZIF-8, and place it in a magnetic stirrer at room temperature for enzyme immobilization. During enzyme loading, add GA in two batches to activate the carrier and cross-link with the enzyme protein. A3. After enzyme immobilization, the product was separated by freeze centrifugation, washed three times with Tris-HCl buffer to remove excess lipase, and then freeze-dried to obtain the immobilized enzyme CRL-N-PVP / HZIF-8.

9. An immobilized enzyme prepared by the method described in claim 8.

10. The application of the immobilized enzyme as described in claim 9 as a biocatalyst in the catalytic synthesis of phytosterol esters.

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