A macroporous MOF encapsulated enzyme complex, and a preparation method and application thereof

Abstract

The application discloses a macroporous MOF encapsulated enzyme composite and a preparation method and application thereof. 2+ The application introduces transition metal iron as a site to controllably synthesize macroporous MOFs-Fe by using metal competition coordination and oxidation pore-forming strategies, and by controlling the incorporation amount of Fe, the pore structure of the composite material between 2-50 nm and 50-100 nm can be accurately adjusted. Compared with the enzyme composite beta-G@MOF which is mainly microporous, the macroporous enzyme composite beta-G@MOF-Fe has higher enzyme activity and better heat resistance. The application provides a simple and mild method for constructing an enzyme composite with controllable macroporous structure, good biological activity and durability, and can provide inspiration for the design and construction of macroporous catalysts for catalytic conversion of macromolecular substrates.

Description

Technical Field

[0001] This invention belongs to the field of high-temperature enzyme catalysis, specifically relating to a macroporous MOF-encapsulated enzyme complex and a method for preparing the macroporous MOF-encapsulated enzyme complex through competitive coordination and oxidative pore-forming strategies, and its application in achieving high-temperature and efficient catalytic conversion of macromolecular cellulose in ionic liquids. Background Technology

[0002] Enzymes are proteins with specific spatial structures and catalytic functions, characterized by high catalytic efficiency, high substrate specificity, and high product selectivity. However, factors such as temperature, solution pH, ionic strength, and organic solvents can disrupt the spatial structure of proteins, causing denaturation and reducing catalytic activity. This often results in lower enzyme catalytic temperatures, thus affecting the rate of catalytic reactions. Recent reports indicate that MOF encapsulation can effectively protect native enzymes from harsh external environments, thereby improving their thermal stability, reusability, and even activity. Metal-organic frameworks (MOFs) are porous framework materials composed of ordered coordination of metal ion nodes and organic ligand units, possessing high specific surface area, customizable pore structures, and rigid organic frameworks. However, the micropores of MOFs themselves are smaller than 2 nm, limiting the entry of large substrate molecules into the MOF channels and their binding to the encapsulated enzyme, thus restricting the conversion of large substrate molecules. The inventors prepared a mesoporous flower-like enzyme complex β-G@MOF(Cu-PABA) by in-situ encapsulating β-glucosidase (β-G) in MOFs using an enzyme-induced method. This complex can enzymatically hydrolyze cellobiose, cellulose, and other substances to produce glucose at 110℃. However, due to the wide molecular weight distribution of cellulose, with some molecules concentrated in the range of 2900–3600 g / mol (approximately 68–84 nm in diameter), large cellulose molecules have difficulty entering the pores of the MOF(Cu-PABA), resulting in an enzymatic hydrolysis rate of only 50% (CN202110679442.3, ChemSusChem, 2022, 1-9). Therefore, it is necessary to prepare MOFs with macroporous (50–100 nm) structures to encapsulate β-glucosidase to achieve high-temperature and efficient enzymatic hydrolysis of cellulose.

[0003] In recent years, to enhance mass transfer and diffusion of macromolecules within the pores of MOFs, various methods have been employed to prepare MOFs with macroporous structures, such as ligand extension, template methods, and defect methods. Ligand extension involves lengthening the link length of organic ligands to synthesize MOFs with larger pore sizes. For example, linking a long ligand, 3”-dihydroxy-[1,1':4',1”-terphenyl]-4,4”-dicarboxylic acid, to the ligand of MOF-74 can synthesize IRMOF-74-III, a mesoporous structure with a diameter of 2.2–2.6 nm (ChemSusChem 2018, 21, 3751-3757). Furthermore, by extending the organic ligands of MOF-74 from a single styrene ring chain to two to eleven, a series of MOF-74s with different pore structures can be synthesized, with the largest pore size reaching 9.8 nm (Science). (2012, 336, 1018-1023). While ligand extension can prepare mesoporous MOFs, the mechanical and chemical stability of the framework gradually decreases as the size of the organic linker increases, often leading to structural collapse. This results in MOFs that are prone to enzyme leakage, reducing their protective effect on enzymes. Furthermore, MOFs constructed via ligand extension have pore sizes ranging from 2 to 20 nm (Angew. Chem. 2015, 127, 12939-12946; Angew. Chem., Int. Ed. 2015, 54, 149-154; Acc. Chem. Res. 2014, 47, 296-307), which is insufficient for mass transfer in high molecular weight cellulose (approximately 68-84 nm in diameter). The template method involves introducing a "template" of a specific structural size into the MOF structure. In the template method, the template is then removed to prepare MOF materials with a predetermined pore size. For example, ordered macro / microporous MOF single crystal materials were prepared using the three-dimensional structure of polystyrene microspheres (PS) as a template, with macropore diameters reaching approximately 190 nm to 470 nm (Science, 2018, 359, 206-210). Using hydrogel as a template for the synthesis of ZIF-67, after template removal, Zn-based H-ZIF-67 with a mesoporous structure of 16–27 nm was obtained (Small, 2019, 15, 1902927-1902937). While the template method can construct pores with a wide range of sizes, from tens to hundreds of nanometers, it requires template removal before enzyme encapsulation, making in-situ enzyme encapsulation difficult. The defect method introduces another metal ion or ligand during MOF preparation to create competing coordination, thereby generating defects in the MOF structure.For example, by adding a long-chain fatty acid (dodecanic acid) to a mixture containing ZrCl4 and BDC (terephthalic acid), a MOF with a pore size of 5.5 nm was synthesized at 90 °C for 12 hours via competitive coordination between the fatty acid and BDC (Angew. Chem., Int. Ed. 2017, 56, 563-567). By adding a smaller linker fragment (R-isopentanoic acid) to a synthetic mixture of triphenyltetracarboxylic acid (TPTC) and copper nitrate hexahydrate, a mesoporous PCN-125 with a pore size of 20 nm was synthesized via defective structures generated through competitive coordination between ligands (J. Am. Chem. Soc. 2012, 134, 20110-20116). Besides ligand competition generating defects, the introduction of metal ions can also induce pore defects in MOFs. For example, Zn was introduced into a MOF with triazine groups as ligands. 2+ By changing Zn 2+ and Ni 2+ HP-MOFs with mesopore sizes of 20–50 nm can be synthesized at 120 °C using fluoroboric acid as a solvent (Inorg. Chem. 2021, 60, 7, 5122-5130). This can be achieved by introducing Cu(BF4)2 as an etchant and reacting it with existing metal ions (Al) at 120 °C. 3+ Competitive coordination was generated to prepare defective mesoporous MOFs (Al-bpydc) with a pore size of 35 nm (Angew. Chem., Int. Ed. 2019, 31, 1904969-1904978). The mesoporous MOFs prepared by the above defect method were synthesized under high temperature, acid, and organic solvent conditions. Although defective pore MOFs can be prepared, the preparation process easily leads to enzyme inactivation, making in-situ enzyme encapsulation during MOF preparation difficult. Therefore, constructing macroporous MOFs that can in-situ encapsulate native enzymes while simultaneously enhancing their catalytic activity and stability under mild conditions is a challenge. Summary of the Invention

[0004] To address the limitation of existing technologies that can only enzymatically hydrolyze about 50% of microcrystalline cellulose, the primary objective of this invention is to provide a method for preparing macroporous MOF-encapsulated enzyme complexes with large pores under mild conditions through metal competitive coordination and an oxidation pore-forming strategy, thereby enabling more cellulose molecules to be hydrolyzed by the enzyme.

[0005] Another object of the present invention is to provide a macroporous MOF-encapsulated enzyme complex prepared by the above method. The prepared MOFs can form defective macropores (50-100 nm) in situ without sacrificing their structural stability, enabling the encapsulated enzyme to catalyze reactions at higher temperatures.

[0006] Another object of the present invention is to provide the application of the above-mentioned macroporous MOF-encapsulated enzyme complex in the high-temperature catalytic hydrolysis of macromolecular substrate cellulose.

[0007] By using transition state metals (Fe) 2+ Fe serves as a metal site for constructing macroporous MOFs. 2+ It is a metal ion that can promote the activity of β-glucosidase, significantly promoting the formation of MOFs around the enzyme through interaction with the exposed carboxyl or amino groups of the enzyme protein. Simultaneously added Fe... 2+ It will react with the original metal Cu 2+ Competitive coordination occurs, and based on the coordination characteristics of MOFs, Fe... 2+ It will preferentially coordinate with amino groups to form Fe 2+ -N bond, and due to Fe 2+ It is converted into Fe under the action of oxygen. 3+ Fe 3+ The weak coordination with N results in some Fe being... 3+ -N bond breaking forms a defective β-G@MOFs-Fe composite material. Compared to the microporous enzyme complex β-G@MOF, the macroporous enzyme complex β-G@MOF-Fe exhibits higher enzyme activity (1.4 times that of β-G@MOF in a 100℃ buffer system). In particular, when using macromolecular cellulose as a substrate, the macroporous enzyme complex β-G@MOF-Fe shows a higher conversion rate, reaching 90% after 64 hours of enzymatic hydrolysis, while the cellulose conversion rate of β-G@MOF under the same conditions is only 50%. This enhanced activity is due to the larger pore size of the constructed macroporous MOFs, which allows for faster diffusion rates and increased accessibility of the substrate to the enzyme active site. Furthermore, Fe... 2+ The formation of -N bonds gives β-G@MOF-Fe a more rigid structure, which not only has resistance to ionic liquids but also good heat resistance (the heat resistance temperature can reach 120℃).

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for preparing a macroporous enzyme complex β-G@MOF-Fe includes the following steps:

[0010] Soluble ferrous salt, β-glucosidase, benzoic acid compounds and buffer solution were mixed evenly to obtain a mixed solution. Soluble copper salt was then added, mixed evenly, and allowed to stand at room temperature. Solid-liquid separation was performed, and the solid phase was washed to obtain macroporous enzyme complex (β-G@MOF-Fe).

[0011] Preferably, the molar ratio of ferrous iron in the soluble ferrous salt to copper in the soluble copper salt is 0.05 to 0.4:1.

[0012] Preferably, the mass ratio of the soluble ferrous salt, β-glucosidase, benzoic acid compound and soluble copper salt is 0.1-5:0.02-5:0.1-4:1-9; more preferably 0.3-4:0.1-2:0.5-3:2-5; and most preferably 0.35-1.34:0.1:1:5.

[0013] Preferably, the soluble ferrous salt is at least one of ferrous nitrate and ferrous sulfate.

[0014] Preferably, the benzoic acid compound is at least one of p-aminobenzoic acid and terephthalic acid.

[0015] Preferably, the soluble copper salt is at least one of copper acetate and copper nitrate.

[0016] Preferably, the β-glucosidase needs to be purified before use, and the purification method is one of ultrafiltration and ion exchange chromatography.

[0017] Preferably, the mass ratio of the soluble ferrous salt to the volume ratio of the buffer solution is 1 g: 300-3000 mL; the buffer solution is an acetate-sodium acetate buffer solution with a pH of 6-8.

[0018] Preferably, the mixed solution further contains polyvinylpyrrolidone, and the mass ratio of β-glucosidase to polyvinylpyrrolidone is 1:0.1 to 0.5; the molecular weight of polyvinylpyrrolidone is 40 to 120 kDa.

[0019] Preferably, the room temperature refers to 10–40°C.

[0020] Preferably, the mixing time is 0.5 to 3 hours; more preferably, it is 0.5 to 2 hours.

[0021] Preferably, the settling time is 1 to 12 hours; more preferably, it is 4 to 10 hours.

[0022] Preferably, the centrifugal speed for solid-liquid separation is 6500–10000 rpm.

[0023] Preferably, the washing solid phase is washed with an acetate-sodium acetate buffer solution with pH = 4.5 to 6.5, and the obtained β-G@MOF-Fe enzyme complex is dried at 25 to 40°C for 1 to 14 hours.

[0024] Preferably, the method for preparing the macroporous enzyme complex β-G@MOF-Fe includes the following steps:

[0025] (1) Dissolve soluble ferrous salt and β-glucosidase in a buffer solution to obtain mixed solution 1;

[0026] (2) Mix the benzoic acid compound or benzoic acid compound solution with mixed solution 1 to obtain mixed solution 2;

[0027] (3) Mix the soluble copper salt or soluble copper salt solution with mixed solution 2 to obtain a mixed solution. Let it stand at room temperature, then separate the solid and liquid phases and wash the solid phase to obtain the macroporous enzyme complex (β-G@MOF-Fe).

[0028] More preferably, the mass ratio of the soluble ferrous salt in step (1) to the volume ratio of the buffer solution is 1 g: 10-86 mL.

[0029] More preferably, the mixed solution 1 in step (1) is obtained by mixing a soluble ferrous salt buffer solution and a β-glucosidase buffer solution, wherein the mass ratio of soluble ferrous salt to buffer solution in the soluble ferrous salt solution is 1g:8-100mL, and the protein concentration in the β-glucosidase buffer solution is 0.1-1mg / mL; the mass ratio of polyvinylpyrrolidone in the β-glucosidase buffer solution to buffer solution is 1g:100-500mL.

[0030] More preferably, in the benzoic acid compound solution in step (2), the mass ratio of the benzoic acid compound to the volume ratio of the acetate-sodium acetate buffer solution is 1g:100-1000mL.

[0031] More preferably, in the soluble copper salt solution of step (3), the mass ratio of the soluble copper salt to the volume ratio of the buffer solution is 1g:100-250mL.

[0032] A macroporase complex β-G@MOF-Fe was prepared by the above method.

[0033] The above-mentioned macroporous enzyme complex β-G@MOF-Fe is used in the enzymatic hydrolysis of cellulose.

[0034] The specific application is as follows:

[0035] The macroporous enzyme complex β-G@MOF-Fe was added to an ionic liquid or buffer solution with cellulose or cellobiose as the substrate, and reacted at 50-130℃ to obtain a saccharified hydrolysate. The enzymatic hydrolysate was separated from the solid and liquid, and after cooling, the macroporous enzyme complex β-G@MOF-Fe was centrifuged for recycling.

[0036] Preferably, the mass ratio of enzyme to cellulose or cellobiose in the macroporous enzyme complex β-G@MOF-Fe is 0.1–5 mg:1 g.

[0037] Preferably, the ionic liquid is at least one selected from imidazole acetate, imidazole ethyl sulfate, imidazole chlorate, imidazole sulfonate, and imidazole sulfate.

[0038] Preferably, the macroporase complex β-G@MOF-Fe is added to an ionic liquid or buffer solution with cellulose or cellobiose as a substrate and reacted at 50–130°C, wherein the concentration of the substrate is 2–20 mg / mL.

[0039] Preferably, the centrifugation time is 2 to 10 minutes and the rotation speed is 5000 to 10000 rpm.

[0040] The mechanism of this invention is as follows:

[0041] Fe 2+ Ferrous ions (Fe2+) are metal ions that can promote the activity of β-glucosidase. They significantly promote the formation of metal-organic fat-free (MOFs) around the enzyme through interaction with the exposed carboxyl or amino groups of the enzyme protein. Based on the coordination characteristics of MOFs, during the coordination process of organic ligands containing both amino and carboxyl groups, ferrous ions compete with copper ions for coordination with the amino group, altering the original coordination pattern and thus creating new Fe2+ groups. 2+ The formation of -N bonds. Fe 2+ After competitive coordination with Cu, and due to Fe 2+ The unstable element is converted into Fe under the action of oxygen. 3+ Fe 3+ The weak coordination with N results in some Fe being... 3+ -N bonds break to form defect structures, which in turn form macroporous structures. Then, β-G@MOF-Fe is used to hydrolyze the macromolecular substrate cellulose. Cellulose is dissolved by the ionic liquid, transforming from its original crystalline structure into dissolved chain-like cellulose molecules. These chain-like cellulose molecules diffuse into the porous structure of the MOFs, where β-G catalyzes the formation of glucose from the glycosidic bonds at the ends of the cellulose chains.

[0042] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0043] 1. This invention is the first to utilize transition metals (Fe) under mild conditions. 2+ As a metal site, a simple and novel metal competitive coordination and oxidation pore-forming strategy is reported to create macroporous MOF-Fe.

[0044] 2. The strategy proposed in this invention enables MOF to encapsulate β-glucosidase in situ under mild conditions, thereby preparing the macroporous enzyme complex β-G@MOF-Fe.

[0045] 3. The enzyme complex (β-G@MOF-Fe) prepared in this invention can be controlled by adjusting the Fe content. 2+The amount of incorporation was adjusted to control the pore structure of the composite material in the ranges of 2–50 nm and 50–100 nm.

[0046] 4. The enzyme complex prepared in this invention exhibits excellent heat resistance, with a heat resistance temperature up to 120℃, due to the formation of Fe-N bonds during coordination and the limitation of porous structure.

[0047] 5. The enzyme complex (β-G@MOF-Fe) prepared in this invention facilitates the diffusion of reactants and products due to the enlargement of pores and the shortening of diffusion paths.

[0048] 6. The macroporous enzyme complex (β-G@MOF-Fe) prepared in this invention exhibits higher bioactivity and stronger catalytic performance for cellulose. In a 100°C buffer system, the enzyme activity of β-G@MOF-Fe is 1.4 times that of β-G@MOF. After 64 hours of enzymatic hydrolysis, β-G@MOF-Fe achieves a cellulose conversion rate of 90%, while β-G@MOF only achieves 50%. Therefore, the synthetic strategy provided by this invention can inspire the design and construction of macroporous catalysts for the catalytic transformation of macromolecular substrates. Attached Figure Description

[0049] Figure 1 This is a schematic diagram illustrating the synthesis of the complex obtained in the embodiments of the present invention.

[0050] Figure 2 (a) is an electron microscope image of β-G@MOF obtained in Comparative Example 2 of the present invention; (be) is an electron microscope image of the enzyme complex obtained in Examples 1-4 of the present invention; (f) is an electron microscope image (left) of the enzyme complex obtained in Example 3 of the present invention and (g) is a mapping spectrum (right).

[0051] Figure 3 (a) Nitrogen adsorption-desorption curves of the β-G@MOF obtained in Comparative Example 2 and the enzyme complexes obtained in Examples 1-4 of the present invention; (b) Pore size distribution diagrams of the β-G@MOF obtained in Comparative Example 2 and the enzyme complexes obtained in Examples 1-4 of the present invention.

[0052] Figure 4 (a) is the XPS total spectrum of the β-G@MOF obtained in Comparative Example 2 of the present invention and the enzyme complexes obtained in Examples 1-4 of the present invention; (b) N 1s; (c) Cu 2p; (df) Fe 2p of the enzyme complexes obtained in Examples 1-3.

[0053] Figure 5 (a) shows the overall XPS spectrum of the MOF obtained in Comparative Example 1 and the composite obtained in Comparative Examples 6-8 of the present invention; (b) N 1s; (c) Cu 2p; (df) Fe 2p of the composite obtained in Comparative Examples 6-8.

[0054] Figure 6 (a) Scanning electron microscope image of MOF-Fe(Ⅲ) obtained in Comparative Example 3; (b) Scanning electron microscope image of β-G@MOF-Fe(Ⅲ) obtained in Comparative Example 4; and (c) Mapping spectrum of MOF-Fe(Ⅲ) obtained in Comparative Example 3.

[0055] Figure 7 (a) XRD spectra of pure MOF obtained in Comparative Example 1, MOF-Fe(Ⅲ) obtained in Comparative Example 3, and β-G@MOF-Fe(Ⅲ) obtained in Comparative Example 4; (b) Infrared spectrum of MOF-Fe(Ⅲ) obtained in Comparative Example 3.

[0056] Figure 8 (a) Preparation process of the composite material MOF-Fe(II) obtained in Comparative Example 5 (under anaerobic conditions); (b) Scanning electron micrograph of the composite material MOF-Fe(II) obtained in Comparative Example 5.

[0057] Figure 9 The XRD spectra of the pure MOF obtained in Comparative Example 1 and the MOF-Fe(II) obtained in Comparative Example 5 are shown.

[0058] Figure 10 (a) is a diagram of the enzymatic hydrolysis process of the enzyme complexes obtained in Examples 1-4 of the present invention; (bg) is a diagram of the enzymatic hydrolysis test performance; (b) Enzyme activity test: substrate cellobiose (buffer system reaction for 24 hours, 50℃~100℃) (the enzyme activity of the free enzyme in the buffer solution at 50℃ for 24 hours is 100% control); (c) Enzyme activity test: substrate cellobiose (ionic liquid reaction for 24 hours, 50℃~130℃) (the enzyme activity of the free enzyme in the ionic liquid at 50℃ for 24 hours is 100% control); (d) Sugar yield of the enzyme complex β-G@MOF-Fe obtained in Examples 1-3 and β-G@MOF obtained in Comparative Example 2 at different times, with cellobiose as the substrate; (e) Sugar yield of the enzyme complex β-G@MOF-Fe obtained in Examples 1-3 and β-G@MOF obtained in Comparative Example 2 at different times, with cellulose as the substrate.

[0059] Figure 11 The images show (a) cycle stability test results for the enzyme complex β-G@MOF-Fe (sample 2) obtained in Example 3 of this invention and β-G@MOF (sample 1) obtained in Comparative Example 2, and (b) storage stability test results for the enzyme complex β-G@MOF-Fe obtained in Example 6 of this invention (24 hours, 120°C ionic liquid, cellobiose as substrate). Detailed Implementation

[0060] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0061] Unless otherwise specified in the embodiments of this invention, the conditions shall be performed according to conventional conditions or conditions recommended by the manufacturer. All raw materials and reagents used, unless otherwise specified, are commercially available conventional products.

[0062] Comparative Example 1

[0063] 9.8 g of copper acetate and 2.4 g of p-aminobenzoic acid were dissolved separately in 500 mL of acetate-sodium acetate buffer solution (pH=7). The solutions were then mixed and incubated at room temperature for 8 h. The resulting solution was centrifuged three times at 9000 rpm for 20 min each time. The solid phase was then filtered and washed with 2 L of sodium acetate solution (pH=5) to obtain a solid MOF. This MOF was dried at 36 °C for 8 h. The dried sample was then ground and stored at room temperature for later use.

[0064] Comparative Example 2

[0065] 9.8 g of copper acetate was dissolved in 500 mL of acetate-sodium acetate buffer solution (pH=7). Then, 2 g of β-glucosidase was added to 10 mL of acetate-sodium acetate buffer solution (pH=7) containing 1.5 g of PVP (40 kDa). The solutions were mixed and sonicated for 15 min. Next, 2.4 g of p-aminobenzoic acid was dissolved in 500 mL of acetate-sodium acetate buffer solution (pH=7), and then added to the mixture. The mixture was stirred for 40 min. The mixture was then allowed to stand at room temperature for 8 h. The solution was centrifuged three times at 9000 rpm for 20 min each time. The solid phase after centrifugation was then filtered and washed with 2 L of sodium acetate solution (pH=5) to obtain the β-G@MOF solid phase. This solid phase was dried at 36 °C for 8 h. The dried sample was then ground and stored at room temperature for later use.

[0066] Comparative Example 3

[0067] 5 g of copper acetate and 1 g of p-aminobenzoic acid were dissolved separately in 500 mL of acetate-sodium acetate buffer solution (pH 7.0), and 0.69 g of ferric sulfate was dissolved in 25 mL of acetate-sodium acetate buffer solution (pH 7.0). The ferric sulfate solution and p-aminobenzoic acid solution were then mixed, followed by the addition of copper acetate solution. The resulting reaction solution was stirred for 30 min, then allowed to stand for 8 h. The mixture was then centrifuged three times at 9000 rpm for 20 min each time. The solid phase after centrifugation was filtered and washed with 2 L of pH 5 sodium acetate solution to obtain the solid phase MOF-Fe(Ⅲ). The sample was dried at 36 °C for 8 h, ground, and stored at room temperature for later use.

[0068] Comparative Example 4

[0069] 0.1 g of β-glucosidase was added to 5 mL of acetate-sodium acetate buffer (pH=7) containing 0.02 g PVP (40 kDa). 1.34 g of ferric sulfate was dissolved in 25 mL of acetate-sodium acetate buffer (pH=7). The ferric sulfate solution was then mixed with the enzyme solution. Next, 1 g of p-aminobenzoic acid was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and this mixture was added to the p-aminobenzoic acid solution. Then, 5 g of copper acetate was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and the copper acetate solution was added to the mixture. The mixture was stirred for 30 min, and then allowed to stand at room temperature for 8 h. The solution was then centrifuged three times at 9000 rpm for 20 min each time. The solid phase after centrifugation was then filtered and washed with 2 L of sodium acetate solution at pH 5 to obtain solid phase β-G@MOF-Fe(Ⅲ). The solid phase was dried at 36℃ for 8 h. The dried sample was then ground and stored at room temperature for later use.

[0070] Comparative Example 5

[0071] 5 g of copper acetate and 1 g of p-aminobenzoic acid were dissolved separately in 500 mL of acetate-sodium acetate buffer solution (pH 7.0). 0.69 g of ferrous sulfate heptahydrate was dissolved in 25 mL of acetate-sodium acetate buffer solution (pH 7.0). The ferrous sulfate heptahydrate solution was then mixed with the p-aminobenzoic acid solution, followed by the addition of copper acetate solution. The resulting reaction solution was stirred for 30 min, then allowed to stand for 8 h. It was then centrifuged three times at 9000 rpm for 20 min each time. The solid phase after centrifugation was then filtered and washed with 2 L of pH 5 sodium acetate solution. All operations were performed in an anaerobic environment to obtain a solid phase MOF-Fe(II). The sample was dried at 36 °C for 8 h, ground, and stored at room temperature for later use.

[0072] Comparative Example 6

[0073] 5 g of copper acetate and 1 g of p-aminobenzoic acid were dissolved separately in 500 mL of acetate-sodium acetate buffer solution (pH 7.0). 0.35 g of ferrous sulfate heptahydrate was dissolved in 25 mL of acetate-sodium acetate buffer solution (pH 7.0). The ferrous sulfate heptahydrate solution was then mixed with the p-aminobenzoic acid solution, followed by the addition of copper acetate solution. The resulting reaction solution was stirred for 30 min, then allowed to stand for 8 h. It was then centrifuged three times at 9000 rpm for 20 min each time. The solid phase after centrifugation was filtered and washed with 2 L of pH 5 sodium acetate solution to obtain a solid phase MOF-Fe (1:0.05). The sample was dried at 36 °C for 8 h, ground, and stored at room temperature for later use.

[0074] Comparative Example 7

[0075] 5 g of copper acetate and 1 g of p-aminobenzoic acid were dissolved separately in 500 mL of acetate-sodium acetate buffer solution (pH 7.0). 0.69 g of ferrous sulfate heptahydrate was dissolved in 25 mL of acetate-sodium acetate buffer solution (pH 7.0). The ferrous sulfate heptahydrate solution was then mixed with the p-aminobenzoic acid solution, followed by the addition of copper acetate solution. The resulting reaction solution was stirred for 30 min, then allowed to stand for 8 h. It was then centrifuged three times at 9000 rpm for 20 min each time. The solid phase after centrifugation was filtered and washed with 2 L of pH 5 sodium acetate solution to obtain a solid phase MOF-Fe (1:0.1). The sample was dried at 36 °C for 8 h, ground, and stored at room temperature for later use.

[0076] Comparative Example 8

[0077] 5 g of copper acetate and 1 g of p-aminobenzoic acid were dissolved separately in 500 mL of acetate-sodium acetate buffer solution (pH 7.0). 1.34 g of ferrous sulfate heptahydrate was dissolved in 25 mL of acetate-sodium acetate buffer solution (pH 7.0). The ferrous sulfate heptahydrate solution was then mixed with the p-aminobenzoic acid solution, followed by the addition of copper acetate solution. The resulting reaction solution was stirred for 30 min, then allowed to stand for 8 h. It was then centrifuged three times at 9000 rpm for 20 min each time. The solid phase after centrifugation was filtered and washed with 2 L of pH 5 sodium acetate solution to obtain a solid phase MOF-Fe (1:0.2). The sample was dried at 36 °C for 8 h, ground, and stored at room temperature for later use.

[0078] Example 1

[0079] 0.1 g of β-glucosidase was added to 5 mL of acetate-sodium acetate buffer (pH=7) containing 0.02 g PVP (40 kDa). 0.35 g of ferrous sulfate heptahydrate was dissolved in 25 mL of acetate-sodium acetate buffer (pH 7.0). The ferrous sulfate heptahydrate solution was then mixed with the enzyme solution. Subsequently, 1 g of p-aminobenzoic acid was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and this mixture was added to the p-aminobenzoic acid solution. Then, 5 g of copper acetate was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and the copper acetate solution was added to the mixture. The mixture was stirred for 30 min, and then allowed to stand at room temperature for 8 h. The solution was then centrifuged three times at 9000 rpm for 20 min each time. The solid phase after centrifugation was then filtered and washed with 2 L of sodium acetate solution at pH 5 to obtain solid phase β-G@MOF-Fe (1:0.05). The solid phase was dried at 36℃ for 8 h. The dried sample was then ground and stored at room temperature for later use.

[0080] Example 2

[0081] 0.1 g of β-glucosidase was added to 5 mL of acetate-sodium acetate buffer (pH=7) containing 0.02 g of PVP (40 kDa). 0.69 g of ferrous sulfate heptahydrate was dissolved in 25 mL of acetate-sodium acetate buffer. The ferrous sulfate heptahydrate solution was then mixed with the enzyme solution. Subsequently, 1 g of p-aminobenzoic acid was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and this mixture was added to the p-aminobenzoic acid solution. Then, 5 g of copper acetate was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and the copper acetate solution was added to the mixture. The mixture was stirred for 30 min, and then allowed to stand at room temperature for 8 h. The solution was then centrifuged three times at 9000 rpm for 20 min each time. The solid phase after centrifugation was then filtered and washed with 2 L of sodium acetate solution at pH 5 to obtain solid phase β-G@MOF-Fe (1:0.1). The solid phase was dried at 36℃ for 8 h. The dried sample was then ground and stored at room temperature for later use.

[0082] Example 3

[0083] 0.1 g of β-glucosidase was added to 5 mL of acetate-sodium acetate buffer (pH=7) containing 0.02 g PVP (40 kDa). 1.34 g of ferrous sulfate heptahydrate was dissolved in 25 mL of acetate-sodium acetate buffer (pH=7). The ferrous sulfate heptahydrate solution was then mixed with the enzyme solution. Subsequently, 1 g of p-aminobenzoic acid was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and this mixture was added to the p-aminobenzoic acid solution. Then, 5 g of copper acetate was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and the copper acetate solution was added to the mixture. The mixture was stirred for 30 min, and then allowed to stand at room temperature for 8 h. The solution was then centrifuged three times at 9000 rpm for 20 min each time. The solid phase after centrifugation was then filtered and washed with 2 L of sodium acetate solution at pH 5 to obtain solid phase β-G@MOF-Fe (1:0.2). The solid phase was dried at 36℃ for 8 h. The dried sample was then ground and stored at room temperature for later use.

[0084] Example 4

[0085] 0.1 g of β-glucosidase was added to 5 mL of acetate-sodium acetate buffer (pH=7) containing 0.02 g of PVP (40 kDa). 2.8 g of ferrous sulfate heptahydrate was dissolved in 25 mL of acetate-sodium acetate buffer. The ferrous sulfate heptahydrate solution was then mixed with the enzyme solution. Subsequently, 1 g of p-aminobenzoic acid was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and this mixture was added to the p-aminobenzoic acid solution. Then, 5 g of copper acetate was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and the copper acetate solution was added to the mixture. The mixture was stirred for 30 min, and then allowed to stand at room temperature for 8 h. The solution was then centrifuged three times at 9000 rpm for 20 min each time. The solid phase after centrifugation was then filtered and washed with 2 L of sodium acetate solution at pH 5 to obtain solid phase β-G@MOF-Fe (1:0.4). The solid phase was dried at 36℃ for 8 h. The dried sample was then ground and stored at room temperature for later use.

[0086] Example 5

[0087] 0.1 g of β-glucosidase was added to 5 mL of acetate-sodium acetate buffer (pH=7) containing 0.02 g of PVP (40 kDa). 1.34 g of ferrous sulfate heptahydrate was dissolved in 25 mL of acetate-sodium acetate buffer, and the ferrous sulfate heptahydrate solution was mixed with the enzyme solution. Then, 1 g of p-aminobenzoic acid was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and the mixture was added to the p-aminobenzoic acid solution. Next, 5 g of copper acetate was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and the copper acetate solution was added to the mixture. The mixture was stirred for 30 min, and then allowed to stand at room temperature for 4 h. The solution was then centrifuged three times at 9000 rpm for 20 min each time. The solid phase after centrifugation was then filtered and washed with 2 L of sodium acetate solution at pH 5 to obtain solid phase β-G@MOF-Fe-4h (1:0.2). The solid phase was dried at 36℃ for 8 h. The dried sample was then ground and stored at room temperature for later use.

[0088] Example 6

[0089] 0.1 g of β-glucosidase was added to 5 mL of acetate-sodium acetate buffer (pH=7) containing 0.02 g of PVP (40 kDa). 1.34 g of ferrous sulfate heptahydrate was dissolved in 25 mL of acetate-sodium acetate buffer, and the ferrous sulfate heptahydrate solution was mixed with the enzyme solution. Then, 1 g of p-aminobenzoic acid was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and the mixture was added to the p-aminobenzoic acid solution. Next, 5 g of copper acetate was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and the copper acetate solution was added to the mixture. The mixture was stirred for 30 min, and then allowed to stand at room temperature for 6 h. The solution was then centrifuged three times at 9000 rpm for 20 min each time. The solid phase after centrifugation was then filtered and washed with 2 L of sodium acetate solution at pH 5 to obtain solid phase β-G@MOF-Fe-6h (1:0.2). The solid phase was dried at 36℃ for 8 h. The dried sample was then ground and stored at room temperature for later use.

[0090] Example 7

[0091] 0.5 g of β-glucosidase was added to 5 mL of acetate-sodium acetate buffer (pH=7) containing 0.02 g PVP (40 kDa). 1.34 g of ferrous sulfate heptahydrate was dissolved in 25 mL of acetate-sodium acetate buffer, and the ferrous sulfate heptahydrate solution was mixed with the enzyme solution. Then, 1 g of p-aminobenzoic acid was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and the mixture was added to the p-aminobenzoic acid solution. Next, 5 g of copper acetate was dissolved in 500 mL of acetate-sodium acetate buffer (pH=7), and the copper acetate solution was added to the mixture. The mixture was stirred for 30 min, and then allowed to stand at room temperature for 10 h. The solution was then centrifuged three times at 9000 rpm for 20 min each time. The solid phase after centrifugation was then filtered and washed with 2 L of sodium acetate solution at pH 5 to obtain solid phase β-G@MOF-Fe-10h (1:0.2). The solid phase was dried at 36℃ for 8 h. The dried sample was then ground and stored at room temperature for later use.

[0092] Test conditions:

[0093] The enzyme activity assay method is as follows: Control group: Prepare 1 mL buffer system (pH 4.8, 50 mmol / L). -1 The enzyme concentration is approximately 10 μg / mL. -1 The substrate (cellobiose or cellulose) concentration is approximately 5 mg / mL. -1 The system was stirred in a constant temperature incubator at 50°C for 24 hours. Glucose content was measured after incubation, and all tests were repeated three times.

[0094] During the enzymatic digestion test, the enzyme concentration is approximately 10 μg / mL in 1 mL buffer solution or ionic liquid. -1 The substrate (cellobiose or cellulose) concentration is approximately 10 mg / mL. -1 The enzyme was incubated in an oven at 50–130°C for 24 hours. After incubation, the glucose content was measured, and all tests were repeated three times. Specific reaction temperatures and times are recorded in subsequent test results. In the enzyme activity assay, the amount of free enzyme added was equal to the amount of enzyme encapsulated in the enzyme complex. The encapsulation rate was the percentage of the β-G content embedded in the MOFs to the initial β-G content, and the encapsulation capacity was the ratio of the β-G content encapsulated in the MOFs to the mass of the enzyme complex.

[0095] Table 1 shows the pore structure parameters of Comparative Examples 1-2, 6-8, and Examples 1-4.

[0096]

[0097]

[0098] Table 2 is a summary table of performance data for Comparative Example 2 and Examples 1-3.

[0099]

[0100] The preparation process of the macroporous enzyme complex of the present invention is as follows: Figure 1 As shown, during the encapsulation process, the enzyme was dispersed in an aqueous solution containing iron, then metal ions (copper acetate) and an organic ligand (PABA) were added, followed by in-situ co-precipitation to synthesize β-G@MOF-Fe (Examples 1-4). Figure 2 As can be clearly seen above, although the enzyme complex β-G@MOF-Fe 1:0.2 (Example 3) prepared in this invention exhibits a similar flower-like morphology to β-G@MOF (Comparative Example 2), Example 3 has a distinct macroporous structure, indicating that the metal competition and oxidation pore-forming strategy proposed in this invention can induce the appearance of a macroporous structure. Figure 3 a in the figure proves that different molar ratios of Fe are added 2+ Subsequently, the obtained composite materials β-G@MOF-Fe (Examples 1-4) exhibited typical type IV and type H hysteresis curves at P / P0 = 0.5–0.9, indicating that the composite material β-G@MOF-Fe prepared in this invention is a macroporous material. Furthermore, Density-Functional-Theory (DFT) calculations further verified that after the addition of Fe, the number of mesopores (20–50 nm) and macropores (50–100 nm) in β-G@MOF-Fe (Examples 1-4) significantly increased, especially the macropores in β-G&MOF-Fe (1:0.2) reaching 90 nm. Figure 3 (b) Furthermore, Table 1 shows that after adding Fe, the mesoporous properties of the enzyme complexes (71.6%, 63.4%, 71.0%, and 64.7%) were all greater than those of the original enzyme complex β-G@MOF (57.8%). The macroporous properties also increased significantly compared to pure MOFs, especially the β-G&MOF-Fe (1:0.2), which had a macroporous property as high as 13.5% (Table 1). This macroporous structure is not only beneficial for the transport of substrates and reactants, but the larger pore size also facilitates the exposure of other pores, resulting in a larger distribution area of ​​enzyme active sites. These findings collectively indicate that Fe… 2+ The introduction of Fe can induce macroporous structures in MOFs, and by controlling Fe 2+ The amount of [agent] added can adjust the pore size of the composite material in the range of 50–100 nm.

[0101] Figure 4 and Figure 5To further explore the formation mechanism of macropores in composite materials, high-resolution N1s spectroscopy was used to study the state of nitrogen species. The results showed that compared to pure MOF (Comparative Example 1), the N1s peaks of MOF-Fe (Comparative Examples 6-8) and β-G@MOF-Fe (Examples 1-4) shifted to lower binding energies (399.6 eV → 399.3 eV and 399.6 eV → 399.5 eV, respectively). Figure 4 b and Figure 5 (b) This confirms that iron coordinated with some nitrogen atoms, thus creating new Fe atoms. 2+ -N bond formation. Furthermore, the Cu 2p binding energy of both MOF-Fe and β-G@MOF-Fe shifts to lower positions after the addition of Fe, indicating that the competitive coordination between Fe and Cu alters the original Cu coordination, leading to Fe... 2+ The formation of -N weakens the coordination between Cu and N. Figure 4 and Figure 5 The Fe 2p peaks are located near 711 eV (2p³ / 2) and 724 eV (2p¹ / 2). Figure 4 and Figure 5 This belongs to the typical octahedral coordination of Fe(III). To clarify Fe... 2+ The unique role of competitive coordination with Cu in the formation of defect macropores led to the design of another experiment to utilize Fe. 2+ Replace with Fe 3+ MOF-Fe(III) was prepared. SEM images showed that both MOF-Fe(III) and the enzyme complex β-G@MOF-Fe(III) exhibited similar flower-like morphology, but no macroporous structure was formed. Figure 6 The SEM images show that adding Fe(III) does not change the crystal size; both MOF-Fe(III) (1:0.1) and pure MOF are around 13 nm, while the crystal size of MOF-Fe(1:0.1) is 22 nm, indicating that Fe... 3+ The addition of Fe does not change the crystal growth orientation. The XRD pattern shows that the diffraction peaks of MOF-Fe(III) perfectly match those of pure MOF, indicating that Fe... 3+ The addition of [a substance] did not affect the crystal structure of the MOF. Figure 7 (a) Furthermore, the infrared spectrum shows no Fe-N formation, indicating that Fe... 3+ It does not compete with Cu for coordination, therefore it cannot form a macroporous structure. Figure 7 (b) Secondly, in order to investigate Fe 2+ Oxidized to Fe 3+ It also plays a key role in the formation of defect structures in MOFs, and is essential for the preparation of MOF-Fe(II) under anaerobic conditions. Figure 8(a) In an anaerobic environment, Fe is added. 2+ During the preparation of the complex, the solution remained green throughout the entire process, indicating that Fe... 2+ No valence state transformation occurred. However, as can be seen from the SEM image, although MOF-Fe(II) also exhibits a flower-like morphology, no macroporous structure was formed. Figure 8 In section b), the structure of the crystalline material also remained unchanged. Figure 9 Therefore, it is reasonable to say that the formation of the defective macroporous structure is regulated in two stages: (1) during the preparation of the composite material by Fe 2+ (2) Fe forms Fe-N bonds through competitive coordination interactions with Cu; 2+ To Fe 3+ Oxidation leads to the breakage of coordination bonds, forming defects and macropores.

[0102] Figure 10 This is a graph showing the enzymatic hydrolysis performance of the samples in this invention. Figure 10 In this context, 'a' refers to the hydrolysis catalytic process in Examples 1-4. For example... Figure 10 As shown in b, comparing the enzyme complex β-G@MOF-Fe with β-G@MOF after the addition of Fe, the enzyme activity increased with increasing temperature. The enzyme activity of β-G@MOF-Fe (1:0.05~1:0.2) was higher than that of β-G@MOF. Among them, the enzyme activity of β-G@MOF-Fe (1:0.2) at 100℃ was 2.8 times that of the aqueous free enzyme β-G at 50℃, and 1.4 times that of β-G@MOF at 100℃. This is mainly because the increased mesopores facilitate the mass transfer of cellobiose, which is consistent with the results of BTE testing. Figure 3 The above results preliminarily indicate that the crystal structure of the β-G@MOF-Fe (1:0.05~1:0.2) enzyme complex prepared in this invention can be maintained at high temperatures. Figure 10 As observed in section c, the β-G@MOF-Fe prepared in this invention not only exhibits excellent resistance to ionic liquids but also demonstrates good high-temperature resistance in ionic liquids. Compared to β-G@MOF, the enzyme complex β-G@MOF-Fe (1:0.1 to 1:0.2) can withstand temperatures up to 120°C in ionic liquids. This enhanced thermal stability is attributed to the formation of Fe-N bonds in β-G@MOF-Fe. To demonstrate the superior macroporous properties of the enzyme complex prepared in this invention, the catalytic performance of samples with different pore structures was tested in the same system. Figure 10As shown in d, β-G@MOF-Fe (1:0.05–1:0.2) reached enzymatic equilibrium after 20 hours of enzymatic hydrolysis, which is faster and faster than β-G@MOF. These results indicate that the larger pores generated in the currently constructed β-G@MOF-Fe result in a faster substrate diffusion rate. Furthermore, when using cellulose as a substrate, the enzymatic hydrolysis efficiency of β-G@MOF-Fe (1:0.1–1:0.2), with a significantly increased macropore size (50–100 nm), continued to increase with time. Figure 10 (e). Among them, β-G@MOF-Fe (1:0.2) achieved a cellulose conversion rate as high as 90% after 64 hours of enzymatic hydrolysis, while β-G@MOF and β-G@MOF-Fe (1:0.05), whose pore size did not increase in the 50-100 nm range, reached a dynamic equilibrium in enzymatic hydrolysis efficiency after 36 hours of reaction (Table 2). This indicates that the increase in macropores in the 50-100 nm range of the enzyme complex has a significant enhancing effect on the mass transfer of the macromolecular substrate cellulose. Figure 11 The stability of this product was demonstrated. The macroporous enzyme complex β-G@MOF-Fe1:0.2 (Example 3) could be easily separated by centrifugation and reused at least five times, while β-G@MOF essentially lost its activity, indicating that β-G@MOF-Fe has higher tolerance. This is because the rigid organic framework constructed by the Fe-N bonds formed by the competitive coordination of Fe and Cu maintains the conformation of the enzyme protein without change. Furthermore, long-term stability is also a key characteristic of the enzyme complex. After 50 days of storage, the enzyme complex β-G@MOF-Fe1:0.2 (Example 3) showed a glucose yield of over 85%. Figure 11 (b) The results above show that the macroporous enzyme complex β-G@MOF-Fe constructed through competitive coordination and oxidative pore-forming exhibits excellent enzyme catalytic performance and stability.

[0103] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a macroporous enzyme complex bG@MOF-Fe, characterized in that, Includes the following steps: Soluble ferrous salt, β-glucosidase, benzoic acid compounds and buffer solution were mixed evenly to obtain a mixed solution. Soluble copper salt was then added, mixed evenly, and allowed to stand at room temperature. Solid-liquid separation was performed, and the solid phase was washed to obtain macroporous enzyme complex. The molar ratio of ferrous iron in the soluble ferrous salt to copper in the soluble copper salt is 0.1–0.2:1; The mass ratio of the soluble ferrous salt, β-glucosidase, benzoic acid compounds, and soluble copper salt is 0.35–1.34:0.1:1:

5. The mass ratio of the soluble ferrous salt to the volume ratio of the buffer solution is 1 g: 300-3000 mL; the buffer solution is an acetate-sodium acetate buffer solution with a pH of 6-8. In the benzoic acid compound solution, the mass ratio of the benzoic acid compound to the volume ratio of the acetate-sodium acetate buffer solution is 1g:100-1000mL; The mixed solution also contains polyvinylpyrrolidone, and the mass ratio of β-glucosidase to polyvinylpyrrolidone is 1:0.1 to 0.5; The mixing time for uniform mixing is 0.5–3 h; the settling time is 1–12 h; and the room temperature refers to 10–40°C. The soluble ferrous salt is at least one of ferrous nitrate and ferrous sulfate; The benzoic acid compound is p-aminobenzoic acid; The soluble copper salt is at least one of copper acetate and copper nitrate.

2. The method for preparing the macroporous enzyme complex bG@MOF-Fe according to claim 1, characterized in that, The molecular weight of the polyvinylpyrrolidone is 40–120 kDa.

3. The method for preparing the macroporous enzyme complex bG@MOF-Fe according to claim 1, characterized in that, The washing solid phase was washed with an acetate-sodium acetate buffer solution with a pH of 4.5–6.5, and the resulting bG@MOF-Fe enzyme complex was dried at 25–40 °C for 1–14 h.

4. The method for preparing the macroporous enzyme complex bG@MOF-Fe according to claim 1, characterized in that, Includes the following steps: (1) Dissolve soluble ferrous salt, β-glucosidase and polyvinylpyrrolidone in a buffer solution to obtain mixed solution 1; (2) Mix the benzoic acid compound or benzoic acid compound solution with mixed solution 1 to obtain mixed solution 2; (3) Mix the soluble copper salt or soluble copper salt solution with mixed solution 2 to obtain a mixed solution. Let it stand at room temperature, then separate the solid and liquid phases and wash the solid phase to obtain the macroporous enzyme complex. In step (1), the mass ratio of the soluble ferrous salt to the volume ratio of the buffer solution is 1g:10-86mL.

5. A macroporous enzyme complex bG@MOF-Fe prepared by the preparation method according to any one of claims 1 to 4.

6. The application of the macroporous enzyme complex bG@MOF-Fe as described in claim 5 in the enzymatic hydrolysis of cellulose.

7. The application of the macroporous enzyme complex bG@MOF-Fe according to claim 6 in the enzymatic hydrolysis of cellulose, characterized in that, The macroporous enzyme complex bG@MOF-Fe was added to an ionic liquid or buffer solution with cellulose or cellobiose as the substrate, and reacted at 50–130 °C to obtain a saccharified hydrolysate. The enzymatic hydrolysate was separated from the solid and liquid and then cooled. The macroporous enzyme complex bG@MOF-Fe was then centrifuged and recycled. The mass ratio of enzyme to cellulose or cellobiose in the macroporous enzyme complex bG@MOF-Fe is 0.1–5 mg:1 g; The ionic liquid is at least one of imidazole acetate, imidazole ethyl sulfate, imidazole chlorate, imidazole sulfonate, and imidazole sulfate. The macroporase complex bG@MOF-Fe is added to an ionic liquid or buffer solution with cellulose or cellobiose as a substrate and reacted at 50–130 °C, wherein the concentration of the substrate is 2–20 mg / mL.

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