A hierarchical encapsulated dual-enzyme MOFs composite and a preparation method and application thereof

Abstract

The application discloses a hierarchical encapsulated double-enzyme MOFs compound and a preparation method and application thereof, and belongs to the field of high-temperature catalysis of enzymes.The GI is encapsulated in ZIF-67 by an in-situ co-precipitation method to form GI@ZIF-67, and GA@IRMOF-9 is in-situ grown on the outer layer by taking the GI@ZIF-67 as a crystal nucleus to form GI@ZIF-67-GA@IRMOF-9;different MOFs have poor tolerance to acid and alkali environments, and a hierarchical encapsulated GI-GA@IRMOF-9 enzyme compound with high enzyme activity and high mass transfer performance is formed by etching ZIF by adding a dilute acid solution.The obtained enzyme compound can directly realize efficient conversion from maltodextrin to fructose at high temperature, and can provide inspiration for design and construction of a multi-enzyme encapsulation carrier.

Description

Technical Field

[0001] This invention belongs to the field of high-temperature enzyme catalysis, specifically involving the preparation of a dual-enzyme@MOFs complex with a hierarchical encapsulation structure through an acid hydrolysis strategy, which enables the efficient catalytic conversion of maltodextrin into fructose through a cascade reaction of dual enzymes at high temperatures. Background Technology

[0002] Among all nutritional sweeteners, sucrose is one of the most important. However, excessive sucrose intake can lead to obesity, dental disease, diabetes, and even accelerate cell aging, calcium deficiency, and osteoporosis. Therefore, excessive sucrose intake is considered a significant health risk factor, especially for diabetics. Fructose, a natural monosaccharide, is 1.2 to 1.8 times sweeter than sucrose and is widely found in fruits and honey. Fructose metabolism does not require insulin, so diabetics can maintain normal metabolism by consuming fructose. Furthermore, oral microorganisms generally do not use fructose as a fermentation substrate, making it potentially helpful in preventing dental diseases in infants and young children. Therefore, from a health perspective, fructose is a more likely and effective alternative to sucrose. It is a nutritional sweetener recognized by the medical community both domestically and internationally for use by diabetics and those with hypoglycemia, and is gradually replacing sucrose as a sweetener in beverage and other food production. Currently, the main methods for industrial fructose production both domestically and internationally utilize starch-rich crops such as corn and rice as raw materials for deep processing, involving amylase, saccharifying enzyme (GA), and glucose isomerase (GI). First, amylase hydrolyzes and liquefies the starch, then saccharifying enzyme hydrolyzes the starch emulsion into glucose, and finally, glucose isomerase converts the glucose into fructose. Since the optimal reaction temperatures for industrial amylase, saccharifying enzyme, and glucose isomerase are 100–120℃, 50–60℃, and 80–90℃ respectively, the reaction process requires continuous temperature adjustment to increase the reaction rate. If the optimal reaction temperatures of all three enzymes could be unified at 90–100℃, not only would the reaction rate be accelerated, but the cumbersome temperature control in production would also be avoided. Among the three enzymes, amylase itself is a thermophilic enzyme, and its reaction with starch is a solid-liquid reaction; therefore, increasing the reaction temperatures of GI and GA becomes crucial for improving efficiency.

[0003] Enzyme immobilization is a simple and effective strategy to improve enzyme activity, stability, and selectivity. It not only facilitates separation and continuous operation but also enhances enzyme stability. Metal-organic frameworks (MOFs), which form three-dimensional network structures through the coordination of metal cations or clusters with organic ligands, offer advantages such as high selectivity, high specific surface area, customizable pore size, high stability, and reduced loss of catalytic activity in immobilized enzymes. After in-situ encapsulation of enzymes with MOFs, their rigid and ordered framework structure protects the conformational stability of the enzyme under extreme conditions, enabling efficient catalysis even at high temperatures, in ionic liquids, or in organic solvents. For example, Liao et al. (Journal of the American Chemical Society, 2017) encapsulated catalase (CAT) in ZIF-8 and then exposed both the encapsulated CAT and free CAT to a denaturing agent (urea) and high temperature (80°C). The results showed that the encapsulated CAT maintained its biological function under urea and at 80°C, while no activity was detected in the free CAT. He et al. (ACS Applied Materials & Interfaces, 2016) encapsulated thermophilic lipase (QLM) into ZIF-8. QLM@ZIF-8 exhibited tolerance to high temperatures and pH, retaining 63% activity after 5 minutes at 70°C. Feng et al. (Advanced Materials, 2019) used biocompatible ZIF-8 and ZIF-90 for rapid loading and efficient release of antibodies, achieving stability during preparation, transportation, and storage. After reacting at 75°C for 20 minutes, the antibody@ZIF retained over 90% activity, and its activity remained above 85% even after repeated freeze-thaw cycles in acetone, methanol, and temperatures ranging from -80°C to 37°C. Furthermore, studies have shown that constructing cascade reactors by encapsulating multiple enzymes using MOFs can achieve good catalytic effects. For example, Wu et al. (Chemical Communications, 2015) immobilized glucose oxidase (GOx) and horseradish peroxidase (HRP) on metal-organic framework (MOF) carriers using an encapsulation method. The immobilized dual-enzyme@MOF composite material exhibited high catalytic efficiency, high selectivity, and enhanced thermal stability. Du et al. (Bioconjugate Chemistry, 2017) immobilized D-amino acid oxidase and catalase on ZIF-8 material using an in-situ aqueous co-precipitation method with MOFs. The immobilized dual-enzyme@MOF material increased the optimal enzymatic hydrolysis temperature by 30 °C. Zhou et al. (Chemical Science, 2016) encapsulated horseradish peroxidase and glucose oxidase in PNC-888, a hierarchical porous material with three pore sizes (2, 5, and 6.2 nm), achieving sequential immobilization of enzyme molecules and demonstrating high catalytic efficiency in enzymatic hydrolysis experiments.

[0004] Therefore, simultaneously immobilizing GI and GA enzymes in the same MOF material and constructing a dual-enzyme cascade reactor can not only achieve efficient enzymatic hydrolysis at high temperatures, but also enable the two enzymes to continuously, stably, and efficiently catalyze the reaction, thus achieving rapid and efficient fructose preparation.

[0005] Although MOF immobilized multi-enzyme technology has made some progress, the activity of immobilized enzymes is usually still lower than that of free enzymes, and the enzymatic hydrolysis efficiency is greatly affected by the pore size of MOFs. Substrate-pore size mismatch will seriously affect the mass transfer rate. Summary of the Invention

[0006] To address the shortcomings and deficiencies of existing technologies, the primary objective of this invention is to provide a method for preparing a layered encapsulated dual-enzyme @MOF complex. GI is encapsulated in ZIF-67 via in-situ co-precipitation to form GI@ZIF-67, which serves as the nucleus for in-situ growth of GA@IRMOF-9 on the outer layer, forming GI@ZIF-67-GA@IRMOF-9. Utilizing the differences in acid and alkali tolerance among different MOFs, a layered encapsulated GI-GA@IRMOF-9 enzyme complex with both high enzyme activity and high mass transfer performance is formed by etching the ZIF with a dilute acid solution, leveraging the differences in the tolerance of different MOFs to acid and alkali environments. The resulting enzyme complex can achieve the conversion of maltodextrin into fructose through a cascade reaction at high temperatures.

[0007] First, larger biphenyl ligands were selected to synthesize IRMOF-9 material, increasing the intrinsic pore size of MOFs and improving the mass transfer performance of maltodextrin substrates. Furthermore, GA was in-situ immobilized during the preparation of IRMOF-9 to enhance its temperature resistance. Second, addressing the issue that GI cannot tolerate the influence of zinc salts on GI activity during the in-situ immobilization of IRMOF-9, making simultaneous encapsulation with IRMOF-9 difficult, GI was first protectively encapsulated in ZIF-67, and then simultaneously encapsulated with GA. This is because cobalt salts do not inhibit GI activity but rather promote it. Finally, to overcome the mass transfer difficulties of GI encapsulated in a ZIF-67 / IRMOF-9 bilayer, taking advantage of GI's good temperature resistance and the fact that it does not require in-situ immobilization, ZIF-67 was etched under dilute acid conditions, allowing GI to exist freely in the hollow structure. The disintegration of ZIF-67 releases cobalt ions that promote GI enzyme activity, thus forming a hierarchical encapsulation structure of a dual-enzyme@MOFs complex with both high enzyme activity and high mass transfer performance.

[0008] Another object of the present invention is to provide a layered encapsulated dual-enzyme@MOFs complex prepared by the above preparation method.

[0009] Another object of the present invention is to provide the application of the above-mentioned layered encapsulation of dual enzymes@MOFs complex.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] A method for preparing a hierarchically encapsulated dual-enzyme@MOFs complex includes the following steps:

[0012] (1) Mix soluble cobalt salt, glucose isomerase and water evenly to obtain solution A; dissolve methylimidazolium compound in water to obtain solution B; mix solutions A and B to obtain mixed solution; after mixing evenly, incubate at room temperature, then separate solid and liquid, wash the solid phase with water to obtain GI@ZIF-67;

[0013] (2) Mix soluble zinc salt, saccharifying enzyme and water evenly to obtain solution C; dissolve biphenyl acid compound in water to obtain solution D; mix GI@ZIF-67, solution C and D, mix evenly and incubate at room temperature, then separate solid and liquid, wash the solid phase with water to obtain GI@ZIF-67-GA@IRMOF-9;

[0014] (3) Add GI@ZIF-67-GA@IRMOF-9 to dilute hydrochloric acid solution, let it stand at room temperature, then separate the solid and liquid phases, wash the solid phase with water to obtain GI-GA@IRMOF-9.

[0015] Preferably, the mass ratio of the soluble cobalt salt, glucose isomerase and methylimidazolium compound in step (1) is 0.3-1.5:0.03-0.5:1.2-3.6; more preferably, it is 0.7-0.9:0.05-0.10:2.2-3.4; and most preferably, it is 0.8:0.05-0.10:2.2-3.4.

[0016] Preferably, the methylimidazolium compound in step (1) is at least one of 2-methylimidazolium and 4-methylimidazolium; and the soluble cobalt salt is at least one of cobalt sulfate, cobalt nitrate and cobalt chloride.

[0017] Preferably, the concentration of soluble cobalt salt in solution A in step (1) is 26–130 mg / ml; more preferably, it is 79 mg / ml.

[0018] Preferably, the concentration of methylimidazolium compounds in solution B in step (1) is 110–170 mg / ml; more preferably, it is 112 mg / ml.

[0019] Preferably, the culture time in steps (1) and (2) is 8 to 24 hours; more preferably, it is 24 hours.

[0020] Preferably, the mixing in steps (1) and (2) refers to stirring for 30 to 60 minutes; the liquid-solid separation in steps (1) and (2) involves filtration for 10 to 30 minutes each time, and filtration is performed 3 to 5 times; the solid phase is washed with water 3 to 5 times.

[0021] Preferably, the mass ratio of the soluble zinc salt, saccharifying enzyme, biphenyl acid compound and GI@ZIF-67 in step (2) is 0.1-0.7:0.03-0.5:0.5-2.4:0.03-0.6; more preferably, it is 0.5-0.6:0.05-0.10:0.7-1.4:0.05-0.20; and most preferably, it is 0.54:0.05-0.10:0.7-1.4:0.05-0.15.

[0022] Preferably, the soluble zinc salt in step (2) is at least one of zinc acetate and zinc nitrate.

[0023] Preferably, the biphenyl acid compound in step (2) is at least one of 4,4'-biphenyldicarboxylic acid and 2,2'-biphenyldicarboxylic acid.

[0024] Preferably, the concentration of soluble zinc salt in solution C in step (2) is 10-70 mg / ml; more preferably, it is 54 mg / ml.

[0025] Preferably, the concentration of biphenyl acid compounds in solution D in step (2) is 36–72 mg / ml; more preferably, it is 36 mg / ml.

[0026] Preferably, the specific steps for mixing GI@ZIF-67, solution C and D in step (2) are as follows: first mix GI@ZIF-67 and solution C, and then add it dropwise to solution D.

[0027] Preferably, the pH value of the dilute hydrochloric acid solution in step (3) is 5.5 to 6; more preferably, it is 5.5.

[0028] Preferably, the settling time in step (3) is 0.5 to 3 hours; more preferably, it is 1 hour.

[0029] Preferably, the solids obtained in steps (1), (2) and (3) are dried at 32–44°C for 4–16 hours.

[0030] The above preparation method yields a layered encapsulated dual-enzyme@MOFs complex.

[0031] The above-mentioned hierarchical encapsulation of a dual-enzyme@MOFs complex is used to realize the catalytic hydrolysis of maltodextrin in a dual-enzyme cascade reaction.

[0032] Preferably, the catalytic reaction temperature for the hydrolysis of maltodextrin by the dual-enzyme cascade reaction is 50–100°C; more preferably, it is 70–90°C.

[0033] The mechanism of this invention is as follows:

[0034] The enzymatic hydrolysis performance of enzyme@MOF composites is greatly affected by the substrate mass transfer performance. Appropriate MOFs are selected for enzyme immobilization based on substrate size. This invention employs a synergistic method of acid etching ZIF-67 and increasing ligand size to improve the mass transfer performance of MOFs. Different MOFs exhibit varying acid and alkali stability. ZIF-67's acid instability causes its structure to collapse and dissolve rapidly under dilute acid conditions. Cobalt ions and methylimidazole, due to their small molecular radii, easily diffuse outside the MOFs or re-immobilize in the larger IRMOF-9 ligand. However, the enzyme's larger molecular structure makes diffusion difficult, ultimately remaining within the internal cavity of the MOFs. This forms an outer IRMOF-9 layer encapsulating the saccharifying enzyme, while the inner hollow structure encapsulates the free glucose isomerase, thus enhancing mass transfer performance.

[0035] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0036] 1. The raw materials used in this invention are simple and readily available, the preparation cost is low, the reaction conditions are mild, and the operation is simple and efficient; the activities of glucose isomerase and glucoamylase can be well maintained in extreme environments;

[0037] 2. The outer biphenyl ligand of the enzyme complex material prepared by this invention makes the intrinsic pore size larger, and the hollow inner structure can reduce mass transfer resistance, promote the interaction between enzyme and substrate molecules, and provide a more convenient channel for the transport of substances.

[0038] 3. The enzyme complex material prepared by this invention can achieve the orderly degradation of maltodextrin, enzymatically hydrolyzing maltodextrin into glucose in the outer layer and converting glucose into fructose in the inner layer;

[0039] 4. The outer layer of the enzyme complex material prepared by this invention has poor temperature tolerance to saccharifying enzymes. MOFs are used for in-situ fixation to keep its structure stable at high temperatures.

[0040] 5. This invention constructs a layered encapsulated GI-GA@IRMOF-9 inner hollow composite material, realizing the cascade reaction from maltodextrin to fructose; and after acid etching of ZIF-67, the mesopores and macropores of the hollow GI-GA@IRMOF-9 structure are significantly improved, the mass transfer performance is increased, and the fructose production reaches 1.82 mg / mL at 90℃. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the synthesis of GI-GA@IRMOF-9 as described in the example.

[0042] Figure 2SEM comparison images of GI-GA@IRMOF-9-1(f) prepared in Example 1, GI-GA@ZIF-67(a) prepared in Comparative Example 1, GI-GA@IRMOF-9(b) prepared in Comparative Example 2, GI@ZIF-67(c)+GA@ZIRMOF-9(d) prepared in Comparative Example 3, and GI@ZIF-67-GA@IRMOF-9(e) prepared in Comparative Example 4.

[0043] Figure 3 The XRD pattern is a comparison of GI-GA@IRMOF-9-1 prepared in Example 1 and GI@ZIF-67-GA@IRMOF-9 prepared in Comparative Example 4.

[0044] Figure 4 A comparison of the pore size distribution of GI-GA@IRMOF-9-1 prepared in Example 1, GI@ZIF-67+GA@IRMOF-9 prepared in Comparative Example 3, and GI@ZIF-67-GA@IRMOF-9 prepared in Comparative Example 4.

[0045] Figure 5 The enzymatic hydrolysis comparison diagrams of GI-GA@IRMOF-9-1 prepared in Example 1, GI-GA@IRMOF-9-2 prepared in Example 2, GI-GA@IRMOF-9-3 prepared in Example 3, GI-GA@IRMOF-9-4 prepared in Example 4, and GI-GA@IRMOF-9-5 prepared in Example 5, compared with GI-GA@ZIF-67 prepared in Comparative Example 1, GI-GA@IRMOF-9 prepared in Comparative Example 2, GI@ZIF-67+GA@IRMOF-9 prepared in Comparative Example 3, and GI@ZIF-67-GA@IRMOF-9 prepared in Comparative Example 4 at different temperatures are shown in Figure a, where glucose content and fructose content are shown in Figure b. Detailed Implementation

[0046] 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.

[0047] 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.

[0048] Comparative Example 1

[0049] Weigh out 0.79 g of CoSO4·7H2O and dissolve it in 8 mL of water to obtain a cobalt sulfate solution; weigh out 2.24 g of 2-methylimidazole and dissolve it in 20 mL of water to obtain a 2-methylimidazole solution. After both are completely dissolved, dissolve 50 mg of GI and 50 mg of GA in 2 mL of water to obtain a GI and GA enzyme solution. Then, slowly add the GI and GA enzyme solution dropwise to the cobalt sulfate solution and mix thoroughly. Then, slowly add the cobalt sulfate, GI, and GA enzyme mixture dropwise to the 2-methylimidazole solution and mix evenly. Stir at room temperature for 30 min and then incubate at room temperature for 24 h. After incubation, filter the mixture for liquid-solid separation, wash three times with water to obtain a purple solid. Dry the solid and grind it thoroughly into powder (GI-GA@ZIF-67) and store it at room temperature for later use.

[0050] Comparative Example 2

[0051] Weigh 0.54g of zinc nitrate and dissolve it in 8mL of water to obtain a zinc nitrate solution; weigh 0.72g of 4,4'-biphenyl dicarboxylic acid and dissolve it in 20mL of water to obtain a 4,4'-biphenyl dicarboxylic acid solution. After both are completely dissolved, dissolve 50mg of GI and 50mg of GA in 2mL of water to obtain a GI / GA mixed enzyme solution. Then, slowly add the GI / GA mixed enzyme solution dropwise to the zinc nitrate solution and mix thoroughly. Then, slowly add the zinc nitrate, GI, and GA enzyme mixed solution dropwise to the 4,4'-biphenyl dicarboxylic acid solution and mix evenly. Stir at room temperature for 30min and then incubate at room temperature for 24h. After incubation, filter the mixture for liquid-solid separation, wash three times with water to obtain a white block. Dry the solid and grind it thoroughly into powder (GI-GA@IRMOF-9), and store it at room temperature for later use.

[0052] Comparative Example 3

[0053] Weigh 0.79 g of CoSO4·7H2O and dissolve it in 8 mL of water to obtain a cobalt sulfate solution; weigh 2.24 g of 2-methylimidazole and dissolve it in 20 mL of water to obtain a 2-methylimidazole solution. After both are completely dissolved, dissolve 100 mg of GI in 2 mL of water to obtain a GI enzyme solution. Then, slowly add the GI enzyme solution dropwise to the cobalt sulfate solution and mix thoroughly. Then, slowly add the cobalt sulfate and GI enzyme mixture dropwise to the 2-methylimidazole solution and mix well. Stir at room temperature for 30 min, and then incubate at room temperature for 24 h. After incubation, filter the mixture for liquid-solid separation, wash three times with water to obtain a purple solid. Dry the solid and grind it thoroughly into powder (GI@ZIF-67), and store it at room temperature for later use.

[0054] Weigh 0.54g of zinc nitrate and dissolve it in 8mL of water to obtain a zinc nitrate solution; weigh 0.72g of 4,4'-biphenyldicarboxylic acid and dissolve it in 20mL of water to obtain a 4,4'-biphenyldicarboxylic acid solution. After both are completely dissolved, dissolve 100mg of GA in 2mL of water to obtain a GA enzyme solution. Then, slowly add the GA enzyme solution dropwise to the zinc nitrate solution and mix thoroughly. Then, slowly add the zinc nitrate and GA enzyme mixture dropwise to the 4,4'-biphenyldicarboxylic acid solution and mix evenly. Stir at room temperature for 30min, and then incubate at room temperature for 24h. After incubation, filter the mixture for liquid-solid separation, wash three times with water to obtain a white block. Dry the solid and grind it thoroughly into powder (GA@IRMOF-9), and store it at room temperature for later use.

[0055] Take GA@IRMOF-9 and GI@ZIF-67 in a mass ratio of 2.73:1, mix them evenly at room temperature and set aside.

[0056] Comparative Example 4

[0057] Weigh 0.79 g of CoSO4·7H2O and dissolve it in 8 mL of water to obtain a cobalt sulfate solution; weigh 2.24 g of 2-methylimidazole and dissolve it in 20 mL of water to obtain a 2-methylimidazole solution. After both are completely dissolved, dissolve 50 mg of GI in 2 mL of water to obtain a GI enzyme solution. Then, slowly add the GI enzyme solution dropwise to the cobalt sulfate solution and mix thoroughly. Then, slowly add the cobalt sulfate and GI enzyme mixture dropwise to the 2-methylimidazole solution and mix evenly. Stir at room temperature for 30 min, and then incubate at room temperature for 24 h. After incubation, filter the mixture for liquid-solid separation, wash three times with water to obtain a purple solid. Dry the solid and grind it thoroughly into powder (GI@ZIF-67), and store it at room temperature for later use. Weigh 0.54g of zinc nitrate and dissolve it in 8mL of water to obtain a zinc nitrate solution; weigh 0.72g of 4,4'-biphenyldicarboxylic acid and dissolve it in 20mL of water to obtain a 4,4'-biphenyldicarboxylic acid solution. After both are completely dissolved, dissolve 50mg of GA in 2mL of water to obtain a GA enzyme solution. Then, slowly add the GA enzyme solution dropwise to the zinc nitrate solution to obtain a mixed solution of zinc nitrate and GA enzyme. Disperse GI@ZIF-67 evenly into the mixed solution of zinc nitrate and GA enzyme, mix thoroughly, and then slowly add the mixed solution of GI@ZIF-67, zinc nitrate, and GA enzyme to the 4,4'-biphenyldicarboxylic acid solution. Mix well, stir at room temperature for 30min, and then incubate at room temperature for 24h. After incubation, filter the mixture for liquid-solid separation, wash three times with water to obtain a white block. Dry the solid and grind it thoroughly into powder (GI@ZIF-67-GA@IRMOF-9), and store it at room temperature for later use.

[0058] Example 1

[0059] Weigh 0.79 g of CoSO4·7H2O and dissolve it in 8 mL of water to obtain a cobalt sulfate solution; weigh 2.24 g of 2-methylimidazole and dissolve it in 20 mL of water to obtain a 2-methylimidazole solution. After both are completely dissolved, dissolve 50 mg of glucose isomerase in 2 mL of water to obtain a GI enzyme solution. Then, slowly add the GI enzyme solution dropwise to the cobalt sulfate solution and mix thoroughly. Then, slowly add the cobalt sulfate and GI enzyme mixture dropwise to the 2-methylimidazole solution and mix well. Stir at room temperature for 30 min, and then incubate at room temperature for 24 h. After incubation, filter the mixture for liquid-solid separation, wash three times with water to obtain a purple solid. Dry the solid and grind it thoroughly into powder (GI@ZIF-67), and store it at room temperature for later use. Weigh 0.54g of zinc nitrate and dissolve it in 8mL of water to obtain a zinc nitrate solution; weigh 0.72g of 4,4'-biphenyldicarboxylic acid and dissolve it in 20mL of water to obtain a 4,4'-biphenyldicarboxylic acid solution. After both are completely dissolved, dissolve 50mg of saccharifying enzyme in 2mL of water to obtain a GA enzyme solution. Then, slowly add the GA enzyme solution dropwise to the zinc nitrate solution to obtain a mixed solution of zinc nitrate and GA enzyme. Take 50mg of GI@ZIF-67 powder and evenly disperse it in the mixed solution of zinc nitrate and GA enzyme. After thorough mixing, slowly add the mixed solution of GI@ZIF-67, zinc nitrate, and GA enzyme to the 4,4'-biphenyldicarboxylic acid solution. Mix well and stir at room temperature for 30min. Then, incubate at room temperature for 24h. After incubation, filter the mixture for liquid-solid separation. Wash three times with water to obtain a white block. Dry the solid and grind it thoroughly into powder (GI@ZIF-67-GA@IRMOF-9), and store it at room temperature for later use. The solid GI@ZIF-67-GA@IRMOF-9 was added to a dilute hydrochloric acid solution at pH 5.5 and allowed to stand at room temperature for 1 hour. Then, the solid and liquid phases were separated, and the solid phase was washed with water to obtain GI-GA@IRMOF-9-1.

[0060] Example 2

[0061] The preparation steps of GI-GA@IRMOF-9-2 in this embodiment are the same as those in Example 1, except that the cobalt salt is cobalt nitrate, the zinc salt is zinc acetate, the amount of GI@ZIF-67 added is 100 mg, and the pH of the dilute hydrochloric acid is 6.

[0062] Example 3

[0063] The preparation steps of GI-GA@IRMOF-9-3 in this embodiment are the same as those in Example 1, except that the cobalt salt is cobalt chloride and the amount of GI@ZIF-67 added is 150 mg.

[0064] Example 4

[0065] The preparation steps of GI-GA@IRMOF-9-4 in this embodiment are the same as those in Example 1, except that the ligands are 4-methylimidazole and 2,2'-biphenyldicarboxylic acid, respectively.

[0066] Example 5

[0067] The preparation steps of GI-GA@IRMOF-9-5 in this embodiment are the same as those in Example 1, except that the amount of 2-methylimidazole added is 3.36g, the amount of 4,4'-biphenyldicarboxylic acid added is 1.44g, and the amount of enzyme added is 100mg of GI and 100mg of GA.

[0068] Example Effect Description

[0069] Figure 1 Using GI@ZIF-67 as the crystal nucleus, GA@IRMOF-9 is grown on the outer layer to form a layered encapsulated GI@ZIF-67-GA@IRMOF-9 composite material. In order to improve its mass transfer performance, the layered encapsulated composite material is acid etched to finally form the GI-GA@IRMOF-9 composite material.

[0070] Figure 2 The SEM images show that in Comparative Example 3, GI@ZIF-67 is a regular dodecahedron of 1-1.5 μm. In Comparative Example 1, the structure of ZIF-67 becomes irregular after encapsulation with two enzymes, indicating that MOF materials need to be properly screened according to the enzymes during the immobilization process. In Comparative Example 4, the MOF materials become spherical particles with a size of about 3 μm after layer-by-layer encapsulation. In Example 1, it can be seen that ZIF-67 becomes a hollow sphere after acid etching, and the size remains basically unchanged.

[0071] Figure 3 As can be seen from the XRD pattern shown, compared with Comparative Example 4 and Example 1, the ZIF-67 crystal structure in Example 1 was destroyed after the addition of acid. The XRD characteristic peaks of the ZIF-67 crystal disappeared, and only the characteristic peaks of IRMOF-9 were retained, indicating that the IRMOF-9 structure was intact.

[0072] Table 1 shows the specific surface areas of Example 1 and Comparative Examples 3 and 4.

[0073]

[0074] Figure 4As shown in the pore size distribution diagram and Table 1, it can be seen that GI@ZIF-67 in Comparative Example 3 is mostly microporous with a large specific surface area, while GA@IRMOF-9 is mostly mesoporous and macroporous. In Comparative Example 4, it can be seen that GI@ZIF-67-GA@IRMOF-9 has both the microporous structure of ZIF-67 and the mesoporous structure of IRMOF-9, and its specific surface area is between that of ZIF-67 and IRMOF-9. The results of Example 1 show that after acid etching, the micropores of GI-GA@IRMOF-9 are reduced, and the specific surface area is closer to that of IRMOF-9, indicating that ZIF-67 is etched away under dilute acid conditions.

[0075] Enzymatic hydrolysis performance tests were conducted on different enzyme@MOF materials prepared in the comparative examples and embodiments, using them as catalysts: GA catalyzed the production of glucose from maltodextrin, and GI further catalyzed the production of fructose from glucose. The peak position of glucose in HPLC was approximately 10.835, and the peak position of fructose was approximately 11.420. Specific test conditions were as follows: enzyme@MOF materials and maltodextrin were added to 5 mL of water, with an enzyme concentration of approximately 0.2 mg / mL and a maltodextrin concentration of approximately 4 mg / mL. The mixture was incubated in a 50-100℃ oven for 1 h. After incubation, the amounts of glucose and fructose produced were measured, and all results were repeated three times.

[0076] Figure 5 The enzymatic hydrolysis performance of different enzymes @MOF materials at different temperatures was demonstrated. In Comparative Examples 1, 3, and 4, the poor mass transfer performance of ZIF-67, due to the predominantly micropore size, resulted in low fructose production. In Comparative Example 1, both glucose and fructose production were low, indicating that maltodextrin had difficulty entering the pores of ZIF-67. In Comparative Example 2, glucose production was high, but fructose production was low, indicating poor GI enzymatic hydrolysis. This was because GI underwent coordination with zinc salts during mixing, leading to enzyme inactivation. In Examples 1-5, it was observed that after acid etching of ZIF-67, mass transfer performance increased, and fructose production increased with increasing temperature, reaching a maximum value at 90°C. In Example 1, the highest fructose production of 1.82 mg / mL was achieved at 90°C.

[0077] 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 hierarchically encapsulated dual-enzyme@MOFs complex, characterized in that, Includes the following steps: (1) Mix soluble cobalt salt, glucose isomerase and water evenly to obtain solution A; dissolve methylimidazolium compound in water to obtain solution B; mix solutions A and B to obtain mixed solution; after mixing evenly, incubate at room temperature, then separate solid and liquid, wash the solid phase with water to obtain GI@ZIF-67; (2) Mix the soluble zinc salt, saccharifying enzyme and water evenly to obtain solution C; A biphenyl acid compound was dissolved in water to obtain solution D; GI@ZIF-67, solution C and D were mixed and incubated at room temperature, then the solid and liquid phases were separated and the solid phase was washed with water to obtain GI@ZIF-67-GA@IRMOF-9; (3) Add GI@ZIF-67-GA@IRMOF-9 to dilute hydrochloric acid solution, let it stand at room temperature, then separate the solid and liquid phases, wash the solid phase with water to obtain GI-GA@IRMOF-9.

2. The method for preparing a layered encapsulated dual-enzyme@MOFs complex according to claim 1, characterized in that, The mass ratio of the soluble cobalt salt, glucose isomerase, and methylimidazolium compound in step (1) is 0.3–1.5: 0.03–0.5: 1.2–3.6; The mass ratio of the soluble zinc salt, saccharifying enzyme, biphenyl acid compound and GI@ZIF-67 in step (2) is 0.1-0.7: 0.03-0.5: 0.5-2.4: 0.03-0.

6.

3. The method for preparing a layered encapsulated dual-enzyme@MOFs complex according to claim 1 or 2, characterized in that, The mass ratio of the soluble cobalt salt, glucose isomerase, and methylimidazolium compound in step (1) is 0.7–0.9:0.05–0.10:2.2–3.4; The mass ratio of the soluble zinc salt, saccharifying enzyme, biphenyl acid compound and GI@ZIF-67 in step (2) is 0.5-0.6: 0.05-0.10: 0.7-1.4: 0.05-0.

20.

4. The method for preparing a layered encapsulated dual-enzyme@MOFs complex according to claim 1 or 2, characterized in that, The methylimidazolium compound in step (1) is at least one of 2-methylimidazolium and 4-methylimidazolium; the soluble cobalt salt is at least one of cobalt sulfate, cobalt nitrate and cobalt chloride; The soluble zinc salt mentioned in step (2) is at least one of zinc acetate and zinc nitrate; The biphenyl acid compound mentioned in step (2) is at least one of 4,4'-biphenyldicarboxylic acid and 2,2'-biphenyldicarboxylic acid.

5. The method for preparing a layered encapsulated dual-enzyme@MOFs complex according to claim 1 or 2, characterized in that, In solution A described in step (1), the concentration of soluble cobalt salt is 26–130 mg / ml; In solution B described in step (1), the concentration of methylimidazolium compounds is 110–170 mg / ml; In solution C described in step (2), the concentration of soluble zinc salt is 10–70 mg / ml; In step (2), the concentration of biphenyl acid compounds in solution D is 36–72 mg / ml.

6. The method for preparing a layered encapsulated dual-enzyme@MOFs complex according to claim 1 or 2, characterized in that, The pH value of the dilute hydrochloric acid solution in step (3) is 5.5-6; The incubation time for steps (1) and (2) is 8–24 hours. The settling time in step (3) is 0.5 to 3 hours.

7. The method for preparing a layered encapsulated dual-enzyme@MOFs complex according to claim 1 or 2, characterized in that, The mixing in steps (1) and (2) refers to stirring for 30 to 60 minutes; the liquid-solid separation in steps (1) and (2) involves filtration for 10 to 30 minutes each time, and filtration is performed 3 to 5 times; the solid phase is washed with water 3 to 5 times. The specific steps for mixing GI@ZIF-67, solution C and D in step (2) are as follows: first mix GI@ZIF-67 and solution C, and then add it dropwise to solution D; The solids obtained in steps (1), (2) and (3) are dried at 32–44°C for 4–16 hours.

8. A layered encapsulated dual-enzyme@MOFs complex prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the hierarchical encapsulation dual-enzyme@MOFs complex of claim 8 in realizing the catalytic hydrolysis of maltodextrin via a dual-enzyme cascade reaction.

10. The application according to claim 9, characterized in that, The catalytic reaction temperature for the hydrolysis of maltodextrin by the dual-enzyme cascade reaction is 50–100℃ or 70–90℃.

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