High-temperature-resistant beta-glucosidase inclusion body (at) MOF catalyst as well as preparation method and application thereof

Abstract

The invention discloses a high-temperature-resistant beta-glucosidase inclusion body (at) MOF (Metal Organic Framework) catalyst as well as a preparation method and application thereof. The preparation method comprises the following steps: firstly, preparing a catalytic activity inclusion body (TsBgl1 CatIBs) of high-temperature-resistant beta-glucosidase, and then introducing the TsBgl1 CatIBs at the initial stage of synthesis of an iron-based MOF carrier under the conditions of a water phase and good biocompatibility, so that MOF crystals grow around an enzyme aggregate and encapsulate the enzyme aggregate in situ, thereby obtaining the high-temperature-resistant beta-glucosidase. Therefore, the immobilized enzyme catalyst with high enzyme activity retention rate, excellent thermal stability and good recoverability is obtained. The enzyme loading capacity of the TsBgl1 CatIBs coated MIL-100 (Fe) prepared by the invention can reach 12mg / g, and the encapsulation efficiency at the moment can reach 55%. The enzyme activity at 100 DEG C is 92.5% of the optimum enzyme activity of free TsBgl1CatIBs, and the equivalent enzyme activity is still kept after the TsBgl1CatIBs is stored at 4 DEG C for 40 days.

Description

Technical Field

[0001] This invention belongs to the field of β-glucosidase immobilization, specifically relating to a high-temperature resistant β-glucosidase inclusion body@MOF catalyst, its preparation method, and its application. Background Technology

[0002] β-glucosidase (EC 3.2.1.21) is a hydrolase that specifically cleaves the non-reducing β-1,4 glycosidic bonds of oligosaccharides or glycoside derivatives, releasing glucose and the corresponding glycosidic aglycone. In the enzymatic saccharification of lignocellulosic biomass, it hydrolyzes cellobiose to reduce the product inhibition of exonucleases and release fermentable sugars, making it a crucial step in the process. β-glucosidase is indispensable in the refining of biofuels such as cellulosic ethanol and is also widely used in various industrial sectors, including food and pharmaceuticals, in key processes such as glycoside modification, flavor release, and activation of bioactive molecules.

[0003] Despite their high selectivity and catalytic efficiency under mild conditions, β-glucosidases face several challenges in industrial applications, including poor thermostability, susceptibility to inhibition by hydrolysis products, and difficulties in recovery and reuse. To address these issues, extensive research has focused on discovering and modifying thermostable β-glucosidases derived from thermophilic microorganisms, which exhibit enhanced substrate tolerance and catalytic activity at high temperatures. However, these thermostable variants are expressed in low quantities and require complex purification processes, hindering large-scale industrial production.

[0004] In recent years, catalytically active inclusion bodies (CatIBs) have attracted attention as a recombinant protein expression form. Unlike traditional inclusion bodies, CatIBs consist of insoluble protein aggregates that retain their correctly folded structure and significant biological activity due to the formation of ordered supramolecular nanostructures through non-covalent interactions. CatIBs combine the activity of soluble enzymes with the stability and separability of insoluble aggregates. They do not require complex refolding steps, can be purified by gentle washing, and can be easily recovered by centrifugation (Int. J. Biol. Macromol. 199 (2022) 358–371). However, these active inclusion bodies may still denature under prolonged high-temperature industrial conditions, highlighting the need for effective enzyme stabilization and reuse strategies.

[0005] Immobilizing enzymes on solid supports has become a promising strategy for improving stability and facilitating reusability. Traditional supports such as mesoporous silica, polymers, and hydrogels have been extensively studied, but immobilization in these inhomogeneous and low-crystallinity solid matrices has resulted in low protein loading efficiency, limited stability at high temperatures, and / or enzyme leaching. Metal-organic frameworks (MOFs), with their crystalline porous structure, high specific surface area, and chemical tunability, provide an attractive platform for enzyme encapsulation. Currently, various strategies for immobilizing enzymes with MOFs can be employed, including surface adsorption, covalent bonding, cross-linking, diffusion, and in-situ encapsulation. In-situ encapsulation introduces the enzyme during support synthesis, and MOF crystallization occurs around the enzyme, encapsulating the enzyme molecule within an ordered MOF crystal (Chem. Rev. 2021, 121, 1077−1129).

[0006] Among numerous MOFs, MIL-100(Fe)—a rigid iron-based framework with two types of permanent mesopores—exhibits excellent thermodynamic and water stability, maintaining its structural integrity at temperatures up to 350°C and even after prolonged exposure to boiling water for weeks. However, MIL-100(Fe) synthesized in aqueous phase suffers from poor crystallinity and low specific surface area, resulting in very limited research reports on in-situ enzyme encapsulation using MIL-100(Fe) under aqueous conditions. Summary of the Invention

[0007] To address the shortcomings and deficiencies of existing technologies, the primary objective of this invention is to provide a method for preparing a high-temperature resistant β-glucosidase inclusion body@MOF catalyst. The core of this method lies in: firstly, preparing a high-temperature resistant catalytically active inclusion body for β-glucosidase (TsBgl1 CatIBs); then, under aqueous and biocompatible conditions, introducing TsBgl1 CatIBs into the initial synthesis of an iron-based MOF support, allowing MOF crystals to grow around the enzyme aggregates and encapsulate them in situ, thereby obtaining an immobilized enzyme catalyst that combines high enzyme activity retention, excellent thermal stability, and good recyclability.

[0008] The amino acid sequence of thermostable β-glucosidase (TsBgl1) was obtained by searching the NCBI database and then analyzed based on Escherichia coli (E. coli). E. coliThe coding sequence was optimized based on codon preference, and the optimized TsBgl1 gene was then synthesized. The recombinant plasmid pET-28a(+)-TsBgl1 was constructed using pET-28a(+) as the expression vector and transformed into *E. coli* for recombinant expression. Active inclusion bodies TsBgl1 CatIBs were isolated and purified. The active inclusion bodies TsBgl1 CatIBs were in situ encapsulated using MIL-100(Fe). The prepared TsBgl1 CatIBs@MIL-100(Fe) had an enzyme loading of up to 12 mg / g, with an encapsulation efficiency of 55%. The enzyme activity at 100℃ was 92.5% of the optimal activity of free TsBgl1 CatIBs, and it retained considerable activity after 40 days of storage at 4℃.

[0009] Another object of the present invention is to provide a high-temperature resistant β-glucosidase inclusion body@MOF catalyst obtained by the above preparation method.

[0010] Another object of the present invention is to provide the application of the above-mentioned high-temperature resistant β-glucosidase inclusion body@MOF catalyst.

[0011] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a high-temperature resistant β-glucosidase inclusion body@MOF catalyst, comprising the following steps: (1) Insert the TsBgl1 gene fragment into the pET-28a(+) plasmid vector to construct pET-28a(+)-TsBgl1; (2) pET-28a(+)-TsBgl1 was transferred into Escherichia coli to prepare engineered bacteria; (3) After the engineered bacteria were activated in LB medium, they were expanded in TB medium and then isopropyl-β-D-thiogalactoside (IPTG) was added to induce expression and obtain culture medium. The precipitate obtained by centrifugation of the culture medium was resuspended and washed to obtain bacterial cells. The cells were then sonicated and centrifuged to obtain recombinant β-glucosidase active inclusion bodies (TsBgl1 CatIBs). (4) Mix the recombinant β-glucosidase active inclusion body solution with the soluble ferrous salt solution, then add the sodium trimellitate compound solution, mix and let stand at constant temperature, separate the solid and liquid, wash the solid phase and dry to obtain the β-glucosidase inclusion body@MOF catalyst.

[0012] Preferably, in step (1), the TsBgl1 gene is optimized and synthesized according to the codon preference of Escherichia coli, with an NcoI restriction endonuclease recognition sequence (CCATGG) introduced at its 5′ end and an XhoI recognition sequence (CTCGAG) introduced at its 3′ end.

[0013]

[0014] Preferably, in step (1), the TsBgl1 gene is cloned into the expression vector pET-28a(+) by double digestion with NcoI and XhoI to construct the recombinant plasmid pET-28a(+)-TsBgl1.

[0015] Preferably, the Escherichia coli in step (2) is E. coli BL21(DE3).

[0016] Preferably, the conditions for activating the engineered bacteria in LB medium in step (3) are: 37±0.5℃, 200±20rpm for 12-14h.

[0017] Preferably, the LB culture medium in step (3) contains 90-110 μg / mL kanamycin sulfate.

[0018] Preferably, in step (3), the culture is expanded in TB medium to OD600 = 0.6 to 0.8.

[0019] Preferably, the TB culture medium in step (3) contains 90–110 μg / mL kanamycin sulfate.

[0020] Preferably, in step (3), the temperature for expansion culture in TB medium is 37±0.5℃, and the culture is carried out on a shaker at 200±20rpm. The ratio of the bacterial culture obtained after activation of the engineered bacteria in LB medium to the inoculation volume of TB medium is 1±0.1:100.

[0021] Preferably, the concentration of isopropyl-β-D-thiogalactoside in step (3) is 0.5±0.1 mmol / L; after adding isopropyl-β-D-thiogalactoside, the mixture is cultured for 20-22 h at 18±0.5℃ and 180±20 rpm.

[0022] Preferably, the centrifugation speed of the culture medium in step (3) is 4000±500 rpm and the time is 20 to 30 min.

[0023] Preferably, the solvent for resuspension in step (3) is a MES buffer solution with pH=6±0.5.

[0024] Preferably, the ultrasonic breaking power in step (3) is 450±10kW, with ultrasonic waves for 2±0.5s, pause for 4±0.5s, and repeated 200 to 250 times.

[0025] Preferably, in step (3), the centrifugation speed for taking the precipitate is 4000±500 rpm, and the centrifugation time is 10 to 15 minutes.

[0026] Preferably, in step (4), the soluble ferrous salt is ferrous chloride tetrahydrate.

[0027] Preferably, in step (4), the pyromellitic acid compound is sodium pyromellitic acid.

[0028] Preferably, the concentration of the recombinant β-glucosidase active inclusion body solution in step (4) is 2.8–5.6 mg / mL, and the solvent is MES buffer with pH = 6 ± 0.5.

[0029] Preferably, the concentration of the soluble ferrous salt solution in step (4) is 75±5 mmol / L, and the solvent is water.

[0030] Preferably, the concentration of the sodium pyromellitic acid compound solution in step (4) is 65±5 mmol / L, and the solvent is water.

[0031] Preferably, in step (4), the ratio of recombinant β-glucosidase active inclusion body to soluble ferrous salt is 2.8–5.6 mg: 0.9 mmol.

[0032] Preferably, in step (4), the molar ratio of the soluble ferrous salt to the sodium pyromellitic acid compound is 4.6 ± 0.1:1.

[0033] Preferably, the mixing and stirring time after adding the trimesic acid compound solution in step (4) is 0.5 to 2 h, more preferably 0.5 ± 0.16 h; the stirring speed is 0 to 200 r / min, more preferably 100 ± 5 r / min.

[0034] Preferably, the temperature of the constant temperature settling in step (4) is 50-80℃, more preferably 70±2℃; and the settling time is 8-10h.

[0035] Preferably, the washing liquid used in step (4) is deionized water, and the washing process is carried out with deionized water 3 to 5 times; the drying temperature is 50 to 60 ℃, and the drying time is 12±2 h.

[0036] Secondly, the present invention provides a high-temperature resistant β-glucosidase inclusion body@MOF catalyst obtained by the above preparation method.

[0037] The high-temperature resistant β-glucosidase inclusion body@MOF catalyst described in this invention has the characteristics of easy recovery and high temperature resistance.

[0038] Thirdly, the present invention provides the application of a high-temperature resistant β-glucosidase inclusion body@MOF catalyst in the catalytic hydrolysis of cellobiose.

[0039] Preferably, the application specifically involves mixing cellobiose substrate with β-glucosidase inclusion body @MOF catalyst and reacting at 80–100°C for 0.5–6 h to achieve the hydrolysis of cellobiose to produce glucose.

[0040] More preferably, the substrate is cellobiose, the solvent is 20±0.5 mmol / L MES buffer, pH 6.0±0.5, and the mass concentration of the substrate in the solvent is 20±1%.

[0041] More preferably, the mass ratio of the β-glucosidase inclusion body@MOF catalyst to the substrate is 42±1 mg:0.3 g.

[0042] The beneficial effects of this invention are: This invention inserts the TsBgl1 gene sequence into Escherichia coli, and under specific pH and temperature conditions, it makes it into micron-sized aggregates that are easy to precipitate, thereby enabling the recombinant TsBgl1 enzyme to self-precipitate. It can be rapidly separated and purified by low-speed centrifugation. This purification method is simple to operate, saves time, and has a large processing capacity. The obtained TsBgl1 CatIBs have catalytic activity and are easy to store.

[0043] The mechanism of this invention is as follows: During the expression and purification of the thermostable β-glucosidase TsBgl1, excessive recombinant protein production led to the formation of insoluble aggregates. Unlike typical inclusion bodies, TsBgl1 CatIBs within these inclusion bodies still contain a large amount of correctly folded protein, resulting in an enzyme activity that is only 59.2% of that of soluble TsBgl1 at the same concentration. Compared to TsBgl1, TsBgl1 CatIBs offer advantages such as higher yield and easier separation and recovery. TsBgl1 CatIBs were specifically expressed and purified. Under biocompatible conditions, a highly water- and thermally stable metal-organic framework, MIL-100(Fe), was synthesized to in situ encapsulate the TsBgl1 CatIBs, with the TsBgl1CatIBs enzyme cluster acting as a dynamic scaffold. This scaffold adsorbs positively charged trinuclear ferrite clusters ([Fe3O(COO)6)). 3+ On the other hand, these precursors are locally concentrated, and MIL-100(Fe) crystal nuclei are promoted to form directly on the protein surface through non-classical means. Under the combined action of directional attachment and Ostwald ripening, crystallization occurs around the enzyme, thereby encapsulating the enzyme molecules in an ordered MOF crystal.

[0044] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The TsBgl1 CatIBs prepared (expressed and purified) by this invention have advantages such as high expression level and convenient separation, which increases the possibility of subsequent industrial application of TsBgl1 CatIBs@MIL-100(Fe).

[0045] 2. The MIL-100(Fe) prepared in this invention is synthesized under aqueous conditions and has high crystallinity and high specific surface area.

[0046] 3. The TsBgl1 CatIBs@MIL-100(Fe) prepared in this invention allows the enzyme activity of TsBgl1 CatIBs to be well preserved under high temperature conditions, which is beneficial for expanding the application of the enzyme in the field of high temperature aqueous phase catalysis.

[0047] 4. The TsBgl1 CatIBs@MIL-100(Fe) prepared in this invention exhibits excellent catalytic performance, with the highest enzyme activity retention rate reaching 92.5% of the optimal enzyme activity of free TsBgl1 CatIBs. Furthermore, after incubation at 100℃ for 9 hours, the catalyst still retains 89% of the optimal enzyme activity of free TsBgl1 CatIBs. Simultaneously, TsBgl1CatIBs@MIL-100(Fe) also demonstrates excellent storage and cycling stability. In the application of hydrolyzing cellobiose, the glucose yield can reach 78.4% after a reaction at 100℃ for 6 hours. Attached Figure Description

[0048] Figure 1 This is the pET-28a-TsBgl1 spectrum in Embodiment 1 of the present invention.

[0049] Figure 2 The image shows an SDS-PAGE of purified TsBgl1 CatIBs from this invention. Lane M is the marker, lane 1 is the cell lysate, lane 2 is the supernatant after centrifugation, and lane 3 is the precipitate.

[0050] Figure 3 shows the effects of different (a) metal ligand molar ratios, (b) stirring speeds, (c) synthesis temperatures, and (d) stirring times on the XRD of the complexes obtained in exploratory experiments 1-4 of this invention.

[0051] Figure 4 In Example 1, (a) is the optimal reaction temperature (pH=6) for TsBgl1 CatIBs, and (b) is the optimal reaction pH (80°C) for TsBgl1 CatIBs.

[0052] Figure 5 This is a schematic diagram of the preparation process of the TsBgl1 CatIBs@MIL-100(Fe) enzyme complex obtained in Examples 6-8 and Comparative Examples 6-7 of the present invention.

[0053] Figure 6The enzyme loading and relative enzyme activity at pH=6 and 80℃ of the TsBgl1 CatIBs@MIL-100(Fe) materials obtained in Examples 6-8 (enzyme addition amounts of 2.8, 4 and 5.6 mg, corresponding to enzyme loadings of 12, 5.156 and 5.656 mg / g, respectively) and the TsBgl1 CatIBs@MIL-100(Fe) materials obtained in Comparative Examples 6-7 of the present invention (enzyme addition amounts of 0.8 and 1.6 mg, corresponding to enzyme loadings of 5 and 7.89 mg / g, respectively) are given, with the enzyme activity of TsBgl1 CatIBs at pH=6 and 80℃ as the baseline (i.e., 100%).

[0054] Figure 7 This is a comparison of the relative enzyme activity of TsBgl1 CatIBs@MIL-100(Fe) obtained in Example 6 and TsBgl1@MIL-100(Fe) obtained in Comparative Example 8 of the present invention at pH=6 and 80℃. The relative enzyme activity of TsBgl1 CatIBs@MIL-100(Fe) obtained in Example 6 at pH=6 and 80℃ is used as the baseline (i.e., 100%). The comparison is made between the relative enzyme activity of TsBgl1@MIL-100(Fe) obtained in Comparative Example 8 and TsBgl1CatIBs@MIL-100(Fe) obtained in Example 6.

[0055] Figure 8 shows (a) SEM images, (b) CLSM images, and (c) TEM-EDS images of the TsBgl1 CatIBs@MIL-100(Fe) materials obtained in Examples 6-7 and Comparative Examples 6-7 of this invention at different time points during the synthesis process.

[0056] Figure 9 The 77k N2 adsorption-desorption curves are for MIL-100(Fe) obtained in Example 4 and TsBgl1 CatIBs@MIL-100(Fe) obtained in Example 6.

[0057] Figure 10 The enzyme activity retention rates of the TsBgl1 CatIBs obtained in Example 1 and the TsBgl1 CatIBs@MIL-100(Fe) obtained in Example 6 are shown in (a) pH=6 and under different temperature conditions, and (b) at 80℃ and under different pH conditions.

[0058] Figure 11The following are the results of the following tests on the TsBgl1 CatIBs materials obtained in Example 1 and the TsBgl1 CatIBs@MIL-100(Fe) materials obtained in Example 6: (a) pH=6, high temperature stability (TsBgl1 CatIBs), (b) pH=6, high temperature stability (TsBgl1CatIBs@MIL-100(Fe)), (c) pH=6, 80°C cycling stability (TsBgl1 CatIBs@MIL-100(Fe)), and (d) pH=6, storage stability at a storage temperature of 4°C and an enzyme activity test temperature of 80°C (TsBgl1 CatIBs and TsBgl1CatIBs@MIL-100(Fe)).

[0059] Figure 12 The conversion rates of TsBgl1 CatIBs obtained in Example 1 and TsBgl1 CatIBs@MIL-100(Fe) materials obtained in Example 6 at different temperatures and times under different conditions were: (a) 80℃, (b) 90℃, and (c) 100℃.

[0060] Figure 13 The conversion rates of cellobiose hydrolyzed by TsBgl1 CatIBs@MIL-100(Fe) obtained in Example 6 and TsBgl1 CatIBs@ZIF-8 obtained in Comparative Example 9 were compared after 6 hours of continuous reaction at pH=6 and different temperatures. Detailed Implementation

[0061] 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.

[0062] 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.

[0063] The nucleotide sequences of TsBgl1 described in the embodiments and comparative examples of this invention are as shown in SEQ ID NO.1.

[0064] Exploration Experiment 1 Table 1. Effect of different metal ligand molar ratios on MIL-100 (Fe)

[0065] According to the table above, ferrous chloride tetrahydrate aqueous solution and sodium trimellitate aqueous solution were mixed and stirred at a volume ratio of 4:1. The stirring temperature of the above solution was 70℃. After stirring, the solution was kept at a constant temperature to achieve crystallization. The crystallization conditions were a constant temperature incubator at 70℃. After the reaction was completed, the solid and liquid phases were separated by filtration. The solid phase was washed three times with deionized water to obtain MOF-MIL-100(Fe) with different molar ratios of metal ligands. The solution was stored at room temperature for later use.

[0066] Exploration Experiment 2 Table 2 Effect of different stirring speeds on MIL-100 (Fe)

[0067] According to the table above, ferrous chloride tetrahydrate aqueous solution and sodium trimellitate aqueous solution were mixed and stirred at a volume ratio of 4:1. The stirring temperature of the above solution was 70℃. After stirring, the solution was kept at a constant temperature to achieve crystallization. The crystallization conditions were a constant temperature incubator at 70℃. After the reaction was completed, the solid and liquid phases were separated by filtration. The solid phase was washed three times with deionized water to obtain MOF-MIL-100(Fe) with different stirring speeds. The solutions were stored at room temperature for later use.

[0068] Exploration Experiment 3 Table 3 Effect of different synthesis temperatures on MIL-100 (Fe)

[0069] According to the table above, ferrous chloride tetrahydrate aqueous solution and sodium trimellitate aqueous solution were mixed and stirred at a volume ratio of 4:1. The stirring temperature of the above solution was 30-80℃. After stirring, the solution was kept at a constant temperature to achieve crystallization. The crystallization conditions were a constant temperature incubator at 30-80℃. After the reaction was completed, the solid and liquid phases were separated by filtration. The solid phase was washed three times with deionized water to obtain MOF-MIL-100(Fe) synthesized at different temperatures. The solutions were stored at room temperature for later use.

[0070] Exploration Experiment 4 Table 4. Effects of different stirring times on MIL-100 (Fe)

[0071] According to the table above, ferrous chloride tetrahydrate aqueous solution and sodium trimellitate aqueous solution were mixed and stirred at a volume ratio of 4:1. The stirring temperature of the above solution was 70℃. After stirring, the solution was kept at a constant temperature to achieve crystallization. The crystallization conditions were a constant temperature incubator at 70℃. After the reaction was completed, the solid and liquid were separated by filtration. The solid phase was washed three times with deionized water to obtain MOF-MIL-100(Fe) with different stirring times, which were stored at room temperature for later use.

[0072] Comparative Example 1 12 mL of 50 mmol / L ferrous chloride tetrahydrate aqueous solution and 3 mL of 50 mmol / L sodium trimellitate aqueous solution were mixed and stirred at 70 °C for 0.5 h at a stirring speed of 100 r / min. The mixture was then incubated in a 70 °C incubator for 8 h. Solid-liquid separation was then performed by filtration. The remaining solid phase was washed three times with deionized water to obtain MIL-100(Fe). The solid phase was dried at 60 °C for 12 h. The dried sample was then ground and stored at 4 °C for later use.

[0073] Comparative Example 2 12 mL of 60 mmol / L ferrous chloride tetrahydrate aqueous solution and 3 mL of 80 mmol / L sodium trimellitate aqueous solution were mixed and stirred at 70 °C for 0.5 h at a stirring speed of 100 r / min. The mixture was then incubated at 70 °C for 8 h. Solid-liquid separation was then performed by filtration. The remaining solid phase was washed three times with deionized water to obtain MIL-100(Fe). The solid phase was dried at 60 °C for 12 h. The dried sample was then ground and stored at 4 °C for later use.

[0074] Comparative Example 3 12 mL of 90 mmol / L ferrous chloride tetrahydrate aqueous solution and 3 mL of 100 mmol / L sodium trimellitate aqueous solution were mixed and stirred at 70 °C for 0.5 h at a stirring speed of 100 r / min. The mixture was then incubated in a 70 °C incubator for 8 h. Solid-liquid separation was then performed by filtration. The remaining solid phase was washed three times with deionized water to obtain MIL-100(Fe). The solid phase was dried at 60 °C for 12 h. The dried sample was then ground and stored at 4 °C for later use.

[0075] Comparative Example 4 12 mL of 75 mmol / L ferrous chloride tetrahydrate aqueous solution and 3 mL of 65 mmol / L sodium trimellitate aqueous solution were mixed and stirred at 70 °C for 0.5 h at a stirring speed of 100 r / min. The mixture was then incubated at 30 °C for 8 h. Solid-liquid separation was then performed by filtration. The remaining solid phase was washed three times with deionized water to obtain MIL-100(Fe). The solid phase was dried at 60 °C for 12 h. The dried sample was then ground and stored at 4 °C for later use.

[0076] Comparative Example 5 12 mL of 75 mmol / L ferrous chloride tetrahydrate aqueous solution and 3 mL of 65 mmol / L sodium trimellitate aqueous solution were mixed and stirred at 70 °C for 0.5 h at a stirring speed of 100 r / min. The mixture was then incubated at 40 °C for 8 h. Solid-liquid separation was then performed by filtration. The remaining solid phase was washed three times with deionized water to obtain MIL-100(Fe). The solid phase was dried at 60 °C for 12 h. The dried sample was then ground and stored at 4 °C for later use.

[0077] Comparative Example 6 Take 1 mL of TsBgl1 CatIBs solution dispersed in MES buffer solution at pH=6 with a protein concentration of 0.8 mg / mL, mix it thoroughly with 12 mL of 75 mmol / L ferrous chloride tetrahydrate aqueous solution, then add 3 mL of 65 mmol / L sodium trimellitate aqueous solution and mix. Stir at 70 °C for 0.5 h, and incubate at 70 °C for 8 h. Then, perform vacuum filtration to achieve solid-liquid separation, wash three times with deionized water to obtain solid phase TsBgl1 CatIBs@MIL-100(Fe), dry at 60 °C for 12 h, grind the dried sample, and store at 4 °C for later use.

[0078] Comparative Example 7 Take 1 mL of TsBgl1 CatIBs solution dispersed in MES buffer solution at pH=6 with a protein concentration of 1.6 mg / mL, mix it thoroughly with 12 mL of 75 mmol / L ferrous chloride tetrahydrate aqueous solution, then add 3 mL of 65 mmol / L sodium trimellitate aqueous solution and mix. Stir at 70 °C for 0.5 h, and incubate at 70 °C for 8 h. Afterwards, perform vacuum filtration to achieve solid-liquid separation, wash three times with deionized water to obtain solid phase TsBgl1 CatIBs@MIL-100(Fe), dry at 60 °C for 12 h, grind the dried sample, and store at 4 °C for later use.

[0079] Comparative Example 8 Take 1 mL of TsBgl1 solution dispersed in MES buffer at pH 6 with a protein concentration of 2.8 mg / mL, mix it thoroughly with 12 mL of 75 mmol / L ferrous chloride tetrahydrate aqueous solution, then add 3 mL of 65 mmol / L sodium trimellitate aqueous solution and mix. Stir at 70 °C for 0.5 h, and incubate at 70 °C for 8 h. Afterwards, perform vacuum filtration to achieve solid-liquid separation, wash three times with deionized water to obtain the solid phase TsBgl1@MIL-100(Fe), dry at 60 °C for 12 h, grind the dried sample, and store at 4 °C for later use.

[0080] Comparative Example 9 Take 1 mL of TsBgl1 CatIBs solution dispersed in MES buffer at pH 6 with a protein concentration of 2.8 mg / mL, mix it thoroughly with 12 mL of 1.92 mmol / L 2-methylimidazole aqueous solution, and then add the above solution dropwise to 6 mL of 240 mmol / L zinc acetate aqueous solution. Stir at 70 °C for 0.5 h, and incubate at 70 °C for 8 h. Afterward, perform vacuum filtration to achieve solid-liquid separation, wash three times with deionized water to obtain the solid phase TsBgl1 CatIBs@ZIF-8, dry at 60 °C for 12 h, grind the dried sample, and store it in a refrigerator at 4 °C for later use.

[0081] Example 1 A gene fragment of β-glucosidase was inserted into the pET-28a plasmid vector to construct a structure as follows: Figure 1 pET-28a-TsBgl1 is shown.

[0082] The target gene was cloned into the pET-28a expression vector after double digestion with NcoⅠ and XhoⅠ, and transformed into *E. coli* BL21(DE3) competent cells. The transformed recombinant strain was inoculated into LB liquid medium containing 100 μg / mL kanamycin sulfate and activated at 37°C and 200 rpm for 12 hours. The next day, the bacterial culture was inoculated into fresh TB medium containing 100 μg / mL kanamycin sulfate and cultured with shaking at 37°C and 200 rpm. When the OD600 reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.5 mmol / L was added to induce protein expression, and the culture was continued at 18°C ​​and 180 rpm for 20 h. After induction, the bacterial cells were collected by centrifugation at 4000 rpm for 20 min at room temperature and resuspended in MES buffer (20 mmol / L, pH 6.0). Cells were resuspended and sonicated on ice (450kW, 2s sonication, 4s pause, 200 cycles). The resulting lysate was centrifuged at 4000 rpm for 10 min to separate the precipitate. The precipitate was thoroughly washed with MES buffer, centrifuged again, and collected. The precipitate was the purified recombinant β-glucosidase active inclusion body protein (TsBgl1CatIBs).

[0083] Example 2 12 mL of 75 mmol / L ferrous chloride tetrahydrate aqueous solution and 3 mL of 65 mmol / L sodium trimellitate aqueous solution were mixed and stirred at 70 °C for 0.5 h at a stirring speed of 100 r / min. The mixture was then incubated at 50 °C for 8 h. Solid-liquid separation was then performed by filtration. The remaining solid phase was washed three times with deionized water to obtain MIL-100(Fe). The solid phase was dried at 60 °C for 12 h. The dried sample was then ground and stored at 4 °C for later use.

[0084] Example 3 12 mL of 75 mmol / L ferrous chloride tetrahydrate aqueous solution and 3 mL of 65 mmol / L sodium trimellitate aqueous solution were mixed and stirred at 70 °C for 0.5 h at a stirring speed of 100 r / min. The mixture was then incubated at 60 °C for 8 h. Solid-liquid separation was then performed by filtration. The remaining solid phase was washed three times with deionized water to obtain MIL-100(Fe). The solid phase was dried at 60 °C for 12 h. The dried sample was then ground and stored at 4 °C for later use.

[0085] Example 4 12 mL of 75 mmol / L ferrous chloride tetrahydrate aqueous solution and 3 mL of 65 mmol / L sodium trimellitate aqueous solution were mixed and stirred at 70 °C for 0.5 h at a stirring speed of 100 r / min. The mixture was then incubated in a 70 °C incubator for 8 h. Solid-liquid separation was then performed by filtration. The remaining solid phase was washed three times with deionized water to obtain MIL-100(Fe). The solid phase was dried at 60 °C for 12 h. The dried sample was then ground and stored at 4 °C for later use.

[0086] Example 5 12 mL of 75 mmol / L ferrous chloride tetrahydrate aqueous solution and 3 mL of 65 mmol / L sodium trimellitate aqueous solution were mixed and stirred at 70 °C for 0.5 h at a stirring speed of 100 r / min. The mixture was then incubated at 80 °C for 8 h. Solid-liquid separation was then performed by filtration. The solid phase was washed three times with deionized water to obtain MIL-100(Fe). The solid phase was dried at 60 °C for 12 h. The dried sample was then ground and stored at 4 °C for later use.

[0087] Example 6 Take 1 mL of TsBgl1 CatIBs solution dispersed in MES buffer solution at pH=6 with a protein concentration of 2.8 mg / mL, mix it thoroughly with 12 mL of 75 mmol / L ferrous chloride tetrahydrate aqueous solution, then add 3 mL of 65 mmol / L sodium trimellitate aqueous solution and mix. Stir at 70 °C for 0.5 h, and incubate at 70 °C for 8 h. Then, perform vacuum filtration to achieve solid-liquid separation, wash three times with deionized water to obtain solid phase TsBgl1 CatIBs@MIL-100(Fe), dry at 60 °C for 12 h, grind the dried sample, and store at 4 °C for later use.

[0088] Example 7 Take 1 mL of TsBgl1 CatIBs solution dispersed in MES buffer solution at pH=6 with a protein concentration of 4 mg / mL, mix it thoroughly with 12 mL of 75 mmol / L ferrous chloride tetrahydrate aqueous solution, then add 3 mL of 65 mmol / L sodium trimellitate aqueous solution and mix. Stir at 70 °C for 0.5 h, and incubate at 70 °C for 8 h. Afterwards, perform vacuum filtration to achieve solid-liquid separation, wash three times with deionized water to obtain solid phase TsBgl1 CatIBs@MIL-100(Fe), dry at 60 °C for 12 h, grind the dried sample, and store at 4 °C for later use.

[0089] Example 8 Take 1 mL of TsBgl1 CatIBs solution dispersed in MES buffer solution at pH=6 with a protein concentration of 5.6 mg / mL, mix it thoroughly with 12 mL of 75 mmol / L ferrous chloride tetrahydrate aqueous solution, then add 3 mL of 65 mmol / L sodium trimellitate aqueous solution and mix. Stir at 70 °C for 0.5 h, and incubate at 70 °C for 8 h. Then, perform vacuum filtration to achieve solid-liquid separation, wash three times with deionized water to obtain solid phase TsBgl1 CatIBs@MIL-100(Fe), dry at 60 °C for 12 h, grind the dried sample, and store at 4 °C for later use.

[0090] Test conditions: I. Assay for β-glucosidase activity Under suitable conditions, 4-nitrophenyl-β-D-glucopyranoside, as a substrate, can be degraded by β-glucosidase into yellow p-nitrophenol (pNP). The enzyme activity of β-glucosidase can be calculated by detecting the absorbance of the product pNP. The specific steps are as follows: Prepare a 1 mL buffer system (MES buffer solution, pH=6.0, 20 mmol / L) with an enzyme concentration of 0.1 mg / mL and a substrate concentration of 100 mmol / L for 4-nitrophenyl-β-D-glucopyranoside. React at 50, 60, 70, 80, 90, and 100 °C for 10 min. After the reaction, add 1 mol / L Na₂CO₃ solution to stop the reaction. 10 min later, measure the absorbance of p-nitrophenol at 410 nm using a microplate reader. Substitute this data into a pNP standard curve plotted under the same conditions, with pNP concentration (μmol / L) as the x-axis and absorbance at 410 nm as the y-axis, to calculate the p-nitrophenol content. All tests were repeated three times. Specific reaction temperatures 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.

[0091] The β-glucosidase activity unit (U) is defined as the amount of enzyme required to catalyze the hydrolysis of a substrate to produce 1.0 μmol of p-nitrophenol in 1 min at 80 °C and pH 6.0, with 1 mg of free enzyme / immobilized enzyme. The formula for β-glucosidase activity is: (1) Where X is β-glucosidase activity (U); C is pNP concentration (μmol / L); V is final reaction volume (L); T is reaction time (min); and M is enzyme dosage (mg).

[0092] II. Calculation of Enzyme Encapsulation Efficiency and Enzyme Loading The Bradford method was used to determine the content of β-glucosidase, with purchased BSA protein as a standard curve. The specific procedure of the Bradford method is as follows: 5 μL of standard enzyme sample or test sample is mixed with 250 μL of Bradford working solution and reacted at 37℃ for 10 min. The absorbance at A595 nm is then measured using an ELISA reader. The standard curve and the concentration of the test sample are obtained based on the relationship between absorbance and concentration.

[0093] Enzyme encapsulation efficiency is defined as the mass percentage of the enzyme encapsulated in the MOF carrier relative to the original amount of free enzyme added. The formula is as follows: (2) Where m represents the mass (mg) of the original free enzyme added; c is the concentration of lipase in the supernatant after encapsulation (mg / mL); and V is the total volume (mL) of the supernatant after encapsulation.

[0094] Enzyme loading is defined as the percentage of the enzyme mass encapsulated within the MOF carrier relative to the total mass of the entire enzyme@MOF complex catalyst. The formula is as follows: (3) Where m represents the mass (mg) of the original free enzyme added; c is the concentration of β-glucosidase in the supernatant after encapsulation (mg / mL); V is the total volume (mL) of the supernatant after encapsulation; and M represents the total mass of the enzyme@MOF complex catalyst.

[0095] III. Determination of the thermal stability of β-glucosidase and β-glucosidase@MOF enzyme activities Under suitable conditions, 4-nitrophenyl-β-D-glucopyranoside, as a substrate, can be decomposed into yellow p-nitrophenol (pNP) by β-glucosidase. By detecting the absorbance of the product pNP, the enzyme activities of β-glucosidase and β-glucosidase@MOF can be calculated. The specific steps are as follows: A 1 mL buffer system (MES buffer solution, pH=6.0, 20 mmol / L) was prepared, with an enzyme concentration of 0.1 mg / mL. The system was incubated at 60, 70, 80, 90, and 100 °C for 0, 0.5, 1, 3, 6, and 9 hours, respectively. After incubation, 100 mmol / L of the substrate 4-nitrophenyl-β-D-glucopyranoside was added to the reaction system. After 10 min of reaction, the reaction was stopped by adding 1 mol / L Na₂CO₃ solution. The absorbance of p-nitrophenol at 410 nm was measured using a microplate reader after 10 min. This data was then used to plot a pNP standard curve under the same conditions, with pNP concentration (μmol / L) on the x-axis and absorbance at 410 nm on the y-axis, to calculate the p-nitrophenol content. All tests were repeated three times. The specific reaction temperatures are described in the subsequent test results. In the enzyme activity stability assay, the amount of free enzyme added was equal to the amount of enzyme encapsulated in the enzyme complex.

[0096] The β-glucosidase activity unit (U) is defined as the amount of enzyme required to catalyze the hydrolysis of a substrate to produce 1.0 μmol of p-nitrophenol in 1 min at 80 °C and pH 6.0, with 1 mg of free enzyme / immobilized enzyme. The formula for β-glucosidase activity is: (4) Where X is β-glucosidase activity (U); C is pNP concentration (μmol / L); V is final reaction volume (L); T is reaction time (min); and M is enzyme dosage (mg).

[0097] IV. β-glucosidase@MOF cycle stability test After one round of reaction with PNPG, the reaction solution of TsBgl CatIBs@MIL-100(Fe) was immediately centrifuged at 4°C and 10,000 rpm for 2 min to separate the supernatant and precipitate. The absorbance of the supernatant was measured after adding Na₂CO₃ solution. The precipitate (TsBgl CatIBs@MIL-100(Fe)) was then recovered, washed with MES buffer, and resuspended with the same volume of MES buffer before adding fresh PNPG substrate for the next round of reaction. The enzyme activity in the first round of reaction (cycle 0) was defined as 100%, and the relative enzyme activity in each round was calculated.

[0098] V. Stability test of β-glucosidase and β-glucosidase@MOF storage TsBgl1 CatIBs and TsBgl CatIBs@MIL-100(Fe) were stored together at 4°C, and samples were taken every five days for residual enzyme activity testing. The enzyme activity of the reaction before storage (day 0 of storage) was defined as 100%, and the relative enzyme activity of each round of reaction was calculated.

[0099] VI. Hydrolysis of cellobiose and detection of products Add 1.5 mL of 20 wt% cellobiose solution (dissolved in 20 mmol / L MES buffer, pH 6.0) to a 5 mL EP tube and pre-incubate at 80, 90, and 100 °C in a shaker for 10 min. Then add 42 mg of TsBgl1CatIBs@MIL-100(Fe) and 42 mg of TsBgl1@MIL-100(Fe), respectively, and react at 80, 90, 100 °C and 500 rpm for 0.5, 1, 3, and 6 hours. Alternatively, add 42 mg of TsBgl1CatIBs@MIL-100(Fe) and 42 mg of TsBgl1CatIBs@ZIF-8, respectively, and react at 100 °C and 500 rpm for 6 hours. After the reaction, centrifuge the mixture at 10000 rpm for 5 min, and collect the supernatant for glucose quantification by HPLC. Chromatographic analysis was performed using a Bio-Rad HPX-87H column with a mobile phase of 5 mM H2SO4, a flow rate of 0.5 mL / min, a column temperature of 50℃, and a total run time of 15 minutes.

[0100] (5) in, C Glu V represents the glucose concentration in the supernatant, in mmol / L; V represents the volume of the supernatant, in L; n represents the initial number of moles of cellobiose in the reaction mixture, in mmol.

[0101] This invention investigated the molecular weights of the precipitates from cell lysis buffer, centrifuged supernatant, and MES buffer dispersed at pH 6. Figure 2 The results showed that most of the target protein was present in the precipitate, with a small amount remaining in the supernatant. TsBgl1 CatIBs were successfully separated and purified by low-speed centrifugation.

[0102] This invention investigated the effect of different reaction conditions on the crystallinity of MIL-100(Fe), and the results are shown in Figure 3. The MIL-100(Fe) synthesized at a metal ion to ligand molar ratio of 4.6:1 showed good agreement with the standard XRD pattern. MIL-100(Fe) could not be successfully synthesized at synthesis temperatures of 30 and 40℃, while samples synthesized at temperatures of 50, 60, 70, and 80℃ successfully matched the standard XRD pattern. The crystallinity of MIL-100(Fe) did not show a significant trend with increasing stirring speed and time. Therefore, the optimal synthesis conditions for subsequent enzyme encapsulation were determined: a stirring speed of 100 r / min, a stirring time of 0.5 h, a synthesis temperature of 70℃, and a metal ligand molar ratio of 4.6:1.

[0103] This invention investigated the optimal reaction temperature and optimal reaction pH of TsBgl1 CatIBs, and the results are shown in […]. Figure 4 In Figure (b), the optimal reaction temperature for TsBgl1 CatIBs is 80℃ and the optimal reaction pH is 6.

[0104] The preparation process of the TsBgl1 CatIBs@MIL-100(Fe) enzyme complex obtained in this invention is as follows: Figure 5 As shown.

[0105] This invention investigated the effects of different amounts of TsBgl1 CatIBs added on enzyme loading and encapsulation efficiency. The results are shown in […]. Figure 6In Figure (a), it can be seen that when the amount of TsBgl1 CatIBs added during the enzyme encapsulation process is 0.8 mg, the enzyme loading of MIL-100(Fe) on TsBgl1 CatIBs is 5 mg / g, and the encapsulation efficiency is 70.3%; when the amount of TsBgl1 CatIBs added during the enzyme encapsulation process is 1.6 mg, the enzyme loading of MIL-100(Fe) on TsBgl1 CatIBs is 7.89 mg / g, and the encapsulation efficiency is 67.3%; when the amount of TsBgl1 CatIBs added during the enzyme encapsulation process is 2.8 mg, the enzyme loading of MIL-100(Fe) on TsBgl1 CatIBs is 12 mg / g, and the encapsulation efficiency is 55%; when the amount of TsBgl1 CatIBs added during the enzyme encapsulation process is 4 mg, the enzyme loading of MIL-100(Fe) on TsBgl1 CatIBs is 5 mg / g, and the encapsulation efficiency is 55%; when the amount of TsBgl1 CatIBs added during the enzyme encapsulation process is 4 mg, the enzyme loading of MIL-100(Fe) on TsBgl1 CatIBs is 5 mg / g, and the encapsulation efficiency is 70.3 ... TsBgl1 CatIBs is 5 mg / g, and the encapsulation efficiency is 70.3%; when the amount of TsBgl1 CatIBs added during the The enzyme loading of CatIBs was 5.156 mg / g, with an encapsulation efficiency of 23.1%. When the amount of TsBgl1 CatIBs added during the enzyme encapsulation process was 5.6 mg, the enzyme loading of TsBgl1 CatIBs in MIL-100(Fe) was 5.656 mg / g, with an encapsulation efficiency of 19.8%. Subsequently, enzyme activity retention tests were performed on TsBgl1CatIBs@MIIL-100(Fe) enzyme catalysts with different enzyme loadings. The results are shown below. Figure 6 In Figure (b), the enzyme activity retained by the enzyme catalyst with an enzyme loading of 5 mg / g is 4.6% of the optimal activity of free TsBgl1 CatIBs; the enzyme activity retained by the enzyme catalyst with an enzyme loading of 7.89 mg / g is 8.9% of the optimal activity of free TsBgl1 CatIBs; the enzyme activity retained by the enzyme catalyst with an enzyme loading of 12 mg / g is 39.4% of the optimal activity of free TsBgl1 CatIBs; the enzyme activity retained by the enzyme catalyst with an enzyme loading of 5.156 mg / g is 25% of the optimal activity of free TsBgl1 CatIBs; and the enzyme activity retained by the enzyme catalyst with an enzyme loading of 5.656 mg / g is 20.1% of the optimal activity of free TsBgl1 CatIBs. In summary, the enzyme catalyst with an enzyme loading of 12 mg / g has the highest enzyme activity retention, which is 39.4% of the optimal activity of free TsBgl1 CatIBs.

[0106] Figure 7 The relative enzyme activities of TsBgl1 CatIBs@MIL-100(Fe) obtained in Example 6 of this invention were compared with those of TsBgl1@MIL-100(Fe) obtained in Comparative Example 8 at 80°C. Figure 7 As shown, the relative enzyme activity of the prepared TsBgl1@MIL-100(Fe) is only 22.58% of that of TsBgl1CatIBs@MIL-100(Fe), indicating that the enzyme activity obtained by encapsulating active inclusion bodies with MIL-100(Fe) is much higher than that obtained by directly encapsulating enzymes with MIL-100(Fe).

[0107] SEM images taken at different times during the synthesis of the enzyme complex (Figure 8(a)) show that TsBgl1 CatIBs underwent a morphological transformation during the formation of the composite material, dissociating from their initial short rod-like shape into smaller, irregular clusters. This simultaneous deformation of the enzyme and the framework indicates a strong interaction during crystallization. Figure 8(b) shows a confocal laser imaging (CLSM) image of the sample, which reveals the fluorescence of TsBgl1CatIBs at corresponding positions in the dark field, proving that TsBgl1 CatIBs have been successfully encapsulated into the carrier material. The mapping diagram of the enzyme complex (Figure 8(c)) shows that Fe, C, and O, as characteristic elements of MIL-100 (Fe), are uniformly distributed, while the characteristic element S of the enzyme is non-uniformly distributed, indicating that the enzyme clusters act as discrete nucleation templates guiding the framework to assemble in a spatially resolved manner.

[0108] Figure 9 The BET test results are for MIL-100(Fe) obtained in Example 4 and TsBgl1 CatIBs@MIL-100(Fe) obtained in Example 6 of this invention. The effect of enzyme encapsulation on the porosity of the material was studied using N2 adsorption-desorption analysis at 77 K. Figure 9 As shown, the TsBgl1 CatIBs@MIL-100(Fe) obtained in Example 6 exhibits a type I isotherm, confirming that its porosity is still predominantly microporous. Quantitative analysis shows a significant difference between it and MIL-100(Fe). The BET specific surface area of ​​TsBgl1 CatIBs@MIL-100(Fe) is 886 m². 2 •g -1 The total pore volume is 0.48 cm³. 3 •g -1 It is lower than the 1573 m of MIL-100(Fe). 2 •g -1 and 0.684cm 3 •g -1 This reduction is primarily attributed to the fact that TsBgl1 CatIBs physically occupy the MOF cavity, thereby reducing the space available for N2 adsorption. Figure 10 This refers to the enzyme activity retention rates under different temperature conditions and different pH conditions in Example 6 of this invention. The enzyme activity of TsBgl1 CatIBs@MIL-100(Fe) prepared using this method was retained (…). Figure 10(a)). Furthermore, compared to free TsBgl1 CatIBs, the optimal reaction temperature in Example 6 increased from 80°C to 100°C, and at 100°C, the enzyme activity retention of the enzyme complex reached 92.5% of the free enzyme. This is attributed to the increased substrate mass transfer rate due to the higher reaction temperature, allowing the substrate to successfully contact the enzyme active sites within the framework, thus releasing the enzyme activity. Furthermore, from Figure 10 As can be seen in (b), TsBgl1 CatIBs@MIL-100(Fe) still retains 20% of its enzyme activity under acidic conditions (pH=4).

[0109] The stability of enzyme complexes is a crucial parameter for evaluating their performance. The protective effect of the MOF-MIL-100(Fe) carrier on β-glucosidase is specifically demonstrated in its high-temperature stability, recyclability, and storage stability. Specific test results can be found in [link to test results]. Figure 11 Regarding high-temperature stability, the prepared Example 6-TsBgl1 CatIBs@MIL-100(Fe) was incubated in aqueous solutions at 60, 70, 80, 90, and 100 °C for 0, 0.5, 1, 3, 6, and 9 h, and its residual enzyme activity was measured. Figure 11 (b)). It can be concluded that TsBgl1CatIBs@MIL-100(Fe) has good high temperature tolerance, retaining 89% of the relative enzyme activity after incubation at 100℃ for 9 hours, while free TsBgl1 CatIBs showed a huge loss of enzyme activity. Figure 11 (a) After incubation at 100℃ for 0.5h, 90% of the enzyme activity was lost. Regarding recycling... Figure 11 In (c) the TsBgl1 CatIBs@MIL-100(Fe) catalyst reaction was carried out at 80℃. After the reaction, the catalyst was recovered by centrifugation, and the enzyme activity retention rate of the sample was tested. The sample was then washed with deionized water for the next cycle. After 10 cycles, the catalyst still maintained a high enzyme activity of 78%. Notably, in the first cycle, the activity of the TsBgl1CatIBs@MIL-100(Fe) enzyme catalyst decreased slightly, with a retention rate of 80%. This decrease can be attributed to the uneven distribution of protein clusters within the MOF. Specifically, some protein clusters were located on the periphery of the framework rather than in the central interior region, making the enzymes at the edges more prone to detachment during cycling, thus reducing the overall activity of the complex. Regarding storage stability (…),… Figure 10In the study (d), the stability of free TsBgl1 CatIBs and TsBgl1 CatIBs@MIL-100(Fe) was tested after 40 days of storage at 4°C. The free enzyme showed a significant decrease in activity after 10 days of storage. After 40 days, its activity retention plummeted to only 7%, significantly lower than that of the encapsulated enzyme. This large difference can be attributed to the inherent instability of inclusion bodies, which are aggregates of misfolded or partially folded proteins formed through hydrophobic interactions and hydrogen bonds. During storage, the protein molecules within these aggregates undergo irreversible chemical damage and tend to slowly but continuously transition to lower-energy, inactive conformations. This process reduces the number of catalytically active molecules, ultimately leading to the sharp loss of activity observed during long-term storage. In contrast, the enzyme catalyst, after 40 days of storage at 4°C, retained 72% of its high activity when tested under the same conditions as the free TsBgl1 CatIBs.

[0110] To evaluate the high-temperature catalytic performance of this catalyst, Example 6-TsBgl1 CatIBs@MIL-100(Fe) was used to catalyze the hydrolysis of cellobiose, with cellobiose as the substrate. The effects of different temperatures and reaction times on the hydrolysis of cellobiose were investigated. Figure 12 It can be seen that immobilization significantly enhances the long-term catalytic stability of TsBgl1 CatIBs at high temperatures. After reacting at 90℃ for 6 hours ( Figure 12 In (b), the glucose conversion rate of free TsBgl1 CatIBs was only 8.2%, while TsBgl1 CatIBs@MIL-100(Fe) achieved a conversion rate of 75%. Its performance under extreme conditions of 100℃ is particularly noteworthy. Figure 12 (c)). Free TsBgl1 CatIBs exhibited negligible activity (4.3% conversion) due to rapid denaturation. In stark contrast, TsBgl1 CatIBs@MIL-100(Fe) achieved a conversion of 74.3% within 3 hours and 78.4% after 6 hours. Although at 80°C ( Figure 12 In (a) of the reaction, the initial reaction rate of TsBgl1 CatIBs@MIL-100(Fe) was slightly lower than that of free TsBgl1 CatIBs, but its stability advantage became apparent over time, ultimately achieving a higher final conversion rate after 6 hours. This superior stability stems from the spatial confinement provided by the MOF-MIL-100(Fe) framework, which effectively inhibits the thermal defolding and aggregation-induced inactivation of enzyme molecules. At the same time, the reaction is further accelerated at high temperatures, resulting in excellent catalytic performance of TsBgl1CatIBs@MIL-100(Fe).

[0111] To evaluate the excellent protective performance of the MOF-MIL-100(Fe) support, a comparative example of 9-TsBgl1 CatIBs@ZIF-8 was used to catalyze the hydrolysis of cellobiose, with cellobiose as the substrate. The conversion rates of the cellobiose hydrolysis reaction at different temperatures for 6 hours were investigated. Figure 13 The results show that ZIF-8 immobilization did not significantly enhance the long-term catalytic stability of TsBgl1 CatIBs at high temperatures. After reacting at 80℃ for 6 hours, the glucose conversion rate of TsBgl1CatIBs@MIL-100(Fe) was 43.4%, while that of TsBgl1 CatIBs@ZIF-8 was only 20%. After reacting at 90℃ for 6 hours, the glucose conversion rate of TsBgl1 CatIBs@MIL-100(Fe) was 75%, while that of TsBgl1 CatIBs@ZIF-8 was only 9%. After reacting at 100℃ for 6 hours, the glucose conversion rate of TsBgl1 CatIBs@MIL-100(Fe) was 78%, while that of TsBgl1 CatIBs@ZIF-8 was only 3.8%, far lower than that of TsBgl1 CatIBs@MIL-100(Fe).

[0112] 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 high-temperature resistant β-glucosidase inclusion body@MOF catalyst, characterized in that, Includes the following steps: (1) Insert the TsBgl1 gene fragment into the pET-28a plasmid vector to construct pET-28a-TsBgl1; (2) pET-28a-TsBgl1 was transferred into Escherichia coli to produce engineered bacteria; (3) After the engineered bacteria were activated in LB medium, they were cultured in TB medium and then isopropyl-β-D-thiogalactoside was added to induce expression and obtain culture medium. The precipitate obtained by centrifugation of the culture medium was resuspended and washed to obtain bacterial cells. The cells were then sonicated, centrifuged and the precipitate was taken to obtain recombinant β-glucosidase active inclusion bodies. (4) Mix the recombinant β-glucosidase active inclusion body solution with the soluble ferrous salt solution, then add the sodium trimellitate compound solution, mix and let stand at constant temperature, separate the solid and liquid, wash the solid phase and dry to obtain the β-glucosidase inclusion body@MOF catalyst.

2. The preparation method according to claim 1, characterized in that, The concentration of the recombinant β-glucosidase active inclusion body solution in step (4) is 2.8–5.6 mg / mL, and the solvent is MES buffer with pH = 6 ± 0.5; And / or, in step (4), the ratio of recombinant β-glucosidase active inclusion bodies to soluble ferrous salt is 2.8–5.6 mg: 0.9 mmol; And / or, in step (4), the molar ratio of the soluble ferrous salt to the sodium pyromellitic acid compound is 4.6 ± 0.1:

1.

3. The preparation method according to claim 1 or 2, characterized in that, In step (4), the soluble ferrous salt is ferrous chloride tetrahydrate; And / or, in step (4), the pyromellitic acid compound is sodium pyromellitic acid; And / or, the concentration of the soluble ferrous salt solution in step (4) is 75±5 mmol / L, and the solvent is water; And / or, the concentration of the sodium pyromellitic acid compound solution in step (4) is 65±5 mmol / L, and the solvent is water.

4. The preparation method according to claim 1 or 2, characterized in that, The temperature for constant temperature settling in step (4) is 50-80℃, more preferably 70±2℃; the settling time is 8-10h. And / or, after adding the pyromellitic acid compound solution in step (4), the mixing and stirring time is 0.5 to 2 h, more preferably 0.5 ± 0.16 h; the stirring speed is 0 to 200 r / min, more preferably 100 ± 5 r / min.

5. The preparation method according to claim 1 or 2, characterized in that, The nucleotide sequence of the TsBgl1 gene fragment in step (1) is shown in SEQ ID NO.1; And / or, the Escherichia coli in step (2) is E. coli BL21(DE3).

6. The preparation method according to claim 1 or 2, characterized in that, The conditions for activating the engineered bacteria in LB medium in step (3) are: 37±0.5℃, 200±20 rpm for 12-14h. And / or, the LB medium in step (3) contains 90–110 μg / mL kanamycin sulfate; And / or, as described in step (3), the culture is expanded in TB medium to an OD600 of 0.6 to 0.8; And / or, the TB culture medium in step (3) contains 90–110 μg / mL kanamycin sulfate; And / or, the temperature for the expansion culture in TB medium in step (3) is 37±0.5℃, and the culture is carried out on a shaker at 200±20rpm. The ratio of the bacterial culture obtained after the engineered bacteria are activated in LB medium to the inoculation volume of TB medium is 1±0.1:

100. And / or, the concentration of isopropyl-β-D-thiogalactoside in step (3) in the system is 0.5±0.1mmol / L; after adding isopropyl-β-D-thiogalactoside, continue to culture at 18±0.5℃ and 180±20 rpm for 20 to 22 h; And / or, the culture medium in step (3) is centrifuged at a speed of 4000±500 rpm for 20 to 30 min; And / or, the solvent for resuspension in step (3) is a MES buffer solution with pH=6±0.5; And / or, the ultrasonic breaking power in step (3) is 450±10kW, ultrasonic for 2±0.5s, pause for 4±0.5s, and cycled 200 to 250 times; And / or, in step (3), the centrifugation speed for taking the precipitate is 4000±500 rpm, and the centrifugation time is 10 to 15 min.

7. A high-temperature resistant β-glucosidase inclusion body@MOF catalyst obtained by the preparation method according to any one of claims 1 to 6.

8. The application of the high-temperature resistant β-glucosidase inclusion body@MOF catalyst of claim 7 in the catalytic hydrolysis of cellobiose.

9. The application according to claim 8, characterized in that, The specific application involves mixing cellobiose substrate with β-glucosidase inclusion bodies @MOF catalyst and reacting at 80–100°C for 0.5–6 h to achieve the hydrolysis of cellobiose to produce glucose.

10. The application according to claim 9, characterized in that, The substrate is cellobiose, and the solvent is 20±0.5 mmol / L MES buffer, pH 6.0±0.5; the mass concentration of the substrate in the solvent is 20±1%. And / or, the mass ratio of the β-glucosidase inclusion body@MOF catalyst to substrate is 42±1 mg:0.3 g.

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