Enzyme protein-metal composite catalyst as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of catalysts, and relates to an enzyme / protein-metal composite catalyst and a preparation method and application thereof. The enzyme / protein-metal composite catalyst is obtained through mixed reaction of enzyme / protein, a monomer and a metal precursor, or obtained through reaction of the monomer and the metal precursor to obtain an organic ligand metal catalyst, and then the organic ligand metal catalyst is combined with the enzyme / protein to obtain the enzyme / protein-metal composite catalyst. According to the invention, the high efficiency of the enzyme / protein-metal composite catalyst is utilized, polymerization can be carried out at normal temperature and medium and low pressure, energy consumption is greatly reduced, rigid steric hindrance provided by an enzyme / protein skeleton can physically limit excessive movement and approaching of a polymer chain, so that the chain transfer reaction is obviously inhibited, and the reaction time is shortened. The preparation method is beneficial to obtaining polymers with narrower molecular weight distribution, the electron withdrawing groups of enzyme / protein molecules can regulate and control the electronic effect of active site metal, and the reaction efficiency and the insertion rate of olefin monomers are improved.

Description

Technical Field

[0001] This application belongs to the field of catalyst technology, and relates to an enzyme / protein-metal composite catalyst, its preparation method and application. Background Technology

[0002] Functionalized polyethylene, as an important class of polymer materials, is widely used in photovoltaics, packaging, automobiles, electronics, pipelines, and many other fields due to its excellent performance in mechanical properties, chemical stability, and processing properties. With the continuous improvement of material performance requirements in downstream applications, the development of high-end functionalized polyethylene products with specific functions and superior performance has become a research hotspot and key direction in the field of polymer materials. Among these, the technical route of controlling the functional properties of polyethylene through copolymerization of ethylene with α-olefins and polar monomers is one of the core pathways for the preparation of high-end functionalized polyethylene. In the synthesis of functionalized polyethylene, the core technology lies in achieving efficient copolymerization of ethylene with olefins or polar monomers. While traditional high-pressure free radical polymerization methods can be used for this type of copolymerization reaction, they have significant drawbacks: on the one hand, the reaction conditions are harsh (requiring high temperature and pressure), resulting in high energy consumption and stringent equipment requirements; on the other hand, the high activity of free radicals during polymerization easily leads to side reactions such as chain transfer and over-branching, resulting in a wide molecular weight distribution of the product and uneven distribution of α-olefins or polar monomers, making it difficult to precisely control product performance and meet the high-quality requirements of high-end applications. Therefore, these methods are gradually being replaced by more efficient catalytic polymerization methods.

[0003] Currently, catalytic systems used for copolymerizing ethylene with olefins or polar monomers mainly include metallocene catalysts and post-transition metal catalysts (such as Pd and Ni-based catalysts). Metallocene catalysts, with their single active center, can precisely control the molecular weight distribution of polyethylene and the insertion selectivity of comonomers, driving the development of the functionalized polyethylene industry. However, these catalysts still have significant shortcomings: they are sensitive to reaction conditions (requiring strict anhydrous and oxygen-free environments and low tolerance to impurities), can only effectively copolymerize with a few weakly polar olefin monomers, and have poor compatibility with monomers containing strongly polar groups; furthermore, the melt strength of products synthesized in some systems is insufficient, making processing difficult. Additionally, the synthesis of catalyst ligands is complex and costly, limiting their application in more functionalized polyethylene scenarios. Post-transition metal catalysts, represented by Pd and Ni, exhibit outstanding advantages in catalyzing the copolymerization of ethylene with α-olefins and polar monomers due to their unique d-electron configuration and flexible coordination number. They can optimize the spatial environment of the active center by regulating the ligands, adapt to α-olefins with different carbon chain lengths to precisely control the branching characteristics of the polymer, and have higher tolerance to polar groups than pre-transition metal catalysts, making it possible to copolymerize polar monomers. In addition, the reaction can achieve efficient polymerization at room temperature and medium to low pressure, reducing equipment and energy costs and minimizing side reactions. However, this type of catalyst still faces three major bottlenecks: First, stability and deactivation issues. Polar monomer heteroatoms, polymer chain ends, or byproducts can interact with the active center, causing irreversible deactivation, which is more prominent in industrial applications. Second, the challenges of copolymerization insertion rate and distribution. When copolymerizing with high-carbon α-olefins, high insertion rates are difficult to achieve due to steric hindrance. When copolymerizing with polar monomers, the large difference in polymerization rates leads to insufficient polar group content and aggregation. Third, chain transfer and β-H elimination issues. The active center has weak binding force on the polymer chain, which easily leads to chain transfer and β-H elimination, reducing polymerization efficiency and affecting product performance.

[0004] Faced with the challenges of balancing high activity and high stability in existing technologies, poor controllability of monomer insertion rate, insufficient precision of chain regulation, complex ligand design, and long improvement cycle, developing a new method that can rapidly optimize catalysts to achieve efficient preparation of functionalized polyethylene is very promising. Summary of the Invention

[0005] This application enhances catalyst stability through ligand electronic / steric hindrance modulation, heterogeneous loading, and multi-site synergistic stabilization. Optimized ligand configuration and heterogeneous systems improve copolymerization insertion rate, while ligand synergy and chiral ligands inhibit chain transfer and β-H elimination. The prepared catalyst can utilize the unique microenvironment of enzymes / proteins to solve the challenges of stability, monomer insertion selectivity, and side reaction suppression in traditional metal catalysts, enabling efficient and controllable synthesis of various high-end functionalized polyethylene materials under mild conditions.

[0006] The first aspect of this application provides an enzyme / protein-metal composite catalyst obtained by reacting an enzyme / protein, a monomer, and a metal precursor. The enzyme includes at least one or any inactive enzyme protein or carrier protein selected from oxidoreductases, hydrolases, transferases, lyases, and isomerases.

[0007] The enzyme protein used in this invention was obtained according to the literature (Engineering Thermostability in Artificial Metalloenzymes to Increase Catalytic Activity), and the gene sequence is shown in SEQ ID NO. 1-6. The whole gene was synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd., cloned into the pET3d vector, and the recombinant plasmid was transformed into Escherichia coli BL21.

[0008] This invention does not impose any specific limitations on the synthesis method of the above-mentioned enzyme protein; conventional methods in the art can be used. The following method is merely an example: 10 mL of medium containing *Escherichia coli* Luria-Bertani (LB) is incubated at 37°C. o Pre-cultured at 1°C for 16 h. Then, the pre-cultured bacterial suspension was transferred at a 1% inoculation rate to 50 mL LB medium containing ampicillin and cultured in a shaker at 37 °C. When the OD600 (optical density of the cell suspension measured at 600 nm) reached 0.6–0.8, IPTG was added to a final concentration of 0.2 mM to induce protein expression, followed by overnight culture. Cells were collected by centrifugation and washed with ice-cold 120 mM NaCl and 16 mM potassium phosphate buffer (pH=7.4). 3.5 g of wet bacterial weight was resuspended in 50 mL of buffer containing 20 mM cyclohexylpropanesulfonic acid (CAPS), 0.5 mM benzalkonium (BA), pH=11.0, and 1 mg / mL lysozyme, 25 µg / mL RNase A, and 10 mM MgCl2 were added. The mixture was shaken at 4ºC for 1.5 hours and then centrifuged to obtain cell extract. Enzyme protein 1 was obtained by passing the extract through an ion exchange column and dialysis.

[0009] In addition, the enzymes used in this invention may also be selected from mutants of enzyme protein 1 and commercially available enzymes or proteins, including: oxidoreductases (e.g., laccase), hydrolases (e.g., lipase BCL), transferases (e.g., glutamine transferase), lyases (e.g., carbonic anhydrase), isomerases (e.g., glucose isomerase), and bovine serum albumin.

[0010] In some preferred embodiments, the metal precursor includes at least one of (1,5-cyclooctadiene)palladium dichloride, anhydrous ferric chloride, anhydrous cobalt chloride, nickel(II) 2-methoxyethyl ether bromide complex, chromium acetylacetone, cuprous bromide, anhydrous titanium tetrachloride, anhydrous zirconium chloride, anhydrous vanadium trichloride, and anhydrous hafnium tetrachloride. The structural formula of the monomer is shown in Formula 1:

[0011] Formula 1.

[0012] In some preferred embodiments, the molar ratio of the enzyme / protein to the metal precursor is 1:(1-1000), and the molar ratio of the monomer to the metal precursor is 1:(0.1-20).

[0013] Preferably, the molar ratio of the enzyme / protein to the metal precursor is 1:(50-200), and the molar ratio of the monomer to the metal precursor is 1:(0.5-3).

[0014] In some preferred embodiments, R 1 -R 12 Substituents are H-substituted or / and C1-C20 alkyl-substituted or / and aromatic hydrocarbons and their derivatives substituted or / and naphthyl or / and alkyl halogens or / and amino-substituted or / and carboxyl-substituted or / and aldehyde-substituted or / and epoxy-substituted.

[0015] The second aspect of this application provides a method for preparing an enzyme / protein-metal composite catalyst, comprising: dissolving the enzyme / protein, monomer and metal precursor in a solvent respectively; mixing and stirring the three solutions under an inert gas atmosphere; separating the solid precipitate; washing and drying to obtain the catalyst.

[0016] In some preferred embodiments, the enzyme / protein may be activated.

[0017] In some preferred embodiments, the enzyme / protein activation method includes: dispersing the enzyme / protein in phosphate buffer, adding EDC and NHS to react, dialyzing using a dialysis bag, and freeze-drying the solution in the dialysis bag to obtain the activated enzyme / protein.

[0018] In some preferred embodiments, the solvent includes at least one of ethyl acetate, acetone, dichloromethane, dimethyl sulfoxide, and N,N-dimethylformamide.

[0019] The third aspect of this application provides an enzyme / protein-metal composite catalyst, which is obtained by first reacting the above-mentioned monomer and metal precursor to obtain an organic ligand metal catalyst, and then combining the organic ligand metal catalyst with an inactive enzyme / protein. The structural formula of the organic ligand metal catalyst is selected from at least one of Formulas 2-6:

[0020] Formula 2

[0021] Formula 3

[0022] Formula 4

[0023] Formula 5

[0024] Formula 6.

[0025] In some preferred embodiments, the molar ratio of the enzyme / protein to the organic ligand metal catalyst is 1:(1-1000).

[0026] In some preferred embodiments, M is a metal ion, including at least one of palladium, iron, cobalt, nickel, chromium, copper, titanium, zirconium, vanadium, and hafnium ions; X is any one of halogens.

[0027] In some preferred embodiments, R 1 -R 12 Substituents are H-substituted or / and C1-C20 alkyl-substituted or / and aromatic hydrocarbons and their derivatives substituted or / and naphthyl or / and alkyl halogens or / and amino-substituted or / and carboxyl-substituted or / and aldehyde-substituted or / and epoxy-substituted.

[0028] The fourth aspect of this application provides the application of the above-mentioned enzyme / protein-metal composite catalyst in catalyzing the copolymerization of ethylene with ethylene and / or with α-olefins and / or with polar monomers.

[0029] In some preferred embodiments, the α-olefin is a C3-C20 α-olefin. At least one of the olefins.

[0030] In some preferred embodiments, the polar monomer is methyl acrylate, ethyl acrylate, vinyl acetate, propylene acetate, methyl norborneol, or 10 Methyl undecenoate, 10 Undecenoic acid, 9 decenol, 5 At least one of hexenol.

[0031] The fifth aspect of this application provides a method for synthesizing polyethylene, the method using the enzyme / protein-metal composite catalyst described above.

[0032] Compared with the prior art, this application achieves at least the following technical effects: 1. Extremely mild reaction conditions: Utilizing the high efficiency of enzyme / protein-metal composite catalysts, polymerization can be carried out at room temperature and medium to low pressure, significantly reducing energy consumption.

[0033] 2. Catalyst Evolvability and Low Cost: By utilizing directed evolution technology, the desired enzyme / protein molecules can be rapidly screened and constructed, enabling rapid iteration and optimization of catalyst performance (activity, selectivity, stability), thus solving the problems of long design cycles and high costs associated with traditional ligands.

[0034] 3. Excellent monomer insertion capability and selectivity control: (1) For α-olefins: By designing amino acids on the surface of enzymes / proteins, the electronic effects of active site metals are regulated to enhance the coordination ability with electronegative α-olefins, thereby significantly improving the insertion rate of high carbon α-olefins and achieving precise control over their insertion sequences. (2) For polar monomers: enzymes / proteins preferentially adsorb and enrich polar monomers through their functional groups, thereby increasing their local concentration near the active site. At the same time, the electronic effects provided by enzymes / proteins reduce the probability of polar monomers directly poisoning the metal center, thus significantly improving the stability of the catalyst and the grafting density of polar monomers.

[0035] 4. Effective suppression of side reactions: (1) Suppression of chain transfer: The rigid steric hindrance provided by the enzyme / protein backbone can physically limit the excessive movement and approach of polymer chains, thereby significantly suppressing chain transfer reactions and facilitating the acquisition of polymers with narrower molecular weight distributions. (2) Suppression of β-H elimination: The three-dimensional structure of the active pocket can stabilize the polymer chains growing on the metal center. Through spatial constraints and weak interactions, it effectively hinders the migration and elimination of β-H to the metal center, reduces the generation of unsaturated end groups, and improves polymerization efficiency. Attached Figure Description

[0036] Figure 1 This is a schematic diagram illustrating the application of enzyme-metal composite catalysts in the polymerization of ethylene.

[0037] Figure 2 The figure shows the effect of the ratio of enzyme to metal precursor on the activity of enzyme-palladium composite catalyst in ethylene polymerization.

[0038] Figure 3 The figure shows the effect of the ratio of metal precursor to monomer on the activity of the composite-palladium catalyst in ethylene polymerization. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0041] Preparation of monomer I: 8.2 mmol of acenaphthene, 30.0 mmol of 2,3,5,6-tetramethyl-p-phenylenediamine, and 50 mL of toluene were added to a 100 mL three-necked flask, followed by dropwise addition of 0.03 mL of sulfuric acid. The mixture was refluxed and stirred for 3 hours. After the reaction was complete, the insoluble residue was filtered off while hot, and the product was dried under vacuum. The dried powder was dissolved in 200 mL of ethyl acetate, and 400 mL of hexane was added. The mixture was stirred for 30 minutes, and then allowed to stand at 15°C for 12 hours. The precipitated product was filtered off, washed with 50 mL of ice-cold hexane, and dried under vacuum overnight to obtain a dry red powder monomer I (the following formula).

[0042] Monomer I Preparation of monomer II: 4.00 mmol of trimethylolamine, 2.0 mmol of acenaphthene, and 30 mL of methanol were added to a two-necked flask. 0.2 mL of formic acid was added under vigorous stirring, and the mixture was refluxed at 50°C for 12 hours. After the reaction was complete and cooled, the resulting orange-red precipitate was separated by filtration. To further purify the product, recrystallization was performed using an ethanol / dichloromethane (volume ratio 13:2) mixed solvent system. The crystalline product was then thoroughly washed with low-temperature ethanol and vacuum dried overnight to finally obtain high-purity monomer II (the following formula).

[0043] Monomer II Preparation of monomer III: 30 mmol of trimethylolpropane, 10 mmol of 9,10-dihydro-9,10-ethyleneanthracene-11,12-dione, 1 g of p-toluenesulfonic acid, and 150 mL of toluene were added to a 500 mL two-necked flask, and the mixture was refluxed and stirred for 24 hours. After the reaction was complete, the insoluble residue was filtered off while hot, and the product was dried under vacuum. The dried powder was purified by silica gel column chromatography (petroleum ether to ethyl acetate volume ratio of 20:1), and finally recrystallized from a petroleum ether / ethyl acetate mixed solvent. The product was then washed with hexane at low temperature and dried under vacuum overnight to obtain high-purity monomer III (the following formula).

[0044] Monomer III Example 1-1 This embodiment provides an enzyme / protein-metal composite catalyst, which is obtained by blending and reacting an activated enzyme or protein, a monomer, and a metal precursor. The preparation method includes: (1) Disperse 50 μmol of enzyme protein 1 in 40 mL of phosphate buffer (pH=6, 100 mM), add 0.7668 g of EDC and 0.1151 g of NHS, react for 1 h, dialyze using a 5 kDa dialysis bag, freeze dry the solution in the dialysis bag to obtain activated enzyme protein 1; (2) Enzyme protein 1, (1,5-cyclooctadiene)palladium dichloride ((COD)PdCl2) and monomer I were dissolved in ethyl acetate at concentrations of 0.5 mmol / L, 0.05 mol / L, and 0.05 mol / L, respectively. The three solutions were mixed with an enzyme to Pd ion molar ratio of 1:100 and a Pd ion to monomer I molar ratio of 1:1. The reaction solution was protected with an inert gas and stirred at room temperature for 12 h using a magnetic stirrer at 600 rpm. After the reaction was completed, hexane was added to precipitate a solid precipitate, which was then centrifuged at 30,000 g. The precipitate was washed twice with hexane and dried under vacuum overnight at 30 °C to obtain the enzyme-palladium composite catalyst with a yield of 84%.

[0045] Examples 1-2 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that acetone is used instead of ethyl acetate, and the yield is 63%.

[0046] Examples 1-3 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that dichloromethane is used instead of ethyl acetate, and the yield is 76%.

[0047] Examples 1-4 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that dimethyl sulfoxide is used instead of ethyl acetate, and the yield is 55%.

[0048] Examples 1-5 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that N,N-dimethylformamide is used instead of ethyl acetate, and the yield is 72%.

[0049] Based on the experimental results of Examples 1-1 to 1-5, ethyl acetate was used as the solvent.

[0050] Example 2-1 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that the molar ratio of enzyme to Pd ion is 1:1 and the molar ratio of Pd ion to monomer I is 1:1.

[0051] Example 2-2 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that the molar ratio of enzyme to Pd ion is 1:50, and the molar ratio of Pd ion to monomer I is 1:1.

[0052] Example 2-3 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that the molar ratio of enzyme to Pd ion is 1:200, and the molar ratio of Pd ion to monomer I is 1:1.

[0053] Examples 2-4 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that the molar ratio of enzyme to Pd ion is 1:1000, and the molar ratio of Pd ion to monomer I is 1:1.

[0054] Example 3-1 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that the molar ratio of enzyme to Pd ion is 1:100, and the molar ratio of Pd ion to monomer I is 1:0.1.

[0055] Example 3-2 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that the molar ratio of enzyme to Pd ion is 1:100, and the molar ratio of Pd ion to monomer I is 1:0.5.

[0056] Example 3-3 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that the molar ratio of enzyme to Pd ion is 1:100, and the molar ratio of Pd ion to monomer I is 1:3.

[0057] Examples 3-4 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that the molar ratio of enzyme to Pd ion is 1:100, and the molar ratio of Pd ion to monomer I is 1:10.

[0058] Examples 3-5 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that the molar ratio of enzyme to Pd ion is 1:100, and the molar ratio of Pd ion to monomer I is 1:20.

[0059] Example 4 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that the enzyme used is non-activating enzyme protein 1, and monomer II is used to replace monomer I.

[0060] Example 5 The specific implementation method of this embodiment is the same as that of Embodiment 1-1, except that the enzyme used is non-activating enzyme protein 1, and monomer III is used instead of monomer I.

[0061] Example 6-1 The specific implementation method of this embodiment is the same as that of embodiment 4, except that the aspartic acid and glutamic acid in enzyme protein 1 used in embodiment 4 are mutated to lysine, and the amino acid sequence is shown in SEQ ID NO.2, which is denoted as enzyme protein 2, to obtain enzyme protein 2@Pd catalyst.

[0062] Example 6-2 The specific implementation method of this embodiment is the same as that of embodiment 4, except that the aspartic acid and glutamic acid in enzyme protein 1 used in embodiment 4 are mutated to arginine, and the amino acid sequence is shown in SEQ ID NO.3, which is denoted as enzyme protein 3, to obtain enzyme protein 3@Pd catalyst.

[0063] Example 6-3 The specific implementation method of this embodiment is the same as that of embodiment 4, except that the aspartic acid and glutamic acid in enzyme protein 1 used in embodiment 4 are mutated to histidine, and the amino acid sequence is shown in SEQ ID NO.4, which is denoted as enzyme protein 4, to obtain enzyme protein 4@Pd catalyst.

[0064] Example 6-4 The specific implementation method of this embodiment is the same as that of embodiment 4, except that the aspartic acid and glutamic acid in enzyme protein 1 used in embodiment 4 are mutated to alanine, and the amino acid sequence is shown in SEQ ID NO.5, which is denoted as enzyme protein 5, to obtain enzyme protein 5@Pd catalyst.

[0065] Example 6-5 The specific implementation method of this embodiment is the same as that of embodiment 4, except that the aspartic acid and glutamic acid in enzyme protein 1 used in embodiment 4 are mutated to leucine, and the amino acid sequence is shown in SEQ ID NO.6, which is denoted as enzyme protein 6, to obtain enzyme protein 6@Pd catalyst.

[0066] Example 7-1 The specific implementation method of this embodiment is the same as that of embodiment 4, except that anhydrous FeCl3 is used to replace (1,5-cyclooctadiene) palladium dichloride.

[0067] Example 7-2 The specific implementation method of this embodiment is the same as that of embodiment 4, except that anhydrous CoCl2 is used instead of (1,5-cyclooctadiene) palladium dichloride.

[0068] Example 7-3 The specific implementation method of this embodiment is the same as that of embodiment 4, except that nickel(II) 2-methoxyethyl ether complex is used instead of (1,5-cyclooctadiene) palladium dichloride.

[0069] Example 7-4 The specific implementation method of this embodiment is the same as that of embodiment 4, except that chromium acetylacetone is used instead of palladium dichloride (1,5-cyclooctadiene).

[0070] Example 7-5 The specific implementation method of this embodiment is the same as that of embodiment 4, except that cuprous bromide is used instead of palladium dichloride (1,5-cyclooctadiene).

[0071] Examples 7-6 The specific implementation method of this embodiment is the same as that of embodiment 4, except that anhydrous titanium tetrachloride is used instead of (1,5-cyclooctadiene) palladium dichloride.

[0072] Example 7-7 The specific implementation method of this embodiment is the same as that of embodiment 4, except that anhydrous zirconium chloride is used to replace (1,5-cyclooctadiene) palladium dichloride.

[0073] Examples 7-8 The specific implementation method of this embodiment is the same as that of embodiment 4, except that vanadium trichloride tetrahydrofuran complex is used instead of (1,5-cyclooctadiene) palladium dichloride.

[0074] Examples 7-9 The specific implementation method of this embodiment is the same as that of embodiment 4, except that hafnium tetrachloride is used instead of palladium dichloride (1,5-cyclooctadiene).

[0075] Example 8-1 This embodiment provides an enzyme-free organic ligand metal catalyst, obtained by reacting a monomer and a metal precursor. The preparation method includes: dissolving (1,5-cyclooctadiene)palladium dichloride and monomer I in ethyl acetate, with concentrations of 0.05 mol / L and 0.05 mol / L, respectively. Adding a (COD)PdCl2 solution to the monomer I solution, with a palladium ion to monomer I molar ratio of 1:1, the reaction mixture is stirred at room temperature for 12 h using a magnetic stirrer at 600 rpm under inert gas protection. After the reaction is complete, hexane is added to precipitate a solid precipitate, which is then centrifuged at 30000 g. The precipitate is washed twice with hexane and dried under vacuum overnight at 30 °C to obtain organic ligand palladium catalyst 1.

[0076] Example 8-2 The specific implementation method of this comparative example is the same as that of Example 8-1, except that monomer I is replaced by monomer II to obtain organic ligand palladium catalyst 2.

[0077] Example 8-3 The specific implementation method of this comparative example is the same as that of Example 8-1, except that monomer I is replaced by monomer III to obtain organic ligand palladium catalyst 3.

[0078] Example 8-4 The specific implementation method of this comparative example is the same as that of Example 8-1, except that anhydrous FeCl3 is used to replace (1,5-cyclooctadiene) palladium dichloride to obtain organic ligand iron catalyst 1.

[0079] Example 8-5 The specific implementation method of this comparative example is the same as that of Example 8-1, except that anhydrous CoCl2 is used to replace (1,5-cyclooctadiene) palladium dichloride to obtain organic ligand cobalt catalyst 1.

[0080] Examples 8-6 The specific implementation of this comparative example is the same as that of Example 8-1, except that nickel(II) 2-methoxyethyl ether complex is used instead of (1,5-cyclooctadiene) palladium dichloride.

[0081] Example 9-1 This embodiment provides an enzyme-metal composite catalyst, which is obtained by blending a monomer and a metal precursor to obtain an organic ligand metal catalyst, and then reacting it with an enzyme. The preparation method includes: dissolving activated enzyme protein 1 and the organic ligand palladium catalyst 1 obtained in Example 8-1 in ethyl acetate, with concentrations of 0.5 mmol / L and 0.05 mol / L, respectively. The molar ratio of enzyme to organic ligand palladium catalyst is 1:100. The reaction solution is protected with an inert gas and stirred at room temperature using a magnetic stirrer at 600 rpm for 12 h. After the reaction is complete, hexane is added to precipitate a solid precipitate, which is then centrifuged at 15000 rpm. The precipitate is washed twice with hexane and dried under vacuum at 30°C overnight to obtain the enzyme-palladium composite catalyst.

[0082] Example 9-2 The specific implementation method of this embodiment is the same as that of embodiment 9-1, except that the enzyme used is non-activated enzyme protein 1, and the organic ligand metal catalyst used is the organic ligand palladium catalyst 2 obtained in 8-2.

[0083] Example 9-3 The specific implementation method of this embodiment is the same as that of Embodiment 9-2, except that the organic ligand metal catalyst used is the organic ligand palladium catalyst 3 obtained in 8-3.

[0084] Example 9-4 The specific implementation method of this embodiment is the same as that of Embodiment 9-2, except that the organic ligand metal catalyst used is the organic ligand iron catalyst 1 obtained in 8-4.

[0085] Example 9-5 The specific implementation method of this embodiment is the same as that of Embodiment 9-2, except that the organic ligand metal catalyst used is the organic ligand cobalt catalyst 1 obtained in 8-5.

[0086] Examples 9-6 The specific implementation method of this embodiment is the same as that of Embodiments 9-2, except that the organic ligand metal catalyst used is the organic ligand nickel catalyst 1 obtained in 8-6.

[0087] Examples 9-7 The specific implementation method of this embodiment is the same as that of embodiments 9-6, except that organic ligand nickel catalyst 2 (the following formula) is used instead of organic ligand nickel catalyst 1.

[0088] Preparation method of organic ligand nickel catalyst 2: (1) 43 mmol of 3,5-di-tert-butyl-2-hydroxybenzaldehyde, 55.9 mmol of 2,6-diisopropylaniline, and 150 mL of methanol were added to a two-necked flask. 0.5 mL of formic acid was added under vigorous stirring, and the mixture was refluxed and stirred at room temperature for 3 days. After the reaction was completed, the precipitate was separated by filtration. The product was dissolved in dichloromethane and sodium sulfate was added and stirred overnight. The dried powder was purified by silica gel column chromatography (petroleum ether to ethyl acetate volume ratio of 20:1). Finally, it was recrystallized from a petroleum ether / ethyl acetate mixed solvent, washed with hexane at low temperature, and dried under vacuum overnight to obtain a high-purity product. The product was further dissolved in dry tetrahydrofuran, and solid NaH was slowly added to the solution. The mixture was stirred for 12 hours, and the mixture was further filtered through dry diatomaceous earth to remove the solvent. The product was dried under vacuum overnight.

[0089] (2) Take 8 mmol of the product from step 1 and 4 mmol of [(allyl)Ni(μ-X)]2 (X=H2CC(CO2Me)CH2) and dissolve it in 150 mL of tetrahydrofuran and stir for 24 hours. Then, remove the solvent by rotary evaporation to obtain organic ligand nickel catalyst 2 (the following formula).

[0090]

[0091] Examples 9-8 The specific implementation method of this embodiment is the same as that of embodiments 9-6, except that organic ligand nickel catalyst 3 (the following formula) is used instead of organic ligand nickel catalyst 1.

[0092] Preparation method of organic ligand nickel catalyst 3: (1) Add 137 mmol of anhydrous benzenesulfonic acid and 400 mL of tetrahydrofuran to a 1 L reaction flask, and heat at 0°C. o A solution of n-butyllithium (174 mL hexane, 274 mmol n-butyllithium) was added under C conditions, and the mixture was brought back to room temperature and stirred continuously for 3 hours. After the reaction was complete, it was further cooled to -78°C. o At temperature C, 125 mmol of diisopropylphosphine chloride was added, and the mixture was then brought back to room temperature and stirred continuously for 15 hours. After the reaction was completed, 137 mmol of trifluoroacetic acid was added to quench the reaction. The solvent was removed by vacuum distillation, and the residue was dissolved in dichloromethane and washed with saturated ammonium chloride solution. The organic layer was dried with anhydrous sodium sulfate and the solvent was removed by vacuum distillation to finally obtain a white powdery 2-(diisopropylphospho)benzenesulfonate.

[0093] (2) Under nitrogen protection, 0.12 mol of 2-(diisopropylphosphino)benzenesulfonate, 0.3 mol of sodium carbonate, and 200 mL of dichloromethane were added to a 500 mL Shrek flask and stirred at room temperature for 4 hours. A [Ni(allyl)Cl]2 solution (50 mL of dichloromethane and 0.072 mol of [Ni(allyl)Cl]2) was added to the reaction system, and stirring was continued for 3 hours. The mixture was filtered through dry diatomaceous earth and the solvent was removed under vacuum. Crystallization was carried out by slow diffusion of n-hexane (100 mL) into a concentrated dichloromethane solution (15 mL). The orange powder was collected by filtration, washed with n-hexane (3 × 20 mL), and then the solvent was removed by rotary evaporation of the mixture to obtain organic ligand nickel catalyst 3 (the following formula).

[0094]

[0095] Example 9-9 The specific implementation method of this embodiment is the same as that of embodiments 9-6, except that organic ligand nickel catalyst 4 (the following formula) is used instead of organic ligand nickel catalyst 1.

[0096] Preparation method of organic ligand nickel catalyst 4: In -20 o Under C conditions, an allyl nickel bromide solution (10 mL of dichloromethane, 0.5 mmol of allyl nickel bromide) was mixed with a (diphenylphosphine)methanethiol solution (5 mL of dichloromethane, 1 mmol of (diphenylphosphine)methanethiol) and stirred vigorously for 30 minutes. A silver hexafluoroantimonate solution (5 mL of dichloromethane, 1 mmol of silver hexafluoroantimonate) was added. The mixture was filtered through dry diatomaceous earth, and the residue was washed with diethyl ether and n-pentane. The solvent was then removed by rotary evaporation to obtain organic ligand nickel catalyst 4 (the following formula).

[0097]

[0098] Examples 9-10 The specific implementation method of this embodiment is the same as that of embodiments 9-6, except that organic ligand nickel catalyst 5 (the following formula) is used instead of organic ligand nickel catalyst 1.

[0099] Preparation of organic ligand nickel catalyst 5 (1) at 0 o Under C and argon protection, a solution of 2,6-di-tert-butylphenol (20 mL anhydrous tetrahydrofuran, 24.2 mmol of 2,6-di-tert-butylphenol) and a sodium hydride suspension (20 mL anhydrous tetrahydrofuran, 26.6 mmol of sodium hydride) were mixed and stirred. Then, under ice bath conditions, 36.3 mmol of chloromethyl ether was slowly added dropwise to the above solution, and the mixture was stirred at 0°C. oThe mixture was stirred at C for 5 hours, then reacted at room temperature for 3 hours. After the reaction was complete, the reaction solution was poured into a saturated ammonium chloride aqueous solution (100 mL). The crude product was extracted twice with 100 mL of diethyl ether, and the combined organic phases were washed with 100 mL of saturated sodium chloride aqueous solution and dried over anhydrous magnesium sulfate. The solvent was removed by vacuum distillation, and the product was dried under vacuum. The residue was dissolved in dichloromethane / n-hexane (1:1) and purified by silica gel column chromatography.

[0100] (2) at 0 o Under C conditions, a solution of the first step product in diethyl ether (30 mL of diethyl ether, 59.5 mmol of the first step product), a solution of N,N,N',N'-tetramethylethylenediamine (30 mL of diethyl ether, 59.5 mmol of N,N,N',N'-tetramethylethylenediamine), and a solution of n-butyllithium (39 mL of hexane, 62.4 mmol of n-butyllithium) were mixed and placed at 0°C. o Stir overnight at C. Then, at 0... o A solution of diisopropylphosphine chloride (15 mL petroleum ether, 59.4 mmol diisopropylphosphine chloride) was added dropwise to the above solution under C conditions, and the mixture was stirred and reacted at room temperature for 15 hours. After the reaction was complete, the solvent was removed by distillation under reduced pressure. The residue was extracted with dichloromethane, and the extract was washed successively with an aqueous solution of sodium dihydrogen phosphate (1 mol / L) and pure water, dried over anhydrous sodium sulfate, and then 80 mL of methanol was added and the mixture was dried over 0. o After C was left to stand for one day, white crystals precipitated. The crystals were then dried under vacuum.

[0101] (3) 14.2 mmol of the second-step product was suspended in 100 mL of methanol, and hydrogen chloride gas was bubbled through it at room temperature until saturation was reached and maintained for 15 h. The mixture was then refluxed for 1 hour to ensure complete pyrolysis. The solvent was removed by vacuum distillation, and the residue was dissolved in 20 mL of methanol, with water added dropwise until the solution became cloudy. The mixture was then heated to 0°C. o After being left to stand at C, white crystals precipitate out. These crystals are then dried under vacuum.

[0102] (4) Add 1.0 mmol of the product from step 3 to tetrahydrofuran, and add 1 mmol of thallium ethanol under stirring conditions, reducing the temperature of the mixture to 0. o C precipitates white crystals, which are then vacuum dried to obtain thallium phosphonophenolate. 0.245 mmol of thallium phosphonophenolate is suspended in 20 mL of tetrahydrofuran and dried at -10 °C. oA solution of (methylallyl nickel bromide) dimer (20 mL tetrahydrofuran, 0.245 mmol (methylallyl nickel bromide) dimer) was added at temperature C. The suspension was slowly heated to room temperature, and solids began to precipitate after 5 minutes. After 4 hours, the solids were filtered and separated, and carefully washed with tetrahydrofuran. The solvent was removed by vacuum distillation of the solids, 5 mL pentane was added, and the insoluble matter was separated and dried under vacuum to obtain organic ligand nickel catalyst 5.

[0103]

[0104] Example 10-1 The specific implementation method of this embodiment is the same as that of embodiment 4, except that laccase (purchased from Sigma-Aldrich) is used instead of enzyme protein 1.

[0105] Example 10-2 The specific implementation method of this embodiment is the same as that of embodiment 4, except that lipase BCL (purchased from Sigma-Aldrich) is used instead of enzyme protein 1.

[0106] Example 10-3 The specific implementation method of this embodiment is the same as that of embodiment 4, except that glutamine transferase (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) is used to replace enzyme protein 1.

[0107] Example 10-4 The specific implementation method of this embodiment is the same as that of embodiment 4, except that carbonic anhydrase (purchased from Sigma-Aldrich) is used instead of enzyme protein 1.

[0108] Examples 10-5 The specific implementation method of this embodiment is the same as that of embodiment 4, except that glucose isomerase (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) is used to replace enzyme protein 1.

[0109] Examples 10-6 The specific implementation method of this embodiment is the same as that of embodiment 4, except that bovine serum albumin (purchased from Sigma-Aldrich) is used instead of enzyme protein 1.

[0110] Experimental Example 1-1 This experimental example prepared branched polyethylene. The preparation method was as follows: A dry, high-pressure resistant reactor (0.5 L) was prepared and transferred to a glove box. 250 mL of n-hexane, 1 μmol of the enzyme-palladium composite catalyst prepared in Example 1-1 (calculated based on palladium content), and 500 eq of methylaluminoxane co-catalyst were added to the reactor. The reaction was maintained at 0.25 MPa, 0.50 MPa, 0.75 MPa, and 1.00 MPa ethylene partial pressure, and reacted at 25 °C for 0.5 h. After the reaction was completed, hydrochloric acid / ethanol solution (V:V = 1:1) was added to terminate the reaction. The product was washed with a large amount of methanol, collected, filtered, washed with methanol, dried, and weighed. The product properties are shown in Table 1.

[0111] Relative activity refers to the mass of polymer generated per mole of metal per hour; M W The weight-average molecular weight (PDI) of a polymer is the ratio of its weight-average molecular weight to its number-average molecular weight, reflecting the molecular weight distribution. The degree of branching refers to the number of branches per 1000 carbon atoms.

[0112] Table 1

[0113] Table 1 shows that the catalytic activity and molecular weight of the catalyst for ethylene polymerization increase with increasing ethylene partial pressure. The highest relative activity and molecular weight are achieved at 1 MPa, reaching 1.51 × 10⁻⁶. 7 g·mol 金属 -1 ·h -1 and 8.23*10 4 g / mol, and in subsequent examples, the preferred ethylene partial pressure is 1 MPa.

[0114] Experimental Examples 1-2 The specific implementation method of this experiment is the same as that of Experiment 1-1, except that the partial pressure of ethylene is 1 MPa, and the reaction is carried out at 25℃, 50℃, 75℃ and 100℃ for 0.5 h respectively. The properties of the product are shown in Table 2.

[0115] Table 2

[0116] As shown in Table 2, the catalytic activity of the catalyst for ethylene polymerization decreases with increasing reaction temperature, and the molecular weight of the product polyethylene decreases. The relative activity and molecular weight are highest at 25°C. Therefore, the preferred reaction temperature in subsequent examples is 25°C.

[0117] Experimental Examples 1-3 The specific implementation method of this experimental example is the same as that of Experiment 1-1, except that the partial pressure of ethylene is 1 MPa, the reaction time is 0.5 h at 25°C, and the catalyst used is different. The catalyst type is the enzyme-palladium composite catalyst prepared in Examples 2-1, 2-2, 2-3, and 2-4, and the activities of each catalyst are as follows: Figure 2 As shown.

[0118] Depend on Figure 2 It is known that the activity of enzyme-palladium catalyst in ethylene polymerization initially remains constant and then decreases as the ratio of enzyme to metal precursor remains constant. This is because the number of amino acids in the enzyme molecule that can regulate the electronic effects of the active site metal is limited. As the proportion of metal precursor increases, the activity decreases. To reduce the amount of enzyme-palladium composite catalyst used, the preferred molar ratio of enzyme to palladium precursor is 1:100.

[0119] Examples 1-4 The specific implementation method of this experimental example is the same as that of Experiment 1-1, except that the partial pressure of ethylene is 1 MPa, the reaction time is 0.5 h at 25°C, and the catalyst used is different. The catalyst is the enzyme-palladium composite catalyst prepared in Examples 3-1, 3-2, 3-3, 3-4, and 3-5, and the activity of each catalyst is as follows: Figure 3 As shown. By Figure 3 It can be seen that the ratio of metal precursor to monomer has little effect on the activity of enzyme-palladium catalyst in ethylene polymerization.

[0120] Examples 1-5 The specific implementation method of this experiment is the same as that of Experiment 1-1, except that the partial pressure of ethylene is 1 MPa, the reaction time is 0.5 h at 25 °C, and a different catalyst is used. The specific catalyst type and product properties are shown in Table 3.

[0121] Table 3

[0122] Compared to Example 8-1, the enzyme-palladium composite catalyst in Example 1-1 showed a 169% increase in activity and a 37% increase in molecular weight, as well as an increase in branching degree, compared to the organic ligand palladium catalyst. This is because the enzyme modulates the electronic effect of the metal, increasing its catalytic activity. Furthermore, the enzyme protein 2@Pd, enzyme protein 3@Pd, and enzyme protein 4@Pd composite catalysts, prepared by mutating the acidic amino acids aspartic acid and glutamic acid in enzyme protein 1 to electron-donating amino acids lysine, arginine, and histidine and then combining them with palladium, showed activities 83%, 41%, and 100% higher than the unmutated enzyme-palladium composite catalyst, respectively. The branching degree reached 101 / 1000C after the mutation to arginine. Further mutation of the acidic amino acids aspartic acid and glutamic acid in the enzyme into nonpolar, sterically hindered alanine and leucine, combined with palladium, resulted in enzyme protein 5@Pd and enzyme protein 6@Pd catalysts with 12% and 89% higher activity, respectively, compared to the unmutated enzyme-palladium composite catalyst. The branching degree of leucine-mutated enzyme protein 6@Pd reached 10⁹ / 1000C, with a 33% increase in branching degree compared to enzyme protein 5@Pd. In addition, the enzyme-iron, enzyme-cobalt, and enzyme-nickel composite catalysts all demonstrated the ability to prepare polyethylene, and their activities were improved compared to organic ligand palladium, organic ligand iron, organic ligand cobalt, and organic ligand nickel catalysts.

[0123] Experimental Example 2 This experimental example prepared ethylene / α-olefin elastomers. The preparation method was as follows: A dry, high-pressure resistant reactor (0.5 L) was prepared and transferred to a glove box. 250 mL of n-hexane, 19.6 mL of 1-octene, 1 μmol of different catalysts (calculated based on metal content), 500 eq of methylaluminoxane co-catalyst, and an ethylene partial pressure (0.5 MPa) were added to the reactor. The reaction was carried out at 25 °C for 0.5 h. After the reaction was completed, hydrochloric acid / ethanol solution was added to terminate the reaction. The product was washed with a large amount of methanol, collected, filtered, washed with methanol, dried, and weighed. The product properties are shown in Table 4.

[0124] Table 4

[0125] Table 4 shows that the enzyme-palladium catalyst exhibits a 213% higher activity, a 62% higher molecular weight, and a 33-fold higher 1-octene insertion rate compared to the organic ligand palladium catalyst in the polymerization of ethylene and 1-octene. Furthermore, the enzyme protein 2@Pd, enzyme protein 5@Pd, and enzyme protein 6@Pd composite catalysts, prepared by mutating the acidic amino acids aspartic acid and glutamic acid in the enzyme to lysine, alanine, and leucine respectively, and then combining them with palladium, show activity increases of 57%, 12%, and 5% respectively compared to the unmutated enzyme-palladium composite catalyst. In addition, the 1-octene insertion rate changes significantly after mutation to alanine and leucine. These findings demonstrate that the insertion rate of α-olefin monomers can be controlled by altering the amino acid type of the enzyme.

[0126] Experimental Example 3 This experimental example prepared an ethylene / polar monomer copolymer. The preparation method was as follows: A dry, high-pressure resistant reactor (0.5 L) was prepared and transferred to a glove box. 250 mL of toluene, 1.23 mL of methyl acrylate, 5 μmol of a metal-based catalyst, 500 eq of methylaluminoxane co-catalyst, and an ethylene partial pressure of 0.5 MPa were added to the reactor. The reaction was carried out at 25 °C for 1 h. After the reaction was completed, hydrochloric acid / ethanol solution was added to terminate the reaction. The product was washed with a large amount of methanol, collected, filtered, washed with methanol, dried, and weighed. The product properties are shown in Table 5.

[0127] Table 5

[0128] Compared with Comparative Example 2, the enzyme-palladium composite catalyst 2 showed a 78% increase in activity, a 72% increase in molecular weight, and a 36% increase in methyl acrylate grafting density in the polymerization of ethylene and methyl acrylate compared with the organic ligand palladium catalyst 2. This is because the enzyme preferentially adsorbs and enriches polar monomers through its functional groups, increasing their local concentration near the active site. At the same time, the electronic effect provided by the enzyme reduces the probability of polar monomers directly poisoning the metal center, thereby significantly improving the stability of the catalyst and the grafting density of polar monomers.

[0129] The applicant declares that this application is illustrated by the above embodiments, but this application is not limited to the above embodiments, that is, it does not mean that this application must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this application, equivalent substitutions of raw materials for the products of this application, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this application.

[0130] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not describe the various possible combinations separately.

Claims

1. An enzyme / protein-metal composite catalyst, characterized in that, It is obtained by the reaction of enzymes or proteins, monomers and metal precursors; The metal precursors include at least one of (1,5-cyclooctadiene)palladium dichloride, anhydrous ferric chloride, anhydrous cobalt chloride, nickel(II) 2-methoxyethyl ether complex, chromium acetylacetone, cuprous bromide, anhydrous titanium tetrachloride, anhydrous zirconium chloride, anhydrous vanadium trichloride, and anhydrous hafnium tetrachloride. Enzymes include at least one of the following: oxidoreductases, hydrolases, transferases, lyases, isomerases, or any inactive enzyme protein or carrier protein. The structural formula of the monomer is shown in Formula 1: Formula 1.

2. The enzyme / protein-metal composite catalyst according to claim 1, characterized in that, The molar ratio of the enzyme / protein to the metal precursor is 1:(1-1000), and / or the molar ratio of the monomer to the metal precursor is 1:(0.1-20).

3. The enzyme / protein-metal composite catalyst according to claim 2, characterized in that, The R 1 -R 12 Substituents are H-substituted or / and C1-C20 alkyl-substituted or / and aromatic hydrocarbons and their derivatives substituted or / and naphthyl or / and alkyl halogens or / and amino-substituted or / and carboxyl-substituted or / and aldehyde-substituted or / and epoxy-substituted.

4. A method for preparing an enzyme / protein-metal composite catalyst according to any one of claims 1-3, characterized in that, include: The enzyme / protein, monomer, and metal precursor are dissolved separately in a solvent. Under an inert gas atmosphere, the three solutions are mixed and stirred. The solid precipitate is separated, washed, and dried to obtain the final product.

5. An enzyme / protein-metal composite catalyst, characterized in that, The organic ligand metal catalyst is first obtained by reacting the monomer and metal precursor as described in any one of claims 1-3, and then the organic ligand metal catalyst is combined with an enzyme / protein to obtain the catalyst. The structural formula of the organic ligand metal catalyst is selected from at least one of Formulas 2-6: Formula 2 Formula 3 Formula 4 Formula 5 Formula 6.

6. The enzyme / protein-metal composite catalyst according to claim 5, characterized in that, The molar ratio of the enzyme / protein and the organic ligand metal catalyst is 1:(1-1000).

7. The enzyme / protein-metal composite catalyst according to claim 6, characterized in that, M is a metal ion, including at least one of palladium, iron, cobalt, nickel, chromium, copper, titanium, zirconium, vanadium, and hafnium ions; X is any one of the halogens.

8. A method for preparing an enzyme / protein-metal composite catalyst according to any one of claims 5-7, characterized in that, include: The enzyme / protein and the organic ligand metal catalyst are dissolved separately in solvents. Under an inert gas atmosphere, the two solutions are mixed and stirred. The solid precipitate is separated, washed, and dried to obtain the final product.

9. The application of the enzyme / protein-metal composite catalyst according to claim 1 or 5, characterized in that, It is used in the catalytic copolymerization of ethylene with ethylene and / or ethylene with α-olefins and / or ethylene with polar monomers.

10. A method for synthesizing functionalized polyethylene, characterized in that, The synthesis method uses the enzyme / protein-metal composite catalyst described in claim 1 or 5.

Images