WO2020113716A1 - 一种高分子-酶-金属复合纳米催化剂及其可控合成方法 - Google Patents

一种高分子-酶-金属复合纳米催化剂及其可控合成方法 Download PDF

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WO2020113716A1
WO2020113716A1 PCT/CN2018/123877 CN2018123877W WO2020113716A1 WO 2020113716 A1 WO2020113716 A1 WO 2020113716A1 CN 2018123877 W CN2018123877 W CN 2018123877W WO 2020113716 A1 WO2020113716 A1 WO 2020113716A1
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polymer
enzyme
metal
lipase
conjugate
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French (fr)
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戈钧
黎晓阳
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清华大学
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
    • B01J31/26—Catalysts comprising hydrides, coordination complexes or organic compounds containing in addition, inorganic metal compounds not provided for in groups B01J31/02 - B01J31/24
    • B01J31/28—Catalysts comprising hydrides, coordination complexes or organic compounds containing in addition, inorganic metal compounds not provided for in groups B01J31/02 - B01J31/24 of the platinum group metals, iron group metals or copper
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J35/00—Catalysts, in general, characterised by their form or physical properties
    • B01J35/30—Catalysts, in general, characterised by their form or physical properties characterised by their physical properties
    • B01J35/391—Physical properties of the active metal ingredient
    • B01J35/393—Metal or metal oxide crystallite size
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J35/00—Catalysts, in general, characterised by their form or physical properties
    • B01J35/40—Catalysts, in general, characterised by their form or physical properties characterised by dimensions, e.g. grain size

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  • the invention belongs to the field of catalyst preparation, and in particular relates to a polymer-enzyme-metal composite nanocatalyst and a controllable synthesis method thereof.
  • the chemical-biological coupling catalytic process coupling heterogeneous catalysis and enzyme catalysis has huge application potential in biomedicine production and chemical green manufacturing industries.
  • the "one-pot" multi-step cascade or cascade chemical-biological reaction catalyzed by the dual-function chemical-biological composite catalyst can avoid the separation and purification steps of reaction intermediate products, and has the advantages of economy, environmental protection and high efficiency.
  • the reaction conditions of the enzyme catalysis process and the noble metal heterogeneous catalysis process do not match in most cases. The respective reaction conditions will lead to the deactivation of the other catalyst, thus seriously restricting the development of the chemical-biological coupling catalytic reaction.
  • reaction conditions for enzyme catalysis are normal temperature, normal pressure, and aqueous solution; on the contrary, most of the heterogeneous catalytic reactions of precious metals need to be carried out under harsh conditions such as high temperature, high pressure, gas protection, and organic solvents. How to prepare a metal-enzyme bifunctional catalyst capable of performing a highly efficient and highly selective catalytic reaction under mild conditions is still a challenge.
  • Patent application 101239295A discloses a preparation method of nanometer micrometer composite micrometers, by chemically bonding nanoparticles with surface functional groups to the surface of functionalized microspheres, and then polymerizing the opposite charges on the surface of nanospheres
  • the nano-micron composite microspheres can be used as catalysts and enzyme carriers. It mainly solves the problem of the stability of nanocomposite microspheres, but does not give the specific combination of nanocomposite microspheres with catalysts and enzymes and the application of metal-enzyme dual-function catalysts in different situations.
  • a feasible scheme for synthesizing metal-enzyme bifunctional catalyst is to use enzyme molecules to assist in-situ synthesis of metal nanoparticles.
  • Patent application CN106729713A discloses a preparation method of protein-coated metal sulfide nanoparticles, which utilizes protein to catalyze metal ions such as Ag + , Bi 3+ , Cd 2+ , Cu 2+ in an alkaline solution to generate protein-coated metal Sulfide nanoparticles.
  • the nanoparticles have an average particle size of 3.0-7.0 nm, and can be used for tumor photothermal therapy reagents.
  • Patent application CN106315663A discloses a method for biosynthesis of ultra-small metal chalcogenide nanocrystals.
  • This method utilizes a series of neutral or acidic proteins with isoelectric point less than 9.0 to reduce metal nanoparticles such as Cu, Cd, Zn and Mo in situ in an alkaline solution to generate metal chalcogenide nanocrystals with a size below 10 nm.
  • the nanocrystal can be used in biological fluorescence imaging and photothermal therapy preparations.
  • Filice et al. used natural lipase as a template to synthesize lipase-palladium, Hybrid aggregates such as lipase-gold and lipase-silver.
  • the lipase-palladium nanoparticle hybrid catalyst can be used for ( ⁇ )-1-phenethylamine dynamic kinetics Split reaction.
  • the present invention provides a polymer-enzyme-metal composite nano-catalyst and a controllable synthesis method thereof.
  • Method so that the prepared metal nanoparticles (sub-nano clusters) with extremely small particle size ( ⁇ 5nm) can be uniformly dispersed inside a single polymer-enzyme nanocomposite, effectively preventing aggregation between metal nanoparticles To achieve controllable backup.
  • the method is simple and easy to operate, and the prepared polymer-enzyme-metal composite nanocatalyst has a wide range of applications, and has good application prospects in the fields of chiral drugs, fine chemicals, pesticides, and photothermal therapeutic preparations.
  • a polymer-enzyme-metal composite nanocatalyst is a metal salt nanoparticle composite catalyst limited to the polymer-enzyme conjugate.
  • the content of the enzyme in the polymer-enzyme conjugate is 5-20 wt%; the mass loading of the metal nanoparticles in the polymer-enzyme-metal composite nanocatalyst is 0.1-30 wt%.
  • the metal nanoparticles are grown inside a single polymer-enzyme conjugate.
  • the polymer-enzyme conjugate is nano-dispersed in the dispersed phase, the particle size distribution is 5-80 nm, and the average particle size is 10-40 nm.
  • the polymer-enzyme-metal composite nanocatalyst is nano-dispersed in the dispersed phase, the particle size distribution is 10-200 nm, and the metal nanoparticle size is 0.2 nm-5 nm.
  • the polymer-enzyme-metal composite nanocatalyst has a surface charge of -0.1--5 at a neutral pH.
  • the particle size of the polymer-enzyme-metal composite nanocatalyst is 20-100 nm.
  • the raw materials for preparing the catalyst include high molecular compounds, enzymes, and metal salts.
  • the polymer compound is a polyether block copolymer containing hydroxyl-terminated polyoxyethylene-polyoxypropylene-polyoxyethylene, specifically with At least one of them.
  • Another object of the present invention is to provide a controllable synthesis method of the above polymer-enzyme-metal composite nanocatalyst, including the following steps:
  • the oxidation reaction time in step (1) is 10-24h; the Schiff base reaction time is 0.5-4h; and the reduction reaction time is 10-20h.
  • the reaction time in step (2) is 10-24h.
  • the oxidant is at least one of manganese dioxide, pyridine chlorochromate, Dess-Martin oxidant and pyridine dichromate;
  • the reducing agent is one or both of sodium borohydride and sodium cyanoborohydride.
  • the mass ratio of the polymer compound to the oxidant is 5-500:5-100.
  • the mass ratio of the polymer compound to the enzyme is 5-500:5-15.
  • the mass ratio of the enzyme to the reducing agent is 5-15:5-50.
  • the mass percentage concentration of the polymer compound in the organic solvent is 1-5%.
  • the organic solvent is at least one of dichloromethane, chloroform, toluene, dimethyl sulfoxide and chloroform.
  • the precipitating agent is at least one of diethyl ether, petroleum ether and n-hexane.
  • the buffer solution is a phosphate buffer solution
  • the pH value is 6-9
  • the concentration is 10-100 mM.
  • the enzyme is Candida antarctica lipase B, porcine pancreatic lipase, Candida rugosa lipase, Rhizopus oryzae lipase, Thermomyces lanuginosus lipase , Cytochrome C, glucose oxidase, Saccharomyces cerevisiae alcohol dehydrogenase, horse liver alcohol dehydrogenase, acetaldehyde dehydrogenase, ketone reductase, formate dehydrogenase, and formaldehyde dehydrogenase.
  • the metal salt is palladium acetate, tetrachloropalladium acid, palladium chloride, palladium nitrate, chloroauric acid, silver nitrate, silver fluoride, silver acetate, ruthenium trichloride, chlorine At least one of platinum acid, potassium tetrachloroplatinate, and potassium hexachloroplatinate.
  • the alcohol is at least one of methanol, ethanol, glycerin, ethylene glycol, isopropanol, n-butanol, and sorbitol; its volume concentration in water solvent is 5 -80%, preferably 5-50%.
  • the mass ratio of the polymer-enzyme conjugate and the metal salt is 1-50:1.
  • the mass ratio of the metal salt to alcohol is 1:100-1000.
  • the molecular weight of the dialysis bag is 12000-14000 Da.
  • the invention includes the construction of a polymer-enzyme conjugate and the in-situ reduction of metal ions within a single polymer-enzyme conjugate.
  • the terminal hydroxyl group of the polymer is oxidized to an aldehyde group under the action of an oxidizing agent, and then the coupling reaction between the aldehyde group of the polymer compound and the amino group of the enzyme occurs in the buffer solution, and then, the carbon-nitrogen formed by the coupling is added with a reducing agent
  • the double bond is reduced to a stable carbon-nitrogen single bond to make a polymer-enzyme conjugate;
  • the polymer-enzyme conjugate is reacted with a metal salt, and the polymer-enzyme conjugate is reduced in situ at room temperature
  • Metal ions and metal nanoparticles grow in a single polymer-enzyme conjugate.
  • a polymer-enzyme-metal composite nanocatalyst can be obtained through dialysis purification and freeze-drying processes.
  • the polymer-enzyme-metal composite nanocatalyst of the present invention can controllably synthesize a metal-enzyme bifunctional catalyst with metal nanoparticles of extremely small size ( ⁇ 5 nm) by simply adjusting the ratio of the reaction precursor.
  • Metal nanoparticles grow in the three-dimensional confined space on the enzyme surface of a single polymer-enzyme conjugate ( ⁇ 80nm), and the polymer-enzyme conjugate has a small amount of negative charge on the surface under neutral pH conditions to effectively avoid the conjugate Between the gathering, the preparation process can be controlled.
  • the polymer-enzyme-metal composite nanocatalyst of the present invention achieves nano-scale dispersion in the organic phase, and has no immobilized carrier, which greatly reduces the reaction mass transfer resistance, and has the advantages of high activity and good stability. Moreover, the polymer-enzyme-metal composite nanocatalyst can be precipitated from an organic solvent at a low temperature and can be used repeatedly.
  • the polymer-enzyme-metal composite nanocatalyst of the present invention has metal particles with extremely small particle diameters, and has high activity under conventional reaction conditions and even at lower reaction temperatures, effectively improving the chemical-biological coupling catalytic reaction of metals and enzymes Compatibility and catalytic efficiency.
  • the polymer-enzyme-metal composite nano-catalyst of the present invention can be prepared in batches, and has a very good application prospect in the fields of chiral drugs, fine chemicals, pesticides, photothermal therapeutic preparations and other fields.
  • Figure 1 is a TEM image of the CALB-Pluronic conjugate prepared in step (2) of Example 1;
  • Example 2 is a HAADF-STEM diagram of the Pd/CALB-Pluronic composite nanocatalyst prepared in Example 1;
  • Example 3 is a TEM image of the Ag/CALB-Pluronic composite nanocatalyst prepared in Example 2;
  • Example 4 is a TEM image of the Au/CALB-Pluronic composite nanocatalyst prepared in Example 3;
  • Example 5 is a TEM image of the Pd/CALB-Pluronic composite nanocatalyst prepared in Example 4.
  • Figure 11 is a comparison of the conversion rates of chiral resolution of 1-phenylethylamine racemate catalyzed by different forms of lipase
  • Figure 12 is a comparison of the efficiency of the racemic reaction of (S)-1-phenethylamine catalyzed by different forms of immobilized palladium nanoparticles;
  • Figure 13 is a comparison of the dynamic kinetic resolution of ( ⁇ )-1-phenethylamine catalyzed by polymer-lipase-palladium composite nanocatalyst and commercial lipase and palladium catalyst (Novozymes 435 and palladium carbon);
  • Figure 14 is a comparison of the catalytic performance of polymer-lipase-palladium composite nano-catalyst recycling.
  • Candida antarctica lipase B (enzyme activity 9U/mg) is 10 parts by weight, polymer compound It is 60 parts by weight, the activator Dess-Martin oxidant is 10 parts by weight, and the reducing agent sodium cyanoborohydride is 5 parts by weight.
  • Step (1) The above proportioned And Dess-Martin oxidant dissolved in dichloromethane, where, The mass fraction in dichloromethane is 1.5%, oxidation reaction is carried out at room temperature for 24 hours, the terminal hydroxyl group is oxidized to an aldehyde group, and then cold ether is added to precipitate to obtain an aldehyde-based polymer compound, which is vacuum dried;
  • Step (2) dissolve the aldehyde-based polymer compound and lipase obtained in step (1) in 10 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 7, and couple the aldehyde group to the amino group at room temperature
  • the reducing agent sodium cyanoborohydride was added to carry out the reduction reaction of the carbon-nitrogen double bond for 18 hours to reduce the carbon-nitrogen double bond formed by the coupling to a stable carbon-nitrogen single bond .
  • the product was lyophilized to obtain CALB-Pluronic, a polymer-lipase conjugate.
  • the polymer-lipase conjugate (containing approximately 10% of lipase CALB) is 28 parts by weight, the divalent palladium complex palladium acetate is 1 part by weight, and methanol is 267 parts by weight.
  • the size of the palladium nanoclusters in the Pd/CALB-Pluronic prepared above is about 0.8 nm, and the mass loading of the metal nanoparticles in the catalyst is 4 wt%
  • the enzymes are Candida antarctica lipase B (enzyme activity is 9U/mg) and Candida rugosa lipase (enzyme activity is 700-1,000U/mg) are 5 parts and 5 parts by weight, polymer compound with It is 60 parts by weight and 40 parts, the activator manganese dioxide oxidizer is 50 parts by weight, and the reducing agent sodium cyanoborohydride is 10 parts by weight.
  • Step (1) The above proportioned with And Dess-Martin oxidant dissolved in chloroform, where, with The mass fraction in chloroform is 5%, the oxidation reaction is carried out at room temperature for 16 hours, the terminal hydroxyl group is oxidized to an aldehyde group, and then a mixed solution of cold ether and petroleum ether is added to precipitate to obtain an aldehyde-based polymer compound, which is vacuum dried;
  • Step (2) Dissolve the aldehyde-based polymer compound obtained in step (1), Candida antarctica lipase B and Candida rugosa lipase in 100 mM disodium phosphate-sodium dihydrogen phosphate buffer at pH 7
  • the coupling reaction of aldehyde group and amino group was carried out at room temperature for 4 hours, and the reducing agent sodium cyanoborohydride was added to perform the reduction reaction of the carbon-nitrogen double bond for 10 hours, so that the carbon-nitrogen double bond formed by the coupling was reduced to stable Carbon-nitrogen single bond.
  • the product was lyophilized to obtain Lipase-Pluronic, a polymer-lipase conjugate.
  • the polymer-lipase conjugate (containing Candida antarctica lipase B and Candida rugosa lipase about 20%) is 25 parts by weight, the precursor silver nitrate is 1 part by weight, and methanol is by weight 50 parts, 50 parts by weight of glycerin.
  • the size of the nano-clusters in the Ag/Lipase-Pluronic prepared above is about 0.5 nm, and the mass loading of the metal nano-particles in the catalyst is 5 wt%
  • the enzyme is Thermomyces lanuginosus lipase (enzyme activity 100,000-180,000 U/g) is 10 parts by weight, polymer compound It is 5 parts by weight, the activator pyridinium chlorochromate oxidant is 5 parts by weight, and the reducing agent sodium borohydride is 5 parts by weight.
  • Step (1) The above proportioned And pyridinium chlorochromate oxidant is dissolved in the mixed solution of dichloromethane and chloroform, where, The mass fraction in dichloromethane and chloroform is 1%, the oxidation reaction is carried out at room temperature for 10 hours, the terminal hydroxyl group is oxidized to an aldehyde group, and then cold petroleum ether is added to precipitate to obtain an aldehyde-based polymer compound, which is vacuum dried;
  • Step (2) Dissolve the aldehyde-based polymer compound obtained in step (1) and Thermomyces lanuginosus lipase in 50 mM disodium phosphate-sodium dihydrogen phosphate buffer at pH 7, room temperature After 0.5 hours of coupling reaction between aldehyde group and amino group, the reducing agent sodium borohydride was added to reduce the carbon-nitrogen double bond for 20 hours, so that the carbon-nitrogen double bond formed by coupling was reduced to a stable carbon-nitrogen single bond . After dialysis, the product was lyophilized to obtain Lipase-Pluronic, a polymer-lipase conjugate.
  • the polymer-lipase conjugate (containing about 10% of Thermomyces lanuginosus lipase) is 50 parts by weight, the precursor chloroauric acid is 1 part by weight, and the methanol is 400 parts by weight. Ethylene glycol is 600 parts by weight.
  • the size of the nano-clusters in the Au/Lipase-Pluronic prepared above is about 0.2 nm, and the mass loading of the metal nano-particles in the catalyst is 0.1 wt%.
  • the enzyme is porcine pancreatic lipase (enzyme activity is 100,000-400,000 U/g), 15 parts by weight, polymer compound It is 500 parts by weight, the activator Dess-Martin oxidant is 100 parts by weight, and the reducing agent sodium cyanoborohydride is 50 parts by weight.
  • Step (1) The above proportioned And Dess-Martin oxidant dissolved in chloroform, where, The mass fraction in chloroform is 5%, the oxidation reaction is carried out at room temperature for 24 hours, the terminal hydroxyl group is oxidized to an aldehyde group, and then cold n-hexane is added to precipitate to obtain an aldehyde-based polymer compound, which is vacuum dried;
  • Step (2) dissolve the aldehyde-based polymer compound and porcine pancreatic lipase obtained in step (1) in a 10 mM disodium phosphate-sodium dihydrogen phosphate buffer solution at pH 6, and perform the aldehyde group and amino group at room temperature After 3 hours of the coupling reaction, the reducing agent sodium cyanoborohydride was added to perform the reduction reaction of the carbon-nitrogen double bond for 16 hours to reduce the carbon-nitrogen double bond formed by the coupling to a stable carbon-nitrogen single bond. After dialysis, the product was lyophilized to obtain Lipase-Pluronic, a polymer-lipase conjugate.
  • the polymer-lipase conjugate (containing about 15% of porcine pancreatic lipase) is 50 parts by weight, the divalent palladium complex palladium acetate is 0.5 parts by weight, and the tetrachloropalladium acid is 0.5 parts by weight.
  • Methanol is 1000 parts by weight.
  • the size of the palladium nanoclusters in the Pd/Lipase-Pluronic prepared above is about 2.5 nm, and the mass loading of the metal nanoparticles in the catalyst is 20 wt%
  • the enzyme is Saccharomyces cerevisiae alcohol dehydrogenase (enzyme activity ⁇ 300U/mg) is 10 parts by weight, polymer compound It is 160 parts by weight, the activator Dess-Martin oxidant is 27 parts by weight, and the reducing agent sodium cyanoborohydride is 16 parts by weight.
  • Step (1) The above proportioned And Dess-Martin oxidant dissolved in dichloromethane, where, The mass fraction in dichloromethane is 5%, the oxidation reaction is carried out at room temperature for 24 hours, the terminal hydroxyl group is oxidized to an aldehyde group, and then cold ether is added to precipitate to obtain an aldehyde-based polymer compound, which is dried in vacuum;
  • Step (2) Dissolve the aldehyde-based polymer compound and alcohol dehydrogenase obtained in step (1) in a 10 mM disodium phosphate-sodium dihydrogen phosphate buffer solution at pH 7, and perform the aldehyde group and amino group at room temperature. After 2 hours of the coupling reaction, sodium cyanoborohydride was added to reduce the carbon-nitrogen double bond for 10 hours to reduce the carbon-nitrogen double bond formed by the coupling to a stable carbon-nitrogen single bond. After dialysis, the product was lyophilized to obtain the polymer-ethanol dehydrogenase conjugate ADH-Pluronic.
  • the polymer-ethanol dehydrogenase conjugate (containing about 5% of alcohol dehydrogenase ADH) is 15 parts by weight, the divalent palladium complex palladium acetate is 1 part by weight, and methanol is 500 parts by weight.
  • the size of the nano clusters in the Pd/ADH-Pluronic prepared above is about 1.8 nm, and the mass loading of the metal nano particles in the catalyst is 25 wt%.
  • the enzyme is glucose oxidase (derived from Aspergillus niger, the enzyme activity is 100,000-250,000U/g) 25 parts by weight, polymer compound with 30 parts, 30 parts and 40 parts by weight, activator Dess-Martin and manganese dioxide oxidant are 40 parts and 60 parts by weight, and the reducing agents sodium cyanoborohydride and sodium borohydride are by weight Counted as 7 and 3 copies.
  • Step (1) The above proportioned with And Dess-Martin and manganese dioxide oxidant dissolved in a mixed solution of chloroform, toluene and dimethyl sulfoxide, where, with The mass fraction in the mixed solution of chloroform, toluene and dimethyl sulfoxide is 4%, the oxidation reaction is carried out at room temperature for 16 hours, the terminal hydroxyl group is oxidized to the aldehyde group, and then the mixed solution of cold ether and petroleum ether is added to precipitate to obtain the aldehyde The basic polymer compound is vacuum dried;
  • Step (2) dissolve the aldehyde-based polymer compound and glucose oxidase obtained in step (1) in a 20 mM disodium phosphate-sodium dihydrogen phosphate buffer solution at pH 8, and couple the aldehyde group with the amino group at room temperature
  • the reducing agents sodium cyanoborohydride and sodium borohydride were added to carry out the reduction reaction of the carbon-nitrogen double bond for 16 hours, so that the carbon-nitrogen double bond formed by the coupling was reduced to a stable carbon-nitrogen single bond.
  • the product was freeze-dried to obtain the polymer-glucose oxidase conjugate GOx-Pluronic.
  • the polymer-glucose oxidase conjugate (containing about 13% of glucose oxidase) is 15 parts by weight, the precursor chloroauric acid is 3 parts by weight, and the methanol is 378 parts by weight.
  • the size of the nano-clusters in the Au/GOx-Pluronic prepared above is about 1.5 nm, and the mass loading of the metal nanoparticles in the catalyst in the catalyst is 15 wt%.
  • Enzyme is cytochrome C (from Saccharomyces cerevisiae) 5 parts by weight, polymer compound It is 50 parts by weight, the activator manganese dioxide oxidant is 70 parts by weight, and the reducing agent sodium cyanoborohydride is 8 parts.
  • Step (1) The above proportioned And manganese dioxide oxidant is dissolved in chloroform, where, The mass fraction in the three-filtered methane is 1%, and the oxidation reaction is carried out at room temperature for 14 hours, the terminal hydroxyl group is oxidized to an aldehyde group, and then cold petroleum ether is added to precipitate to obtain an aldehyde-based polymer compound, which is vacuum dried;
  • Step (2) Dissolve the aldehyde-based polymer compound and cytochrome C obtained in step (1) in a 10 mM disodium phosphate-sodium dihydrogen phosphate buffer solution with a pH of 9, and couple the aldehyde group with the amino group at room temperature
  • the reducing agent sodium cyanoborohydride was added to carry out the reduction reaction of the carbon-nitrogen double bond for 18 hours to reduce the carbon-nitrogen double bond formed by the coupling to a stable carbon-nitrogen single bond.
  • the product was lyophilized to obtain the polymer-cytochrome C conjugate Cyt C-Pluronic.
  • the polymer-cytochrome C conjugate (containing about 20% of cytochrome C) is 1 part by weight, the precursor silver acetate is 1 part by weight, and ethanol is 700 parts by weight.
  • the size of the nano-clusters in the Ag/Gyt C-Pluronic prepared above is about 3 nm, and the mass loading of the metal nano-particles in the catalyst is 30 wt%.
  • the enzyme is glucose oxidase (derived from Aspergillus niger, enzyme activity is 100,000-250,000U/g) is 10 parts by weight, polymer compound 100 parts by weight, activator Dess-Martin, manganese dioxide and pyridine dichromate oxidant were 20 parts, 14 parts and 16 parts by weight, respectively, and the reducing agent sodium borohydride was 5 parts by weight.
  • Step (1) The above proportioned And Dess-Martin, manganese dioxide and pyridinium dichromate oxidant dissolved in dichloromethane solution, where, The mass fraction in dichloromethane is 5%, the oxidation reaction is carried out at room temperature for 20 hours, the terminal hydroxyl group is oxidized to an aldehyde group, and then a solution of cold ether is added to precipitate to obtain an aldehyde-based polymer compound, which is vacuum dried;
  • Step (2) dissolve the aldehyde-based polymer compound and glucose oxidase obtained in step (1) in 80 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7, and couple the aldehyde group with the amino group at room temperature
  • the reducing agent sodium borohydride was added to carry out the reduction reaction of the carbon-nitrogen double bond for 15 hours, so that the carbon-nitrogen double bond formed by the coupling was reduced to a stable carbon-nitrogen single bond.
  • the product was freeze-dried to obtain the polymer-glucose oxidase conjugate GOx-Pluronic.
  • the polymer-glucose oxidase conjugate (containing about 15% of glucose oxidase) is 10 parts by weight, the precursors chloroplatinic acid and potassium tetrachloroplatinate are 1 part and 1 part respectively by weight, ethylene glycol 100 parts by weight.
  • the size of the nanoclusters in the Pt/GOx-Pluronic prepared above is about 5 nm, and the mass loading of the metal nanoparticles in the catalyst is 10 wt%.
  • Candida antarctica lipase B (enzyme activity 9U/mg) is 10 parts by weight, polymer compound It is 70 parts by weight, the activator manganese dioxide is 100 parts by weight, and the reducing agent sodium cyanoborohydride is 10 by weight.
  • Step (1) The above proportioned And manganese dioxide oxidant is dissolved in the mixed solution of dichloromethane and chloroform, where, The mass fraction in the mixed solution of dichloromethane and chloroform is 2%, the oxidation reaction is carried out at room temperature for 12 hours, the terminal hydroxyl group is oxidized to an aldehyde group, and then a solution of cold petroleum ether is added to precipitate to obtain a high aldehyde group. Molecular compounds, vacuum dried;
  • Step (2) Dissolve the aldehyde-based polymer compound and lipase obtained in step (1) in a 50 mM disodium phosphate-sodium dihydrogen phosphate buffer solution at pH 7, and couple the aldehyde group to the amino group at room temperature
  • the reducing agent sodium cyanoborohydride was added to carry out the reduction reaction of the carbon-nitrogen double bond for 14 hours to reduce the carbon-nitrogen double bond formed by the coupling to a stable carbon-nitrogen single bond.
  • the product was lyophilized to obtain CALB-Pluronic, a polymer-lipase conjugate.
  • the polymer-lipase conjugate (containing about 16% of lipase) is 30 parts by weight, the precursor ruthenium trichloride is 1 part by weight, and ethanol and glycerin are 100 parts and 2 parts by weight, respectively.
  • the size of the nanoclusters in the Ru/CALB-Pluronic prepared above is about 3.1 nm, and the mass loading of the metal nanoparticles in the catalyst is 8 wt%
  • Candida antarctica lipase B (enzyme activity 9U/mg) is 28 parts by weight, the divalent palladium complex palladium acetate is 1 part by weight, and methanol is 267 parts by weight.
  • the palladium particles in the composite catalyst are unevenly distributed, with a particle diameter of 3-50 nm ( Figure 6). It can be seen that the direct loading of metal nanoparticles on the protein surface cannot prevent the aggregation between the metal particles, and the controllable preparation of the metal particle size cannot be achieved.
  • the enzyme is Candida antarctica lipase B (enzyme activity is 9U/mg) is 16 parts by weight, polymer compound It is 3 parts by weight, the activator Dess-Martin oxidizer is 0.5 parts by weight, and the reducing agent sodium cyanoborohydride is 8 parts by weight.
  • Step (1) The above proportioned And Dess-Martin oxidant dissolved in dichloromethane, where, The mass fraction in dichloromethane is 1.5%, oxidation reaction is carried out at room temperature for 24 hours, the terminal hydroxyl group is oxidized to an aldehyde group, and then cold ether is added to precipitate to obtain an aldehyde-based polymer compound, which is dried in vacuum;
  • Step (2) dissolve the aldehyde-based polymer compound and lipase obtained in step (1) in 10 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 7, and couple the aldehyde group to the amino group at room temperature
  • the reducing agent sodium cyanoborohydride was added to carry out the reduction reaction of the carbon-nitrogen double bond for 18 hours to reduce the carbon-nitrogen double bond formed by the coupling to a stable carbon-nitrogen single bond .
  • the product was lyophilized to obtain CALB-Pluronic, a polymer-lipase conjugate.
  • the polymer-lipase conjugate (containing about 26% of lipase CALB) is 28 parts by weight, the divalent palladium complex palladium acetate is 1 part by weight, and methanol is 267 parts by weight.
  • the distribution of the palladium nanoparticles is not uniform, and the particle diameter is 0.1-10 nm (see FIG. 7), and the controllable preparation of the metal particle size cannot be achieved.
  • the mass loading of metal nanoparticles in the catalyst is 20 wt%.
  • Candida antarctica lipase B (enzyme activity 9U/mg) is 4 parts by weight, polymer compound It is 600 parts by weight, the activator Dess-Martin oxidant is 100 parts by weight, and the reducing agent sodium cyanoborohydride is 2 parts by weight.
  • Step (1) The above proportioned And Dess-Martin oxidant dissolved in dichloromethane, where, The mass fraction in dichloromethane is 1.5%, oxidation reaction is carried out at room temperature for 24 hours, the terminal hydroxyl group is oxidized to an aldehyde group, and then cold ether is added to precipitate to obtain an aldehyde-based polymer compound, which is dried in vacuum;
  • Step (2) dissolve the aldehyde-based polymer compound and lipase obtained in step (1) in 10 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 7, and couple the aldehyde group to the amino group at room temperature
  • the reducing agent sodium cyanoborohydride was added to carry out the reduction reaction of the carbon-nitrogen double bond for 18 hours to reduce the carbon-nitrogen double bond formed by the coupling to a stable carbon-nitrogen single bond .
  • the product was lyophilized to obtain CALB-Pluronic, a polymer-lipase conjugate.
  • the polymer-lipase conjugate (containing about 2% of lipase CALB) is 28 parts by weight, the divalent palladium complex palladium acetate is 1 part by weight, and methanol is 267 parts by weight.
  • the distribution of the palladium nanoparticles is not uniform, and the particle diameter is 1-50 nm (see FIG. 8), and the controllable preparation of the metal particle size cannot be achieved.
  • the mass loading of metal nanoparticles in the catalyst is 0.05 wt%.
  • Candida antarctica lipase B (enzyme activity 9U/mg) is 10 parts by weight, polymer compound It is 60 parts by weight, the activator Dess-Martin oxidant is 10 parts by weight, and the reducing agent sodium cyanoborohydride is 5 parts by weight.
  • Step (1) The above proportioned And Dess-Martin oxidant dissolved in dichloromethane, where, The mass fraction in dichloromethane is 1.5%, oxidation reaction is carried out at room temperature for 24 hours, the terminal hydroxyl group is oxidized to an aldehyde group, and then cold ether is added to precipitate to obtain an aldehyde-based polymer compound, which is dried in vacuum;
  • Step (2) dissolve the aldehyde-based polymer compound and lipase obtained in step (1) in 10 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 7, and couple the aldehyde group to the amino group at room temperature
  • the reducing agent sodium cyanoborohydride was added to carry out the reduction reaction of the carbon-nitrogen double bond for 18 hours to reduce the carbon-nitrogen double bond formed by the coupling to a stable carbon-nitrogen single bond .
  • the product was lyophilized to obtain CALB-Pluronic, a polymer-lipase conjugate.
  • the polymer-lipase conjugate (containing about 10% of lipase CALB) is 51 parts by weight, the divalent palladium complex palladium acetate is 1 part by weight, and methanol is 267 parts by weight.
  • Candida antarctica lipase B (enzyme activity 9U/mg) is 10 parts by weight, polymer compound It is 60 parts by weight, the activator Dess-Martin oxidant is 10 parts by weight, and the reducing agent sodium cyanoborohydride is 5 parts by weight.
  • Step (1) The above proportioned And Dess-Martin oxidant dissolved in dichloromethane, where, The mass fraction in dichloromethane is 1.5%, oxidation reaction is carried out at room temperature for 24 hours, the terminal hydroxyl group is oxidized to an aldehyde group, and then cold ether is added to precipitate to obtain an aldehyde-based polymer compound, which is vacuum dried;
  • Step (2) dissolve the aldehyde-based polymer compound and lipase obtained in step (1) in 10 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 7, and couple the aldehyde group to the amino group at room temperature
  • the reducing agent sodium cyanoborohydride was added to carry out the reduction reaction of the carbon-nitrogen double bond for 18 hours to reduce the carbon-nitrogen double bond formed by the coupling to a stable carbon-nitrogen single bond .
  • the product was lyophilized to obtain CALB-Pluronic, a polymer-lipase conjugate.
  • the polymer-lipase conjugate (containing about 10% of lipase CALB) is 0.5 parts by weight, the divalent palladium complex palladium acetate is 1 part by weight, and methanol is 267 parts by weight.
  • the natural enzyme CALB and the polymer-lipase conjugate CALB-Pluronic, Novozymes 435 and the natural lipase-palladium conjugate Pd/CALB containing the same quality lipase were reacted under the same conditions, compare ( ⁇ )-1- The catalytic efficiency of chiral resolution of phenethylamine. As shown in FIG. 11, under the same reaction conditions, the catalytic efficiency of the polymer-lipase-palladium composite nanocatalyst is about 3.6 times that of the natural enzyme CALB, and 1.9 times that of commercial immobilized lipase Novozymes 435.
  • the 10 wt% Pd/C containing the same quality palladium nanoparticles and the natural lipase-palladium conjugate Pd/CALB were reacted under the same conditions, and the (S)-1-phenylethylamine racemic catalytic efficiency was compared.
  • the conversion frequency (TOF, turnover frequency) of the polymer-lipase-palladium composite nanocatalyst in this racemic reaction is 6.3 times that of Pd/C, which is the natural enzyme-palladium conjugate Pd/C. 2.2 times CALB.
  • the conversion rate of the catalytic reaction of industrial lipase-palladium catalyst Novozymes 435 and palladium-carbon decreased from 30% to 13%.
  • the polymer-lipase-palladium composite nanocatalyst catalyzes the reaction at 55°C for 8 hours, and the conversion rate can reach 92%, which is 9.9 times that of the industrial lipase-palladium catalyst under the same conditions ( Figure 13).
  • the polymer-lipase-palladium composite nanocatalyst has excellent catalytic performance. As shown in FIG. 14, after repeated use 10 times, the conversion rate of the dynamic kinetic resolution reaction of ( ⁇ )-1-phenylethylamine catalyzed by the composite nanocatalyst was 82%, and the ee value of the reaction product was >99%.

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Abstract

一种高分子-酶-金属复合纳米催化剂及其可控合成方法。该催化剂由高分子化合物、酶、金属盐制备而成，是一种限域于高分子-酶结合物内的金属纳米颗粒复合催化剂，其制备步骤包括高分子-酶结合物的构建和金属离子在单个高分子-酶结合物内部原位还原两个部分，可有效控制合成过程中金属颗粒的尺寸，防止聚集，实现高活性的极小金属纳米颗粒的可控制备，同时该催化剂在水溶液及常规有机溶剂中形成纳米级分散，有利于保持其在反应过程中的酶催化活性和金属催化剂活性，同时，该催化剂在常见有机溶剂中具有温度响应特性，在低温下可通过简单的离心分离，多次重复使用。

Description

一种高分子-酶-金属复合纳米催化剂及其可控合成方法 技术领域
本发明属于催化剂制备领域，具体涉及一种高分子-酶-金属复合纳米催化剂及其可控合成方法。
背景技术
将多相催化与酶催化相耦合的化学-生物耦合催化过程在生物医药生产、化工绿色制造行业具有巨大的应用潜力。由双功能化学-生物复合催化剂催化的“一锅法”多步串联或级联的化学-生物反应，可避免反应中间产物的分离和纯化步骤，具有经济、环保、高效等优点。然而，酶催化过程与贵金属多相催化过程的反应条件在多数情况下并不匹配，各自的反应条件会导致另一方催化剂的失活，因此严重制约了化学-生物耦合催化反应的发展。酶催化通常情况下比较合适的反应条件是常温、常压、水溶液；相反，贵金属多相催化反应大多需要在高温、高压、气体保护、有机溶剂等较为苛刻的条件下进行。如何制备在温和条件下能够进行高效、高选择性催化反应的金属-酶双功能催化剂，至今仍是一个挑战。
专利申请101239295A公开了一种纳米微米复合微米的制备方法，通过化学的方法把带有表面功能团的纳米颗粒键合到功能化的微球表面上，再把带有纳米球表面相反电荷的聚合物吸附到复合纳微米球的表面，从而制得稳定的纳微米复合微球，这些纳微米复合微球可以用于催化剂和酶载体。其主要解决了纳米复合微球的稳定性问题，但是没有给出纳米复合微球和催化剂和酶的具体结合情况以及不同情况下，金属-酶双功能催化剂的作用的应用情况。
合成金属-酶双功能催化剂的一个可行的方案是利用酶分子辅助原位合成金属纳米颗粒，如Xie等发现金离子在碱性环境(pH＝12)下被牛血清蛋白BSA原位还原为金纳米团簇(J.P.Xie,Y.G.Zheng,J.Y.Ying,J.Am.Chem.Soc.,131,888-889(2009))。专利申请CN106729713A公开了一种蛋白质包覆金属硫化物纳米粒子的制备方法，利用蛋白质在碱性溶液中催化Ag +、Bi 3+、Cd 2+、Cu 2+等金属离子，生成蛋白质包覆金属硫化物纳米粒子。该纳米粒子平均粒径为3.0-7.0nm，可用于肿瘤光热治疗试剂。专利申请CN106315663A公开了一种超小金属硫族化合物纳米晶的生物合成方法。该方法利用一系列等电点小于9.0的中性或酸性蛋白质在碱性溶液中原位还原Cu、Cd、Zn和Mo等金属纳米颗粒，生成尺寸在10nm以下金属硫族化合物纳米晶。该纳米晶可用于生物荧光成像、光热治疗制剂。
上述工作及专利中金属-蛋白质复合物的制备均在碱性条件下进行，对于更多的不耐受碱性环境的蛋白质并不适用。而且复合物中的蛋白质没有催化功能。
据文献(M.Filice,M.Marciello,etal.Chem.Commun.,49,6876-6878(2013))报道，Filice等利用天然脂肪酶为模板在中性pH条件下合成了脂肪酶-钯、脂肪酶-金、脂肪酶-银等杂合聚集体。在该方法中，由于仅采用天然脂肪酶作为模板，难以防止金属颗粒的聚集和生长，金属纳米颗粒在生长过程中发生团聚，并且金属纳米颗粒分布不均一，所以该聚集体中金属颗粒的直径分布为1.5-6.8nm，不能可控合成含有极小粒径的金属纳米颗粒的金属-酶双功能催化剂。由于金属颗粒较大并且分布不均一，其催化活性较低，需要在相对较高温度下，70℃，脂肪酶-钯纳米颗粒杂合催化剂可用于(±)-1-苯乙胺动态动力学拆分反应。
发明内容
为解决以上问题，本发明提供了一种高分子-酶-金属复合纳米催化剂及其可控合成方法，该方法是一种用高分子-酶结合物限域原位还原金属纳米颗粒(团簇)的方法，使得所制备的极小粒径(≤5nm)的金属纳米颗粒(亚纳米团簇)能够均匀地分散在单个高分子-酶纳米结合物内部，有效阻止金属纳米颗粒之间的聚集，实现可控制备。该方法操作简便易行、所制备的高分子-酶-金属复合纳米催化剂适用范围广泛，在手性药物、精细化学品、农药、光热治疗制剂合成等领域具有很好的应用前景。
为实现上述目的，本发明采用的技术方案如下：
一种高分子-酶-金属复合纳米催化剂，其为一种限域于高分子-酶结合物内的金属盐纳米颗粒复合催化剂。
优选地，所述高分子-酶结合物中酶的含量为5-20wt％；所述高分子-酶-金属复合纳米催化剂中金属纳米颗粒的质量负载量为0.1-30wt％。
优选地，所述金属纳米颗粒生长在单个高分子-酶结合物内部。
优选地，所述高分子-酶结合物在分散相中呈纳米分散，粒径分布为5-80nm，平均粒径10-40nm。
优选地，所述高分子-酶-金属复合纳米催化剂在分散相中呈纳米分散，粒径分布为10-200nm，金属纳米颗粒尺寸为0.2nm-5nm。
优选地，所述高分子-酶-金属复合纳米催化剂在中性pH下表面电荷为-0.1--5。
优选地，所述高分子-酶-金属复合纳米催化剂粒径为20-100nm。
优选地，所述催化剂的制备原料包括高分子化合物、酶、金属盐。
优选地，所述高分子化合物为含有端羟基的聚氧乙烯-聚氧丙烯-聚氧乙烯的聚醚类嵌段共聚物，具体为
Figure PCTCN2018123877-appb-000001
和
Figure PCTCN2018123877-appb-000002
中的至少一种。
本发明的另一目的在于提供上述高分子-酶-金属复合纳米催化剂的可控合成方法，包括如下步骤：
(1)高分子-酶结合物的制备：将高分子化合物溶于有机溶剂，加氧化剂，氧化反应后，加沉淀剂沉淀，得醛基化的高分子化合物；
将酶和所得醛基化的高分子化合物在缓冲液中进行希夫碱反应后，加还原剂，还原反应后，得到高分子-酶结合物；
(2)将金属盐溶解在含醇的水溶液中，加入所述高分子-酶结合物，反应后，经透析纯化、冷冻干燥得金属-酶纳米高分子-酶-金属复合纳米催化剂。
优选地，步骤(1)中所述氧化反应时间为10-24h；所述席夫碱反应时间为0.5-4h；所述还原反应时间为10-20h。
优选地，步骤(2)中所述反应时间为10-24h。
优选地，步骤(1)中，所述氧化剂为二氧化锰、氯铬酸吡啶、戴斯-马丁氧化剂和重铬酸吡啶中的至少一种；
优选地，所述还原剂为硼氢化钠和氰基硼氢化钠中的一种或两种。
优选地，步骤(1)中，所述高分子化合物与氧化剂的质量比为5-500：5-100。
优选地，步骤(1)中，所述高分子化合物、酶的质量比为5-500：5-15。
优选地，步骤(1)中，所述酶与还原剂的质量比为5-15：5-50。
优选地，步骤(1)中，所述高分子化合物在有机溶剂中的质量百分浓度为1-5％。
优选地，步骤(1)中，所述有机溶剂为二氯甲烷、三氯甲烷、甲苯、二甲基亚砜和氯仿中的至少一种。
优选地，步骤(1)中，所述沉淀剂为乙醚、石油醚和正己烷中的至少一种。
优选地，步骤(1)中，所述缓冲溶液为磷酸缓冲液，pH值为6-9，浓度为10-100mM。
优选地，步骤(1)中，所述酶为南极假丝酵母脂肪酶B、猪胰脂肪酶、褶皱假丝酵母脂肪酶、米黑根毛霉脂肪酶、疏棉状嗜热丝孢菌脂肪酶、细胞色素C、葡萄糖氧化酶、酿酒酵母乙醇脱氢酶、马肝醇脱氢酶、乙醛脱氢酶、酮还原酶、甲酸脱氢酶和甲醛脱氢酶中的至少一种。
优选地，步骤(2)中，所述金属盐为醋酸钯、四氯合钯酸、氯化钯、硝酸钯、氯金酸、硝酸银、氟化银、醋酸银、三氯化钌、氯铂酸、四氯合铂酸钾和六氯合铂酸钾中的至少一种。
优选地，步骤(2)中，所述醇为甲醇、乙醇、甘油、乙二醇、异丙醇、正丁醇、山梨醇的至少一种；其在水溶剂中的体积百分浓度为5-80％，优选为5-50％。
优选地，步骤(2)中，所述高分子-酶结合物、金属盐的质量比为1-50：1。
优选地，步骤(2)中，所述金属盐和醇的质量比为1：100-1000。
优选地，步骤(2)中，所述透析过程中，透析袋分子量为12000-14000Da。
本发明的发明构思：
本发明包括高分子-酶结合物的构建和金属离子在单个高分子-酶结合物内部原位还原两个部分。
首先，在氧化剂的作用下将高分子的端羟基氧化为醛基，之后在缓冲液中高分子化合物的醛基和酶的氨基发生偶联反应，之后，加入还原剂使偶联形成的碳-氮双键还原为稳定的碳-氮单键，制成高分子-酶的结合物；其次，将高分子-酶的结合物与金属盐反应，高分子-酶的结合物在室温下原位还原金属离子，金属纳米颗粒在单个高分子-酶结合物中生长，反应一段时间后，经透析纯化、冷冻干燥处理过程即可得到高分子-酶-金属复合纳米催化剂。
本发明具有以下优点：
(1)本发明的高分子-酶-金属复合纳米催化剂可通过简单调节反应前驱体的比例可控地合成具有极小尺寸(≤5nm)金属纳米颗粒的金属-酶双功能催化剂。金属纳米颗粒在单个高分子-酶结合物(≤80nm)的酶表面的三维限域空间中生长，而高分子-酶结合物在中性pH条件下表面带少量得负电荷可有效避免结合物之间的聚集，制备过程可控。
(2)本发明的高分子-酶-金属复合纳米催化剂在有机相中实现纳米级分散，且无固定化载体，极大地降低了反应传质阻力，具有活性高、稳定性好等优点。而且低温下该高分子-酶-金属复合纳米催化剂可以从有机溶剂中沉淀，多次重复使用。
(3)本发明的高分子-酶-金属复合纳米催化剂具有极小粒径的金属颗粒，在常规反应条件甚至在较低反应温度下活性高，有效提高金属和酶在化学-生物耦合催化反应中的兼容性和催化效率。
(4)本发明的高分子-酶-金属复合纳米催化剂可批量制备，在手性药物、精细化学品、农药、光热治疗制剂合成等领域具有很好的应用前景。
附图说明
图1为实例1步骤(2)所制备的CALB-Pluronic结合物的TEM图；
图2为实例1所制备的Pd/CALB-Pluronic复合纳米催化剂的HAADF-STEM图；
图3为实例2所制备的Ag/CALB-Pluronic复合纳米催化剂的TEM图；
图4为实例3所制备的Au/CALB-Pluronic复合纳米催化剂的TEM图；
图5为实例4所制备的Pd/CALB-Pluronic复合纳米催化剂的TEM图；
图6为对比例1所制备的Pd/CALB复合催化剂的TEM图；
图7-10对比例2-5所制备的Pd/CALB-Pluronic复合纳米催化剂的TEM图；
图11为不同形式的脂肪酶催化1-苯乙胺外消旋体手性拆分的转化率对比；
图12为不同形式的固定化钯纳米颗粒催化(S)-1-苯乙胺外消旋反应效率对比；
图13为高分子-脂肪酶-钯复合纳米催化剂与商业用脂肪酶和钯催化剂(诺维信435和钯碳)催化(±)-1-苯乙胺动态动力学拆分反应动力学对比；
图14为高分子-脂肪酶-钯复合纳米催化剂循环使用催化性能对比。
现结合附图和实施例对本发明作进一步说明：
具体实施方式
下面结合具体实施例对本发明作进一步阐述，但本发明并不限于以下实施例。
实施例1
高分子-脂肪酶-钯复合纳米催化剂的制备
酶为南极假丝酵母脂肪酶B(酶活为9U/mg)以重量计为10份，高分子化合物
Figure PCTCN2018123877-appb-000003
以重量计为60份，活化剂戴斯-马丁氧化剂以重量计为10份，还原剂氰基硼氢化钠以重量计为5份。
步骤(1)：将上述配比的
Figure PCTCN2018123877-appb-000004
及戴斯-马丁氧化剂溶解在二氯甲烷中，其中，
Figure PCTCN2018123877-appb-000005
在二氯甲烷中的质量分数为1.5％，在室温下进行氧化反应24小时，将端羟基氧化为醛基，之后加入冷乙醚沉淀得到醛基化的高分子化合物，真空干燥；
步骤(2)：将步骤(1)所得醛基化高分子化合物和脂肪酶溶解在pH值为7的10mM磷酸氢二钠-磷酸二氢钠缓冲液中，室温进行醛基与氨基的偶联反应(席夫碱反应)2小时后，加入还原剂氰基硼氢化钠进行碳-氮双键的还原反应18小时，使偶联形成的碳-氮双键还原为稳定的碳-氮单键。产物经过透析后，冻干得到高分子-脂肪酶结合物CALB-Pluronic。
高分子-脂肪酶结合物(含脂肪酶CALB约10％)以重量计为28份，二价钯配合物醋酸钯以重量计为1份，甲醇以重量计为267份。
按上述比例通过超声、震荡将二价钯配合物醋酸钯溶解于体积分数为20％的甲醇水溶液中，加入CALB-Pluronic结合物，在室温下磁力搅拌20小时，经透析、冻干后得到高分子-脂肪酶-钯复合纳米催化剂Pd/CALB-Pluronic干粉。
上述制备得到的Pd/CALB-Pluronic中钯纳米团簇大小约为0.8nm，催化剂中金属纳米颗粒的质量负载量为4wt％
实施例2
高分子-脂肪酶-银复合纳米催化剂的制备
酶为南极假丝酵母脂肪酶B(酶活为9U/mg)和褶皱假丝酵母脂肪酶(酶活为700-1,000U/mg)以重量计为5份和5份，高分子化合物
Figure PCTCN2018123877-appb-000006
和
Figure PCTCN2018123877-appb-000007
以重量计为60份和40份，活化剂二氧化锰氧化剂以重量计为50份，还原剂氰基硼氢化钠以重量计为10份。
步骤(1)：将上述配比的
Figure PCTCN2018123877-appb-000008
和
Figure PCTCN2018123877-appb-000009
及戴斯-马丁氧化剂溶解在氯仿中，其中，
Figure PCTCN2018123877-appb-000010
和
Figure PCTCN2018123877-appb-000011
在氯仿中的质量分数为5％，在室温下进行氧化反应16小时，将端羟基氧化为醛基，之后加入冷乙醚和石油醚混合溶液沉淀得到醛基化的高分子化合物，真空干燥；
步骤(2)：将步骤(1)所得醛基化高分子化合物和南极假丝酵母脂肪酶B和褶皱假丝酵母脂肪酶溶解在pH值为7的100mM磷酸氢二钠-磷酸二氢钠缓冲液中，室温进行醛基与氨基的偶联反应4小时后，加入还原剂氰基硼氢化钠进行碳-氮双键的还原反应10小时，使偶联形成的碳-氮双键还原为稳定的碳-氮单键。产物经过透析后，冻干得到高分子-脂肪酶结合物Lipase-Pluronic。
高分子-脂肪酶结合物(含南极假丝酵母脂肪酶B和褶皱假丝酵母脂肪酶约20％)以重量计为25份，前驱体硝酸银以重量计为1份，甲醇以重量计为50份，甘油以重量计为50份。
按上述比例将硝酸银溶解于体积分数为80％的甲醇-甘油水溶液中，加入Lipase-Pluronic结合物，在室温下磁力搅拌24小时，经透析、冻干后得到高分子-脂肪酶-银复合纳米催化剂Ag/Lipase-Pluronic干粉。
上述制备得到的Ag/Lipase-Pluronic中纳米团簇大小约为0.5nm，催化剂中金属纳米颗粒的质量负载量为5wt％
实施例3
高分子-脂肪酶-金复合纳米催化剂的制备
酶为疏棉状嗜热丝孢菌脂肪酶(酶活为100,000-180,000U/g)以重量计为10份，高分子化合物
Figure PCTCN2018123877-appb-000012
以重量计为5份，活化剂氯铬酸吡啶氧化剂以重量计为5份，还原剂硼氢化钠以重量计为5份。
步骤(1)：将上述配比的
Figure PCTCN2018123877-appb-000013
及氯铬酸吡啶氧化剂溶解在二氯甲烷和氯仿的混合溶液中，其中，
Figure PCTCN2018123877-appb-000014
在二氯甲烷和氯仿中的质量分数为1％，在室温下进行氧化反应10小时，将端羟基氧化为醛基，之后加入冷石油醚沉淀得到醛基化的高分子化合物，真空干燥；
步骤(2)：将步骤(1)所得醛基化高分子化合物和疏棉状嗜热丝孢菌脂肪酶溶解在pH值为7的50mM磷酸氢二钠-磷酸二氢钠缓冲液中，室温进行醛基与氨基的偶联反应0.5小时 后，加入还原剂硼氢化钠进行碳-氮双键的还原反应20小时，使偶联形成的碳-氮双键还原为稳定的碳-氮单键。产物经过透析后，冻干得到高分子-脂肪酶结合物Lipase-Pluronic。
高分子-脂肪酶结合物(含疏棉状嗜热丝孢菌脂肪酶约10％)以重量计为50份，前驱体氯金酸以重量计为1份，甲醇以重量计为400份，乙二醇以重量计为600份。
按上述比例将氯金酸溶解在甲醇-乙二醇-水溶液中，加入Lipase-Pluronic结合物，在室温下磁力搅拌10小时，经透析、冻干后得到高分子-脂肪酶-金复合纳米催化剂Au/Lipase-Pluronic干粉。
上述制备得到的Au/Lipase-Pluronic中纳米团簇大小约为0.2nm，催化剂中金属纳米颗粒的质量负载量为0.1wt％。
实施例4
高分子-脂肪酶-钯复合纳米催化剂的制备
酶为猪胰脂肪酶(酶活为100,000-400,000U/g)以重量计为15份，高分子化合物
Figure PCTCN2018123877-appb-000015
以重量计为500份，活化剂戴斯-马丁氧化剂以重量计为100份，还原剂氰基硼氢化钠以重量计为50份。
步骤(1)：将上述配比的
Figure PCTCN2018123877-appb-000016
及戴斯-马丁氧化剂溶解在三氯甲烷中，其中，
Figure PCTCN2018123877-appb-000017
在三氯甲烷中的质量分数为5％，在室温下进行氧化反应24小时，将端羟基氧化为醛基，之后加入冷正己烷沉淀得到醛基化的高分子化合物，真空干燥；
步骤(2)：将步骤(1)所得醛基化高分子化合物和猪胰脂肪酶溶解在pH值为6的10mM磷酸氢二钠-磷酸二氢钠缓冲液中，室温进行醛基与氨基的偶联反应3小时后，加入还原剂氰基硼氢化钠进行碳-氮双键的还原反应16小时，使偶联形成的碳-氮双键还原为稳定的碳-氮单键。产物经过透析后，冻干得到高分子-脂肪酶结合物Lipase-Pluronic。
高分子-脂肪酶结合物(含猪胰脂肪酶约15％)以重量计为50份，二价钯配合物醋酸钯以重量计为0.5份，四氯合钯酸以重量计为0.5份，甲醇以重量计为1000份。
按上述比例通过超声、震荡将二价钯配合物醋酸钯和四氯合钯酸溶解于体积分数为5％的甲醇-水溶液中，加入Lipase-Pluronic结合物，在室温下磁力搅拌20小时，经透析、冻干后得到高分子-脂肪酶-钯复合纳米催化剂Pd/Lipase-Pluronic干粉。
上述制备得到的Pd/Lipase-Pluronic中钯纳米团簇大小约为2.5nm，催化剂中金属纳米颗粒的质量负载量为20wt％
实施例5
高分子-乙醇脱氢酶-钯复合纳米催化剂
酶为酿酒酵母乙醇脱氢酶(酶活为≥300U/mg)以重量计为10份，高分子化合物
Figure PCTCN2018123877-appb-000018
以重量计为160份，活化剂戴斯-马丁氧化剂以重量计为27份，还原剂氰基硼氢化钠以重量计为16份。
步骤(1)：将上述配比的
Figure PCTCN2018123877-appb-000019
及戴斯-马丁氧化剂溶解在二氯甲烷中，其中，
Figure PCTCN2018123877-appb-000020
在二氯甲烷中的质量分数为5％，在室温下进行氧化反应24小时，将端羟基氧化为醛基，之后加入冷乙醚沉淀得到醛基化的高分子化合物，真空干燥；
步骤(2)：将步骤(1)所得醛基化高分子化合物和乙醇脱氢酶溶解在pH值为7的10mM磷酸氢二钠-磷酸二氢钠缓冲液中，室温进行醛基与氨基的偶联反应2小时后，加入还氰基硼氢化钠进行碳-氮双键的还原反应10小时，使偶联形成的碳-氮双键还原为稳定的碳-氮单键。产物经过透析后，冻干得到高分子-乙醇脱氢酶结合物ADH-Pluronic。
高分子-乙醇脱氢酶结合物(含乙醇脱氢酶ADH约5％)以重量计为15份，二价钯配合物醋酸钯以重量计为1份，甲醇以重量计为500份。
按上述比例通过超声、震荡将二价钯配合物醋酸钯溶解于体积分数为80％的甲醇水溶液中，加入ADH-Pluronic结合物，在室温下磁力搅拌10小时，经透析、冻干后得到高分子-乙醇脱氢酶-钯复合纳米催化剂Pd/ADH-Pluronic干粉。
上述制备得到的Pd/ADH-Pluronic中纳米团簇大小约为1.8nm，催化剂中金属纳米颗粒的质量负载量为25wt％。
实施例6
高分子-葡萄糖氧化酶-金复合纳米催化剂
酶为葡萄糖氧化酶(来源于黑曲霉，酶活为100,000-250,000U/g)以重量计为25份，高分子化合物
Figure PCTCN2018123877-appb-000021
和
Figure PCTCN2018123877-appb-000022
分别以重量计为30份、30份和40份，活化剂戴斯-马丁和二氧化锰氧化剂分别以重量计为40份和60份，还原剂氰基硼氢化钠和硼氢化钠分别以重量计为7份和3份。
步骤(1)：将上述配比的
Figure PCTCN2018123877-appb-000023
和
Figure PCTCN2018123877-appb-000024
及戴斯-马丁和二氧化锰氧化剂溶解在氯仿、甲苯和二甲基亚砜的混合溶液中，其中，
Figure PCTCN2018123877-appb-000025
Figure PCTCN2018123877-appb-000026
和
Figure PCTCN2018123877-appb-000027
在氯仿、甲苯和二甲基亚砜混合溶液中的质量分数为4％，在室温下进行氧化反应16小时，将端羟基氧化为醛基，之后加入冷乙醚和石油醚的混合溶液沉淀得到醛基化的高分子化合物，真空干燥；
步骤(2)：将步骤(1)所得醛基化高分子化合物和葡萄糖氧化酶溶解在pH值为8的20mM磷酸氢二钠-磷酸二氢钠缓冲液中，室温进行醛基与氨基的偶联反应1小时后，加入还原剂氰基硼氢化钠和硼氢化钠进行碳-氮双键的还原反应16小时，使偶联形成的碳-氮双键还原为稳定的碳-氮单键。产物经过透析后，冻干得到高分子-葡萄糖氧化酶结合物GOx-Pluronic。
高分子-葡萄糖氧化酶结合物(含葡萄糖氧化酶约13％)以重量计为15份，前驱体氯金酸以重量计为3份，甲醇以重量计为378份。
按上述比例将氯金酸溶解在40％的甲醇-水溶液中，加入GOx-Pluronic结合物，在室温下磁力搅拌15小时，经透析、冻干后得到高分子-葡萄糖氧化酶-金复合纳米催化剂Au/GOx-Pluronic干粉。
上述制备得到的Au/GOx-Pluronic中纳米团簇大小约为1.5nm，催化剂中催化剂中金属纳米颗粒的质量负载量为15wt％。
实施例7
高分子-细胞色素C-银复合纳米催化剂
酶为细胞色素C(来源于酿酒酵母)以重量计为5份，高分子化合物
Figure PCTCN2018123877-appb-000028
以重量计为50份，活化剂二氧化锰氧化剂以重量计为70份，还原剂氰基硼氢化钠为8份。
步骤(1)：将上述配比的
Figure PCTCN2018123877-appb-000029
及二氧化锰氧化剂溶解在三氯甲烷中，其中，
Figure PCTCN2018123877-appb-000030
在三滤甲烷中的质量分数为1％，在室温下进行氧化反应14小时，将端羟基氧化为醛基，之后加入冷石油醚沉淀得到醛基化的高分子化合物，真空干燥；
步骤(2)：将步骤(1)所得醛基化高分子化合物和细胞色素C溶解在pH值为9的10mM磷酸氢二钠-磷酸二氢钠缓冲液中，室温进行醛基与氨基的偶联反应3小时后，加入还原剂氰基硼氢化钠进行碳-氮双键的还原反应18小时，使偶联形成的碳-氮双键还原为稳定的碳-氮单键。产物经过透析后，冻干得到高分子-细胞色素C结合物Cyt C-Pluronic。
高分子-细胞色素C结合物(含细胞色素C约20％)以重量计为1份，前驱体醋酸银以重量计为1份，乙醇以重量计为700份。
按上述比例将醋酸银溶解在60％乙醇-水溶液中，加入Cyt C-Pluronic结合物，在室温下磁力搅拌20小时，经透析、冻干后得到高分子-细胞色素C-银复合纳米催化剂Ag/Gyt C-Pluronic干粉。
上述制备得到的Ag/Gyt C-Pluronic中纳米团簇大小约为3nm，催化剂中金属纳米颗粒的质量负载量为30wt％。
实施例8
高分子-葡萄糖氧化酶-铂复合纳米催化剂
酶为葡萄糖氧化酶(来源于黑曲霉，酶活为100,000-250,000U/g)以重量计为10份，高分子化合物
Figure PCTCN2018123877-appb-000031
以重量计为100份，活化剂戴斯-马丁、二氧化锰和重铬酸吡啶氧化剂分别以重量计为20份、14份和16份，还原剂硼氢化钠以重量计为5份。
步骤(1)：将上述配比的
Figure PCTCN2018123877-appb-000032
及戴斯-马丁、二氧化锰和重铬酸吡啶氧化剂 溶解在二氯甲烷的溶液中，其中，
Figure PCTCN2018123877-appb-000033
在二氯甲烷中的质量分数为5％，在室温下进行氧化反应20小时，将端羟基氧化为醛基，之后加入冷乙醚的溶液沉淀得到醛基化的高分子化合物，真空干燥；
步骤(2)：将步骤(1)所得醛基化高分子化合物和葡萄糖氧化酶溶解在pH值为7的80mM磷酸氢二钠-磷酸二氢钠缓冲液中，室温进行醛基与氨基的偶联反应2小时后，加入还原剂硼氢化钠进行碳-氮双键的还原反应15小时，使偶联形成的碳-氮双键还原为稳定的碳-氮单键。产物经过透析后，冻干得到高分子-葡萄糖氧化酶结合物GOx-Pluronic。
高分子-葡萄糖氧化酶结合物(含葡萄糖氧化酶约15％)以重量计为10份，前驱体氯铂酸、四氯合铂酸钾分别以重量计为1份和1份，乙二醇以重量计为100份。
按上述比例将氯铂酸、四氯合铂酸钾溶解在25％的乙二醇-水溶液中，加入GOx-Pluronic结合物，在室温下磁力搅拌15小时，经透析、冻干后得到高分子-葡萄糖氧化酶-铂复合纳米催化剂Pt/GOx-Pluronic干粉。
上述制备得到的Pt/GOx-Pluronic中纳米团簇大小约为5nm，催化剂中金属纳米颗粒的质量负载量为10wt％。
实施例9
高分子-脂肪酶-钌复合纳米催化剂
酶为南极假丝酵母脂肪酶B(酶活为9U/mg)以重量计为10份，高分子化合物
Figure PCTCN2018123877-appb-000034
以重量计为70份，活化剂二氧化锰以重量计为100份，还原剂氰基硼氢化钠以重量计为10。
步骤(1)：将上述配比的
Figure PCTCN2018123877-appb-000035
及二氧化锰氧化剂溶解在二氯甲烷、三氯甲烷的混合溶液中，其中，
Figure PCTCN2018123877-appb-000036
在二氯甲烷、三氯甲烷的混合溶液中的质量分数为2％，在室温下进行氧化反应12小时，将端羟基氧化为醛基，之后加入冷石油醚的溶液沉淀得到醛基化的高分子化合物，真空干燥；
步骤(2)：将步骤(1)所得醛基化高分子化合物和脂肪酶溶解在pH值为7的50mM磷酸氢二钠-磷酸二氢钠缓冲液中，室温进行醛基与氨基的偶联反应1.5小时后，加入还原剂氰基硼氢化钠进行碳-氮双键的还原反应14小时，使偶联形成的碳-氮双键还原为稳定的碳-氮单键。产物经过透析后，冻干得到高分子-脂肪酶结合物CALB-Pluronic。
高分子-脂肪酶结合物(含脂肪酶约16％)以重量计为30份，前驱体三氯化钌以重量计为1份，乙醇和甘油分别以重量计为100份和2份。
按上述比例将三氯化钌溶解在40％的乙醇-甘油-水溶液中，加入CALB-Pluronic结合物，在室温下磁力搅拌24小时，经透析、冻干后得到高分子-脂肪酶-钌复合纳米催化剂 Ru/CALB-Pluronic干粉。
上述制备得到的Ru/CALB-Pluronic中纳米团簇大小约为3.1nm，催化剂中金属纳米颗粒的质量负载量为8wt％
对比例1(与实施例1相比，不含高分子化合物)
脂肪酶-钯复合催化剂的制备
南极假丝酵母脂肪酶B(酶活为9U/mg)以重量计为28份，二价钯配合物醋酸钯以重量计为1份，甲醇以重量计为267份。
步骤：按上述比例将醋酸钯溶解在20％的甲醇水溶液中，加入南极假丝酵母脂肪酶B，在室温下磁力搅拌20小时，经透析、冻干后得到脂肪酶-钯复合纳米催化剂Pd/CALB干粉。
该复合催化剂中钯颗粒分布不均一，颗粒直径为3-50nm(附图6)。可见，将金属纳米颗粒直接负载在蛋白质表面并不能避免金属颗粒之间的聚集，不能实现金属颗粒粒径的可控制备。
对比例2(与实施例1相比，高分子化合物、酶的比例不同(3:16))
高分子-脂肪酶-钯复合纳米催化剂的制备
酶为南极假丝酵母脂肪酶B(酶活为9U/mg)以重量计为16份，高分子化合物
Figure PCTCN2018123877-appb-000037
以重量计为3份，活化剂戴斯-马丁氧化剂以重量计为0.5份，还原剂氰基硼氢化钠以重量计为8份。
步骤(1)：将上述配比的
Figure PCTCN2018123877-appb-000038
及戴斯-马丁氧化剂溶解在二氯甲烷中，其中，
Figure PCTCN2018123877-appb-000039
在二氯甲烷中的质量分数为1.5％，在室温下进行氧化反应24小时，将端羟基氧化为醛基，之后加入冷乙醚沉淀得到醛基化的高分子化合物，真空干燥；
步骤(2)：将步骤(1)所得醛基化高分子化合物和脂肪酶溶解在pH值为7的10mM磷酸氢二钠-磷酸二氢钠缓冲液中，室温进行醛基与氨基的偶联反应(席夫碱反应)2小时后，加入还原剂氰基硼氢化钠进行碳-氮双键的还原反应18小时，使偶联形成的碳-氮双键还原为稳定的碳-氮单键。产物经过透析后，冻干得到高分子-脂肪酶结合物CALB-Pluronic。
高分子-脂肪酶结合物(含脂肪酶CALB约26％)以重量计为28份，二价钯配合物醋酸钯以重量计为1份，甲醇以重量计为267份。
按上述比例通过超声、震荡将二价钯配合物醋酸钯溶解于体积分数为20％的甲醇水溶液中，加入CALB-Pluronic结合物，在室温下磁力搅拌20小时，经透析、冻干后得到高分子-脂肪酶-钯复合纳米催化剂Pd/CALB-Pluronic干粉。
上述制备得到的Pd/CALB-Pluronic中钯纳米颗粒分布不均一，颗粒直径为0.1-10nm(附图7)，不能实现金属颗粒粒径的可控制备。催化剂中金属纳米颗粒的质量负载量为20wt％。
对比例3(与实施例1相比，高分子化合物、酶的比例不同(600:4))
高分子-脂肪酶-钯复合纳米催化剂的制备
酶为南极假丝酵母脂肪酶B(酶活为9U/mg)以重量计为4份，高分子化合物
Figure PCTCN2018123877-appb-000040
以重量计为600份，活化剂戴斯-马丁氧化剂以重量计为100份，还原剂氰基硼氢化钠以重量计为2份。
步骤(1)：将上述配比的
Figure PCTCN2018123877-appb-000041
及戴斯-马丁氧化剂溶解在二氯甲烷中，其中，
Figure PCTCN2018123877-appb-000042
在二氯甲烷中的质量分数为1.5％，在室温下进行氧化反应24小时，将端羟基氧化为醛基，之后加入冷乙醚沉淀得到醛基化的高分子化合物，真空干燥；
步骤(2)：将步骤(1)所得醛基化高分子化合物和脂肪酶溶解在pH值为7的10mM磷酸氢二钠-磷酸二氢钠缓冲液中，室温进行醛基与氨基的偶联反应(席夫碱反应)2小时后，加入还原剂氰基硼氢化钠进行碳-氮双键的还原反应18小时，使偶联形成的碳-氮双键还原为稳定的碳-氮单键。产物经过透析后，冻干得到高分子-脂肪酶结合物CALB-Pluronic。
高分子-脂肪酶结合物(含脂肪酶CALB约2％)以重量计为28份，二价钯配合物醋酸钯以重量计为1份，甲醇以重量计为267份。
按上述比例通过超声、震荡将二价钯配合物醋酸钯溶解于体积分数为20％的甲醇水溶液中，加入CALB-Pluronic结合物，在室温下磁力搅拌20小时，经透析、冻干后得到高分子-脂肪酶-钯复合纳米催化剂Pd/CALB-Pluronic干粉。
上述制备得到的Pd/CALB-Pluronic中钯纳米颗粒分布不均一，颗粒直径为1-50nm(附图8)，不能实现金属颗粒粒径的可控制备。催化剂中金属纳米颗粒的质量负载量为0.05wt％。
对比例4(与实施例1相比，高分子-酶结合物、金属盐比例不同(51:1))
高分子-脂肪酶-钯复合纳米催化剂的制备
酶为南极假丝酵母脂肪酶B(酶活为9U/mg)以重量计为10份，高分子化合物
Figure PCTCN2018123877-appb-000043
以重量计为60份，活化剂戴斯-马丁氧化剂以重量计为10份，还原剂氰基硼氢化钠以重量计为5份。
步骤(1)：将上述配比的
Figure PCTCN2018123877-appb-000044
及戴斯-马丁氧化剂溶解在二氯甲烷中，其中，
Figure PCTCN2018123877-appb-000045
在二氯甲烷中的质量分数为1.5％，在室温下进行氧化反应24小时，将端羟基氧化为醛基，之后加入冷乙醚沉淀得到醛基化的高分子化合物，真空干燥；
步骤(2)：将步骤(1)所得醛基化高分子化合物和脂肪酶溶解在pH值为7的10mM磷酸氢二钠-磷酸二氢钠缓冲液中，室温进行醛基与氨基的偶联反应(席夫碱反应)2小时后，加入还原剂氰基硼氢化钠进行碳-氮双键的还原反应18小时，使偶联形成的碳-氮双键还原为稳定的碳-氮单键。产物经过透析后，冻干得到高分子-脂肪酶结合物CALB-Pluronic。
高分子-脂肪酶结合物(含脂肪酶CALB约10％)以重量计为51份，二价钯配合物醋酸钯以重量计为1份，甲醇以重量计为267份。
按上述比例通过超声、震荡将二价钯配合物醋酸钯溶解于体积分数为20％的甲醇水溶液中，加入CALB-Pluronic结合物，在室温下磁力搅拌20小时，经透析、冻干后得到高分子-脂肪酶-钯复合纳米催化剂Pd/CALB-Pluronic干粉。
上述制备得到的Pd/CALB-Pluronic中钯颗粒分布不均一，颗粒直径为3-45nm(附图9)。不能实现金属颗粒粒径的可控制备。催化剂中金属纳米颗粒的质量负载量为32wt％。
对比例5(与实施例1相比，高分子-酶结合物、金属盐比例不同(0.5:1))
高分子-脂肪酶-钯复合纳米催化剂的制备
酶为南极假丝酵母脂肪酶B(酶活为9U/mg)以重量计为10份，高分子化合物
Figure PCTCN2018123877-appb-000046
以重量计为60份，活化剂戴斯-马丁氧化剂以重量计为10份，还原剂氰基硼氢化钠以重量计为5份。
步骤(1)：将上述配比的
Figure PCTCN2018123877-appb-000047
及戴斯-马丁氧化剂溶解在二氯甲烷中，其中，
Figure PCTCN2018123877-appb-000048
在二氯甲烷中的质量分数为1.5％，在室温下进行氧化反应24小时，将端羟基氧化为醛基，之后加入冷乙醚沉淀得到醛基化的高分子化合物，真空干燥；
步骤(2)：将步骤(1)所得醛基化高分子化合物和脂肪酶溶解在pH值为7的10mM磷酸氢二钠-磷酸二氢钠缓冲液中，室温进行醛基与氨基的偶联反应(席夫碱反应)2小时后，加入还原剂氰基硼氢化钠进行碳-氮双键的还原反应18小时，使偶联形成的碳-氮双键还原为稳定的碳-氮单键。产物经过透析后，冻干得到高分子-脂肪酶结合物CALB-Pluronic。
高分子-脂肪酶结合物(含脂肪酶CALB约10％)以重量计为0.5份，二价钯配合物醋酸钯以重量计为1份，甲醇以重量计为267份。
按上述比例通过超声、震荡将二价钯配合物醋酸钯溶解于体积分数为20％的甲醇水溶液中，加入CALB-Pluronic结合物，在室温下磁力搅拌20小时，经透析、冻干后得到高分子-脂肪酶-钯复合纳米催化剂Pd/CALB-Pluronic干粉。
上述制备得到的Pd/CALB-Pluronic中钯颗粒分布不均一，颗粒直径为3-50nm(附图10)。不能实现金属颗粒粒径的可控制备。催化剂中金属纳米颗粒的质量负载量为50wt％。
表征及性能试验例
高分子-脂肪酶-钯复合纳米催化剂催化(±)-1-苯乙胺手性拆分
方法：将高分子-脂肪酶-钯复合纳米催化剂(含有3.6mg CALB)干粉重悬在2mL甲苯溶液中，加入26μL消旋化的1-苯乙胺和60μL乙酸乙酯。在70℃、0.1MPa氩气的保护下磁力搅拌反应，每隔1小时取样，底物转化率、产物ee值通过手性HPLC测定。
将含有相同质量脂肪酶的天然酶CALB和高分子-脂肪酶结合物CALB-Pluronic、诺维信435和天然脂肪酶-钯结合物Pd/CALB在相同条件下反应，比较(±)-1-苯乙胺的手性拆分催化效率。如附图11所示，在相同反应条件下，高分子-脂肪酶-钯复合纳米催化剂的催化效率约为天然酶CALB的3.6倍，是商业用固定化脂肪酶诺维信435的1.9倍。
高分子-脂肪酶-钯复合纳米催化剂催化(S)-1-苯乙胺外消旋
方法：将高分子-脂肪酶-钯复合纳米催化剂(含有0.64mg Pd)干粉重悬在2mL甲苯溶液中，加入52μL消旋化的(S)-1-苯乙胺。在70℃、0.1MPa氩气的保护下磁力搅拌反应，每隔1小时取样，每隔1小时取样，底物转化率通过手性HPLC测定。
将含有相同质量钯纳米颗粒的10wt％Pd/C和和天然脂肪酶-钯结合物Pd/CALB在相同条件下反应，比较(S)-1-苯乙胺外消旋催化效率。如附图12所示，高分子-脂肪酶-钯复合纳米催化剂在该外消旋反应中的转换频率(TOF,turnover frequency)是Pd/C的6.3倍，是天然酶-钯结合物Pd/CALB的2.2倍。
高分子-脂肪酶-钯复合纳米催化剂催化(±)-1-苯乙胺动态动力学拆分
方法：将高分子-脂肪酶-钯复合纳米催化剂(含有3.6mg CALB和0.64mg Pd)干粉重悬在2mL甲苯溶液中，加入26μL消旋化的1-苯乙胺和60μL乙酸乙酯。在55℃、0.1MPa氩气的保护下磁力搅拌反应，底物转化率、产物ee值通过手性HPLC测定。
当反应温度从70℃下降到55℃，反应12小时后，工业脂肪酶-钯催化剂诺维信435和钯碳的催化反应的转化率从30％下降到13％。而高分子-脂肪酶-钯复合纳米催化剂在55℃下催化反应8小时，转化率即可达到92％，为同一条件下工业脂肪酶-钯催化剂的9.9倍(附图13)。
高分子-脂肪酶-钯复合纳米催化剂重复使用
方法：将动态动力学拆分反应后的溶液在10,000rpm下离心5min，取沉淀。用甲苯洗涤3次后，将催化剂重悬在2mL甲苯溶液中，加入26μL消旋化的1-苯乙胺和60μL乙酸乙酯。在55℃、0.1MPa氩气的保护下磁力搅拌反应，底物转化率、产物ee值通过手性HPLC测定。
高分子-脂肪酶-钯复合纳米催化剂拥有优异的催化性能。如图14所示，重复使用10次以后，该复合纳米催化剂催化(±)-1-苯乙胺动态动力学拆分反应转化率为82％，反应产物的ee值>99％。
上述详细说明是针对本发明其中之一可行实施例的具体说明，该实施例并非用以限制本发明的专利范围，凡未脱离本发明所为的等效实施或变更，均应包含于本发明技术方案的范围内。

Claims (10)

  1. 一种高分子-酶-金属复合纳米催化剂，其特征在于，所述催化剂为一种限域于高分子-酶结合物内的金属纳米颗粒复合催化剂。
  2. 根据权利要求1所述的高分子-酶-金属复合纳米催化剂，其特征在于，所述高分子-酶结合物中酶的含量为5-20wt％；所述高分子-酶-金属复合纳米催化剂中金属纳米颗粒的质量负载量为0.1-30wt％。
  3. 根据权利要求1所述的高分子-酶-金属复合纳米催化剂，其特征在于，所述金属纳米颗粒生长在单个高分子-酶结合物内部；
    所述高分子-酶结合物在分散相中呈纳米分散，粒径分布为5-80nm，平均粒径10-40nm；
    所述高分子-酶-金属复合纳米催化剂在分散相中呈纳米分散，粒径分布为10-200nm，金属纳米颗粒尺寸为0.2nm-5nm；
    所述高分子-酶-金属复合纳米催化剂在中性pH下表面电荷为-0.1--5。
  4. 根据权利要求1所述的高分子-酶-金属复合纳米催化剂，其特征在于，所述催化剂的制备原料包括高分子化合物、酶、金属盐。
  5. 根据权利要求4所述的高分子-酶-金属复合纳米催化剂，其特征在于，所述高分子化合物为含有端羟基的聚氧乙烯-聚氧丙烯-聚氧乙烯的聚醚类嵌段共聚物，具体为
    Figure PCTCN2018123877-appb-100001
    Figure PCTCN2018123877-appb-100002
    中的至少一种。
  6. 根据权利要求5所述的高分子-酶-金属复合纳米催化剂的可控合成方法，其特征在于，包括如下步骤：
    (1)高分子-酶结合物的制备：将高分子化合物溶于有机溶剂，加氧化剂，氧化反应后，加沉淀剂沉淀，得醛基化的高分子化合物；
    将酶和所得醛基化的高分子化合物在缓冲液中进行希夫碱反应后，加还原剂，还原反应后，得到高分子-酶结合物；
    (2)将金属盐溶解在含醇的水溶液中，加入所述高分子-酶结合物，反应后，经透析纯化、冷冻干燥得高分子-酶-金属复合纳米催化剂。
  7. 根据权利要求6所述的高分子-酶-金属复合纳米催化剂的可控合成方法，其特征在于，步骤(1)中，所述氧化剂为二氧化锰、氯铬酸吡啶、戴斯-马丁氧化剂和重铬酸吡啶中的至少一种；
    所述还原剂为硼氢化钠和氰基硼氢化钠中的一种或两种；
    所述高分子化合物与氧化剂的质量比为5-500：5-100；
    所述高分子化合物、酶的质量比为5-500：5-15；
    所述酶与还原剂的质量比为5-15：5-50。
  8. 根据权利要求6所述的高分子-酶-金属复合纳米催化剂的可控合成方法，其特征在于，步骤(1)中，所述高分子化合物在有机溶剂中的质量百分浓度为1-5％；
    所述有机溶剂为二氯甲烷、三氯甲烷、甲苯、二甲基亚砜和氯仿中的至少一种；
    所述沉淀剂为乙醚、石油醚和正己烷中的至少一种；
    所述缓冲溶液为磷酸缓冲液，pH值为6-9，浓度为10-100mM。
  9. 根据权利要求6所述的高分子-酶-金属复合纳米催化剂的可控合成方法，其特征在于，步骤(1)中，所述酶为南极假丝酵母脂肪酶B、猪胰脂肪酶、褶皱假丝酵母脂肪酶、米黑根毛霉脂肪酶、疏棉状嗜热丝孢菌脂肪酶、细胞色素C、葡萄糖氧化酶、酿酒酵母乙醇脱氢酶、马肝醇脱氢酶、乙醛脱氢酶、酮还原酶、甲酸脱氢酶和甲醛脱氢酶中的至少一种。
  10. 根据权利要求6所述的高分子-酶-金属复合纳米催化剂的可控合成方法，其特征在于，步骤(2)中，所述金属盐为醋酸钯、四氯合钯酸、氯化钯、硝酸钯、氯金酸、硝酸银、氟化银、醋酸银、三氯化钌、氯铂酸、四氯合铂酸钾和六氯合铂酸钾中的至少一种；
    所述醇为甲醇、乙醇、甘油、乙二醇、异丙醇、正丁醇和山梨醇的至少一种，其在水溶剂中的体积百分浓度为5-80％；
    所述高分子-酶结合物、金属盐的质量比为1-50：1；
    所述金属盐和醇的质量比为1：100-1000。
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