CN109266639B

CN109266639B - Dual immobilized enzyme and preparation method and application thereof

Info

Publication number: CN109266639B
Application number: CN201811012619.9A
Authority: CN
Inventors: 罗志刚; 陈永志
Original assignee: South China Institute of Collaborative Innovation
Current assignee: South China Institute of Collaborative Innovation
Priority date: 2018-08-31
Filing date: 2018-08-31
Publication date: 2021-07-30
Anticipated expiration: 2038-08-31
Also published as: CN109266639A

Abstract

The invention belongs to the field of immobilized enzymes, and discloses a method for dual-curing an enzyme by using an in-situ free radical polymerization technology and a carbon nano material. Firstly, dispersing a carbon nano material in a phosphate buffer solution to obtain a carbon nano tube dispersion solution; preparing an enzyme solution, and modifying and protecting enzyme molecules by adopting an in-situ free radical polymerization technology to obtain an enzyme nanocapsule solution; then adding the enzyme nano-capsule solution into the carbon nano-material dispersion liquid, uniformly mixing and standing; and finally, separating and washing the mixed solution to obtain a precipitate, namely the dual immobilized enzyme. The double immobilized enzyme system takes the enzyme nanocapsule as a basic unit, and compared with the traditional immobilized enzyme, the surface of an enzyme molecule is modified with a polymer layer, so that the stability of the enzyme molecule is greatly improved. The double immobilized enzyme obtained by the invention keeps higher enzyme activity, and has higher environmental stability and reusability than free enzyme and traditional immobilized enzyme.

Description

Dual immobilized enzyme and preparation method and application thereof

Technical Field

The invention belongs to the field of immobilized enzymes, and particularly relates to a method for preparing a dual immobilized enzyme by utilizing an in-situ free radical polymerization technology and a carbon nano material and application thereof.

Background

Enzymes are specialized organic substances with catalytic activity and high selectivity, most of which are proteins in their chemical nature. Because of the nature of the protein itself, enzymes are very sensitive to environmental changes, and enzymes may lose their activity at high temperatures, high pressures, heavy metal ions, and at too high or too low a pH. Therefore, it is necessary to immobilize natural enzyme molecules to improve the structural stability of the enzyme and to stably exert the catalytic action.

The traditional immobilized enzyme method mainly comprises a physical adsorption method, a covalent bonding method, a crosslinking method, an embedding method and the like. In order to obtain an ideal immobilized enzyme and improve the activity and stability of the immobilized enzyme, both an efficient immobilization method and an ideal immobilized carrier are selected. As an ideal enzyme immobilization carrier, the carbon nano material has stable physical properties and excellent biocompatibility. On one hand, the stable physical property of the carbon nano material ensures that the carbon nano material has a more stable structure in the environment, and compared with organic carriers such as chitosan, alginate, cellulose nanocrystalline, porous starch and the like, the carbon nano material is not degraded by microorganisms in the environment and provides a good attachment carrier for enzyme; on the other hand, compared with inorganic materials such as silica, glass, diatomite and the like, the excellent biocompatibility can maximally retain the activity of the enzyme with little influence on the environment.

The in-situ free radical polymerization technology is widely applied to the targeted delivery and stabilization of protein drugs. The method is to modify enzyme protein on the molecular level, and polymerize on the surface of enzyme molecules by an in-situ free radical polymerization technology to generate a polymer film, so as to obtain the enzyme nanocapsule. The polymer film is covalently anchored on the surface of the enzyme molecule, so that the conformation of the enzyme molecule can be stabilized, and the influence of the external environment on the enzyme molecule can be reduced.

The carbon nano material has an ideal enzyme immobilized material due to the special structure and performance, and the carbon nano material immobilized enzyme has a wide application prospect in the aspects of chemical production, clean energy development, drug targeting delivery, biosensor preparation and the like, however, the problem of researchers is that how to further improve the immobilized amount and stability of enzyme molecules on the carbon material is always solved, a large number of research reports are focused on selecting different cross-linking agents or adopting a chemical bonding method to immobilize the enzyme on the carbon material, but the methods have large difference in immobilization effects on different carbon materials and enzymes, and the activity of the obtained immobilized enzyme is often difficult to satisfy.

Disclosure of Invention

In order to overcome the disadvantages and shortcomings of the prior art, the invention provides a preparation method of a dual immobilized enzyme.

Another object of the present invention is to provide a dual immobilized enzyme prepared by the above method.

Another object of the present invention is to provide the use of the above dual immobilized enzyme in catalysis.

The purpose of the invention is realized by the following scheme:

a preparation method of a dual immobilized enzyme mainly comprises the following steps:

(1) uniformly dispersing the carbon nano material in a phosphate buffer solution with the pH value of 6-9 to obtain a carbon nano material dispersion liquid;

(2) adding enzyme into phosphate buffer solution with the pH value of 6-9 to form enzyme solution, adding a modifier into the enzyme solution, uniformly mixing, carrying out enzyme modification reaction, adding a monomer and a cross-linking agent into the enzyme solution after the reaction is finished, adding an initiator and a catalyst into the enzyme solution under an anaerobic condition, initiating in-situ polymerization reaction, and purifying the obtained reaction solution after the reaction is finished to obtain enzyme nanocapsule solution;

(3) and (2) adding the enzyme nanocapsule solution prepared in the step (2) into the carbon nanomaterial dispersion liquid in the step (1), uniformly mixing, placing into a refrigerator for standing, and finally separating and washing the mixed liquid to obtain a precipitate, namely the dual immobilized enzyme.

The carbon nano material in the step (1) is preferably one or a mixture of graphene, graphene oxide, reduced graphene oxide, single-walled carbon nanotubes, multi-walled carbon nanotubes, carbon fibers and carbon nanospheres, and is preferably one or a mixture of graphene oxide, reduced graphene oxide and single-walled carbon nanotubes;

the dispersing in the step (1) is performed by at least one of stirring and ultrasonic treatment, preferably mechanical stirring and ultrasonic treatment for 1-5 hours, wherein the speed of the mechanical stirring is 100-1000 r/min, and the ultrasonic power is 100-200W; more preferably, the dispersion is carried out by mechanical stirring and ultrasonic treatment for 2h, wherein the speed of the mechanical stirring is 600r/min and the ultrasonic power is 100W.

The concentration of the carbon nano material dispersion liquid in the step (1) is 1-10 mg/mL;

the enzyme in step (2) includes, but is not limited to, organophosphorus hydrolase, lipase, horseradish peroxidase, laccase, formate dehydrogenase, formaldehyde dehydrogenase, methanol dehydrogenase, glucose oxidase, catalase, glucose isomerase, malic enzyme, alanine dehydrogenase, and the like.

Preferably, the enzyme in step (2) is at least one of organophosphorus hydrolase, lipase, horseradish peroxidase and glucose oxidase;

the enzyme solution in the step (2) is an enzyme solution with the concentration of 1-10 mg/mL;

the modifier in the step (2) is at least one of acrylic acid, acrylate compounds and acrylate compounds, preferably at least one of N-acryloyloxy succinimide (NAS), sodium acrylate (AAS) and Acrylic Acid (AA);

the enzyme modification reaction in the step (2) is carried out at 4-30 ℃ for 2-10 h.

The monomer in the step (2) is a monomer with an unsaturated chemical bond capable of undergoing a polymerization reaction, such as styrene containing a carbon-carbon double bond, acrylamide, an acrylate compound, a methacrylate compound and the like; or at least one of pyrrole, thiophene, pyridine, and the like having a conjugated double bond structure;

preferably, the monomer in the step (2) is at least one of acrylamide and N- (3-aminopropyl) -methacrylamide hydrochloride;

the crosslinking agent in the step (2) is a compound containing a plurality of unsaturated double bonds, preferably N-methylene Bisacrylamide (BIS).

The initiator in the step (2) is at least one of a peroxide initiator, an azo initiator, a redox initiator, and the like, and is preferably Ammonium Persulfate (APS).

The catalyst in the step (2) is tetramethylethylenediamine.

The oxygen-free state in the step (2) refers to that nitrogen is introduced to remove oxygen.

The mass ratio of the enzyme, the modifier, the monomer, the cross-linking agent, the initiator and the catalyst in the step (2) is 1: (0.01-0.2): (1-10): (0.1-1): (0.5-1): (1-2);

the in-situ free radical polymerization reaction in the step (2) is carried out at 4-30 ℃ for 1-5 h;

the purification in the step (2) is dialysis purification by using a dialysis bag with the molecular weight cutoff of 1.0-3.5 kD.

The using amount of the carbon nano tube dispersion liquid and the enzyme nano capsule solution in the step (3) meets the requirement that the mass ratio of the enzyme nano capsule to the carbon nano material is 0.5-3: 1;

the step (3) of placing in a refrigerator for standing refers to standing in a refrigerator at 4 ℃ for 0.5-24 h, preferably standing in a refrigerator at 4 ℃ for 24 h;

the separation in the step (3) is preferably performed by one of the modes of centrifugation, filtration, suction filtration and the like; washing means washing the precipitate for 3-5 times by using a phosphate buffer solution;

a dual immobilized enzyme prepared by the method.

The double immobilized enzyme is applied to catalysis.

Compared with the prior art, the invention has the following advantages and beneficial effects:

according to the invention, the enzyme protein is modified by adopting an in-situ free radical polymerization technology and then is fixed on the carbon nano material through adsorption, and compared with the traditional adsorption and fixation method, the immobilization amount of the enzyme nanocapsule on the carbon nano material is far higher than that of unmodified enzyme molecules.

The double immobilized enzyme obtained by the invention keeps high enzyme activity, overcomes the problem that the catalyst cannot be recovered after the free enzyme and the enzyme nanocapsule are used, and has higher environmental stability and reusability than the free enzyme and the traditional immobilized enzyme and higher activity than the free enzyme due to the synergistic action between the polymer layer on the surface of the enzyme and the carbon nano material.

Drawings

FIG. 1 is a graph of enzyme immobilization and immobilization rates for different enzyme/material mass ratios in example 1;

FIG. 2 is a surface topography of OPH, nOPH, OPH (2) @ GO and nOPH (2) @ GO observed by atomic force microscope in example 1, wherein A-D graphs are surface topography graphs of OPH, nOPH, OPH (2) @ GO and nOPH (2) @ GO in this order;

FIG. 3 is a graph showing the relative values of enzyme activities of OPH, OPH (2) @ GO and nOPH (2) @ GO at 65 ℃ for different incubation times in example 1;

fig. 4 is a graph of the solid-loading amount and solid-loading rate of graphene oxide pairs oph (a) and noph (b) with different degrees of reduction in example 4.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

The reagents used in the examples are commercially available without specific reference.

The organophosphorus hydrolase described in the examples was obtained from Beijing Genobia bioengineering technology, Inc.; horseradish peroxidase (HRP), Glucose Oxidase (GOD), Lipase (LPS) described in the examples were purchased from shanghai leaf biotechnology limited.

Graphene Oxide (GO), Graphene (GN), and single-walled Carbon Nanotubes (CNTs) described in the examples were purchased from tokyo chayote nanomaterials technologies, inc.

Example 1

(1) Accurately weighing a certain amount of Graphene Oxide (GO), preparing a dispersion liquid with the concentration of 10mg/mL by using a phosphate buffer solution with the pH value of 6.0 and 50mmol/L, and treating for 1h by combining mechanical stirring (1000r/min) with ultrasound (100W);

(2) an organophosphorus hydrolase (OPH) solution having a concentration of 10mg/mL was prepared from a phosphate buffer solution of 50mmol/L and pH 6, and a modifier: n-acryloyloxy succinimide (NAS), uniformly mixing, reacting the solution at 4 ℃ for 10 hours, and sequentially adding monomers into the enzyme solution: acrylamide (AAM) and N- (3-aminopropyl) -methacrylamide hydrochloride (APM), crosslinker: n-methylene Bisacrylamide (BIS) and after 3min of nitrogen gas introduction, initiator was added: ammonium Persulfate (APS) and catalyst: tetramethylethylenediamine (TEMED) initiates an aqueous phase in-situ free radical polymerization reaction, wherein the addition ratio (mass ratio) of the components is OPH: NAS: AAM: APM: BIS: APS: TEMED ═ 1:0.1:1:2:0.2:0.5:2, the reaction time is 5h, a 3.5kD dialysis bag is adopted for dialysis and purification to obtain an enzyme nanocapsule (nOPH), and the final concentration of the enzyme nanocapsule is determined by a BCA method.

(3) Transferring 0.25mL of the graphene oxide dispersion liquid prepared in the step (1) into 410 mL centrifuge tubes respectively, and carrying out reaction according to the conditions that nOPH and GO are equal to [0.5: 1; 1: 1; 2: 1; 3:1] (mass ratio) the enzyme solution of step (2), i.e., 0.125mL, 0.25mL, 0.5mL, 0.75mL, was added thereto, and the total volume was made up to 1mL with 50mmol/L of a phosphate buffer solution at pH 6.0. Vortex mixing, transferring to a refrigerator at 4 ℃ for standing for 24h, finally performing 10000r/min centrifugal separation on the mixed solution, washing with phosphate buffer solution for 3 times, collecting supernatant, wherein the obtained precipitate is dual immobilized enzyme nOPH (0.5) @ GO, nOPH (1) @ GO, nOPH (2) @ GO and nOPH (3) @ GO, and the numbers in the parentheses represent the mass ratio of nOPH and GO respectively.

(4) The preparation method of the traditional immobilized enzyme is similar to the preparation method of the double immobilized enzyme, and the difference is that the step (2) is as follows: an organophosphorus hydrolase (OPH) solution having a concentration of 10mg/mL was prepared from 50mmol/L of a phosphate buffer solution having a pH of 6. The remaining operation steps (1) and (3) are the same as the above-described method. Finally obtaining traditional immobilized enzymes OPH (0.5) @ GO, OPH (1) @ GO, OPH (2) @ GO and OPH (3) @ GO.

The protein content in the supernatant was determined by the Brandford method, and the enzyme immobilization amount and the immobilization rate were calculated at different enzyme/material ratios, and the results are shown in fig. 1, and it can be seen from fig. 1 that the immobilization amounts of the proenzyme and the enzyme nanocapsules were gradually increased but the immobilization rate was gradually decreased as the enzyme/material ratio was increased, and when the enzyme/material ratio was 0.5:1, the immobilization rates of OPH and nOPH were 70.0% and 80.0%, respectively; when the enzyme/material ratio was 3:1, the immobilized amount of nOPH reached a maximum of 1.31mg/mg, which was twice the immobilized amount of OPH (0.63 mg/mg). The immobilization amount of the enzyme nanocapsule on the carbon nanomaterial is far higher than that of the unmodified enzyme molecule.

And (3) carrying out enzyme activity test on the obtained traditional immobilized enzyme OPH (2) @ GO, double immobilized enzyme nOPH (2) @ GO, enzyme nano-capsule nOPH and free OPH. Refer to the method of Mulbry with minor modifications. mu.L of the enzyme solution was added to a system containing 5. mu.L of methyl parathion and 900. mu.L of 50mmol/L phosphate buffer solution having pH 8.0 at 37 ℃ to effect reaction for 10min, and then 1mL of 10 vol.% trichloroacetic acid was added immediately to terminate the reaction, and 1mL of 0.1g/mL sodium carbonate solution was added to develop color and absorbance was measured at 410 nm. The results are expressed as relative enzyme activities, and when the relative enzyme activity of the free OPH is set to 100%, the relative enzyme activities of nOPH, OPH (2) @ GO and nOPH (2) @ GO are 84.89%, 80.56% and 107.09%, respectively. The high enzyme activity recovery of the double immobilized enzymes benefits from the synergistic effect of the amino group of the polymer layer on the surface of the enzyme and the carboxyl group on the surface of GO, and a good microenvironment is created for enzyme molecules.

The morphologies of OPH, nOPH, OPH (2) @ GO and nOPH (2) @ GO were observed by atomic force microscope. As shown in FIG. 2, the graphs A to D are surface morphology graphs of OPH, nOPH, OPH (2) @ GO and nOPH (2) @ GO, in that order. As shown in fig. 2A and 2B, after encapsulation, the surface morphology of the enzyme molecule is significantly changed, and the particle size of npoh is significantly larger than that of OPH, which indicates that the enzyme nanocapsule has been successfully prepared. FIGS. 2C and 2D show that OPH and nOPH have been successfully attached to the GO surface, i.e. the traditional immobilized enzymes OPH (2) @ GO and the dual immobilized enzyme nOPH (2) @ GO have been successfully prepared.

Testing the thermal stability of the immobilized enzyme: respectively incubating OPH, OPH (2) @ GO and nOPH (2) @ GO at 65 ℃ for 10min, 20min, 30min, 40min, 60min, 80min, 120min and 180 min, then measuring the enzyme activity, setting the relative activity of the corresponding sample at 37 ℃ to be 100, and taking relative values of the enzyme activities measured at other temperatures, wherein the results are shown in figure 3, and can be seen in figure 3: firstly, the activities of free OPH and traditional immobilized enzyme OPH (2) @ GO are gradually reduced along with the time extension, after incubation for 120min at 65 ℃, the free OPH basically loses all enzyme activities, the enzyme is inactivated because the structure of the enzyme is irreversibly destroyed at higher temperature, and about 35 percent of the enzyme activity is kept after the OPH (2) @ GO immobilized by adopting the traditional method is incubated for 2 h; ② the activity of double immobilized enzyme nOPH (2) @ GO is slowly reduced along with the time extension, after 60min incubation, the enzyme activity is basically kept about 80%, the good thermal stability of the double immobilized enzyme nOPH (2) @ GO is kept because the polymer layer on the surface of nOPH can stabilize the structure, and the damage of high temperature to the enzyme structure is reduced to a certain extent. The double immobilized enzyme has higher environmental stability than the free enzyme and the traditional immobilized enzyme.

And (3) testing reusability of the immobilized enzyme: in order to determine the reusability of the immobilized enzyme, after each reaction, the immobilized enzyme is centrifugally separated and recovered, and then is fully washed for the second enzyme activity determination, with the enzyme activity of the first circulation as 100%, the enzyme activity of OPH (2) @ GO is reserved by about 40% and the enzyme activity of nOPH (2) @ GO is reserved by about 95% after 10 times of circulation. The dual immobilized enzyme has better recycling stability than the traditional immobilized enzyme.

Example 2

(1) Accurately weighing a certain amount of Graphene (GN), preparing a dispersion liquid with the concentration of 5mg/mL by using a phosphate buffer solution with the pH value of 9.0 of 50mmol/L, and treating for 2.5h by combining mechanical stirring (100r/min) and ultrasound (200W);

(2) an organophosphorus hydrolase (OPH) solution having a concentration of 5mg/mL was prepared from a phosphate buffer solution of 50mmol/L and pH 9.0, and a modifier: n-acryloyloxy succinimide (NAS), uniformly mixing, placing the solution at 4 ℃ for reaction for 5 hours, and then sequentially adding monomers into the enzyme solution: acrylamide (AAM) and N- (3-aminopropyl) -methacrylamide hydrochloride (APM), crosslinker: after 3min of nitrogen gas introduction into N-methylene Bisacrylamide (BIS), initiator was added: ammonium Persulfate (APS) and catalyst: tetramethylethylenediamine (TEMED) initiates an aqueous phase in-situ free radical polymerization reaction, wherein the addition ratio (mass ratio) of the components is OPH: NAS: AAM: APM: BIS: APS: TEMED ═ 1:0.02:2:2:0.4:1:2, the reaction time is 2.5h, a 1.0kD dialysis bag is adopted for dialysis and purification to obtain an enzyme nanocapsule (nOPH), and the final concentration of the enzyme nanocapsule is determined by a BCA method.

(3) To the graphene dispersion prepared in step (1), 0.25mL was transferred and placed in a 10mL centrifuge tube, and the enzyme solution of step (2), i.e., 0.5mL was added in accordance with the mass ratio of npoh: GN ═ 2:1, and the total volume was made up to 1mL with a phosphate buffer solution of 50mmol/L and pH ═ 9.0. Mixing by vortex, transferring to refrigerator at 4 deg.C, standing for 0.5h, centrifuging at 10000r/min, washing with phosphate buffer solution for 3 times, and collecting precipitate as dual immobilized enzyme nOPH (2) @ GN.

The enzyme activity was measured for the immobilized enzyme samples nOPH (2) @ GN and free OPH obtained above. The results were expressed as relative enzyme activities, and when the relative enzyme activity of free OPH was set at 100%, the relative enzyme activity of nOPH (2) @ GN was 89.41%, which was higher than 84.89% of the nOPH activity of the enzyme nanocapsule prepared in example 1.

And (3) measuring the protein content in the supernatant by using a Brandford method, and calculating the enzyme immobilization amount and the immobilization rate when the enzyme/material ratio is 2, wherein the immobilization amount of the nOPH reaches 0.91mg/mg, and the immobilization amount of the OPH reaches 0.43 mg/mg. The immobilization amount of the enzyme nanocapsule on the carbon nanomaterial is far higher than that of the unmodified enzyme molecule.

Example 3

(1) Accurately weighing a certain amount of single-walled Carbon Nanotubes (CNT), preparing a dispersion liquid with the concentration of 2mg/mL by using a phosphate buffer solution with the pH value of 7.0 of 50mmol/L, and treating for 2 hours by combining mechanical stirring (600r/min) with ultrasound (150W);

(2) an organophosphorus hydrolase (OPH) solution having a concentration of 2mg/mL was prepared from a phosphate buffer solution having a pH of 7.0, and a modifier: N-Acryloyloxysuccinimide (NAS), after mixing, the solution was reacted at 4 ℃ for 2.5h, and then the monomers were sequentially added to the enzyme solution: acrylamide (AAM) and N- (3-aminopropyl) -methacrylamide hydrochloride (APM), crosslinker: after 3min of nitrogen gas introduction into N-methylene Bisacrylamide (BIS), initiator was added: ammonium Persulfate (APS) and catalyst: tetramethylethylenediamine (TEMED) initiates an aqueous phase in-situ free radical polymerization reaction, wherein the addition ratio (mass ratio) of the components is OPH: NAS: AAM: APM: BIS: APS: TEMED ═ 1:0.02:2:2:0.2:0.5:2, the reaction time is 2 hours, a 3.5kD dialysis bag is adopted for dialysis and purification to obtain an enzyme nanocapsule (nOPH), and the final concentration of the enzyme nanocapsule is determined by a BCA method.

(3) To the single-walled carbon nanotube dispersion prepared in step (1), 0.25mL was transferred and placed in a 10mL centrifuge tube, and the enzyme solution of step (2), i.e., 0.5mL was added in accordance with the mass ratio of nOPH: GN ═ 2:1, and the total volume was made up to 1mL with a phosphate buffer solution of 50mM and pH ═ 9.0. Mixing by vortex, transferring to refrigerator at 4 deg.C, standing for 0.5h, centrifuging at 10000r/min, washing with phosphate buffer solution for 3 times to obtain precipitate as dual immobilized enzyme nOPH (2) @ CNT.

The enzyme activity was measured for the immobilized enzyme samples nOPH (2) @ CNT and free OPH obtained above. The results were expressed as relative enzyme activities, and when the relative enzyme activity of free OPH was set at 100%, the relative enzyme activity of nOPH (2) @ CNT was 92.69%, respectively.

And (3) measuring the protein content in the supernatant by using a Brandford method, and calculating the enzyme immobilization amount and the immobilization rate when the enzyme/material ratio is 2, wherein the immobilization amount of the nOPH reaches 0.88mg/mg, and the immobilization amount of the OPH reaches 0.46 mg/mg. The immobilization amount of the enzyme nanocapsule on the carbon nanomaterial is far higher than that of the unmodified enzyme molecule.

Example 4

(1) At 25 ℃, mixing graphene oxide/ascorbic acid in a mass ratio of 1: 10, adding ascorbic acid into the graphene oxide, and reducing for 2h and 24h respectively to obtain graphene oxide (rGO2h and rGO24h) with different reduction degrees. Accurately weighing certain amounts of GO, rGO2h and rGO24h, preparing into 1mg/mL dispersion solutions respectively by using 50mmol/L phosphate buffer solution with pH of 8.0, and treating for 5h by combining mechanical stirring (300 r/min) and ultrasound (100W);

(2) an organophosphorus hydrolase (OPH) solution having a concentration of 1mg/mL was prepared from a phosphate buffer solution of 50mmol/L and pH 8, and a modifier: n-acryloyloxy succinimide (NAS), uniformly mixing, reacting the solution at 4 ℃ for 2 hours, and sequentially adding monomers into the enzyme solution: acrylamide (AAM) and N- (3-aminopropyl) -methacrylamide hydrochloride (APM), crosslinker: after 3min of nitrogen gas introduction into N-methylene Bisacrylamide (BIS), initiator was added: ammonium Persulfate (APS) and catalyst: tetramethylethylenediamine (TEMED) initiates an aqueous phase in-situ free radical polymerization reaction, wherein the addition ratio (mass ratio) of the components is OPH: NAS: AAM: APM: BIS: APS: TEMED ═ 1:0.02:2:1:0.2:0.5:2, the reaction time is 1h, a 3.5kD dialysis bag is adopted for dialysis and purification to obtain an enzyme nanocapsule (nOPH), and the final concentration of the enzyme nanocapsule is determined by a BCA method.

(3) For the GO, rGO2h and rGO24h dispersions prepared in step (1), 0.25mL of each dispersion was transferred into a 10mL centrifuge tube, 0.5mL of enzyme solution was added according to the nOPH: 2:1 (mass ratio) and the total volume was made up to 1mL with 50mmol/L of phosphate buffer solution at pH 8.0. Mixing uniformly by vortex, transferring to a refrigerator at 4 ℃ for standing for 12h, finally performing centrifugal separation on mixed liquor at 10000r/min, washing for 3 times by phosphate buffer solution, and collecting supernatant to obtain precipitates, namely dual immobilized enzymes nOPH @ GO, nOPH @ rGO2h and nOPH @ rGO24 h.

The protein content in the supernatant was determined by the Brandford method, and the immobilization amounts and the immobilization rates of the graphene oxide with different reduction degrees to OPH and nOPH were calculated, and the results are shown in fig. 4, where fig. 4A shows that the immobilization amounts of OPH on GO, rGO2h and rGO24h are 0.57mg/mg, 0.74mg/mg and 0.79 mg/mg, respectively, and the immobilization rates are 28.5%, 37.0% and 39.5%. FIG. 4B shows that the immobilization of nOPH on GO, rGO2h and rGO24h is 1.21mg/mg, 1.09mg/mg, 0.88mg/mg, respectively, with immobilization rates of 60.5%, 54.5% and 44.0%. On GO with the same reduction degree, the immobilization amount and the immobilization rate of nOPH are always larger than that of OPH, so that the polymer layer on the surface of the nanocapsule contains abundant amino functional groups and can generate hydrogen bond interaction with the material, and only a small amount of amino residues on the surface of the OPH can interact with the material, so that the immobilization amount and the immobilization rate are far lower than that of nOPH. This shows that the enzyme nanocapsules also have a good immobilization effect on carbon nanomaterials with surface functional groups that are not abundant, which has better material adaptability than free enzymes.

The potentials of the carbon nanomaterials GO, rGO2h and rGO24h and free OPH, nOPH @ GO prepared in example 4 were tested using a dynamic light scattering instrument. The potentials of GO, rGO2h and rGO24h are-30.65 mV, -28.90mV, -23.75mV respectively, the surface of the graphene oxide is negatively charged due to a large number of oxygen-containing functional groups on the surface, and the potential of the graphene oxide gradually increases along with the increase of the reduction degree of the graphene oxide, which indicates that part of the oxygen-containing functional groups on the surface of the graphene oxide are reduced, and the conjugated structure of carbon is partially repaired. The potential of free OPH is about-4.55 mV, the potential of nOPH is about 2.16mV, and the potential of the obtained nOPH @ GO immobilized enzyme after immobilization is 1.75mV, which is probably because the nOPH has a certain shielding effect on the surface charge of GO after being attached to the surface of GO. The change in GO surface charge also further indicates that nOPH has been successfully immobilized.

Example 5

(1) Accurately weighing a certain amount of Graphene Oxide (GO), preparing a 2mg/mL dispersion solution by using a phosphate buffer solution with the pH value of 8.0 and the concentration of 50mmol/L, and treating for 2h by combining mechanical stirring (600r/min) with ultrasound (150W);

(2) a 2mg/mL horseradish peroxidase (HRP) solution was prepared in 50mmol/L phosphate buffer at pH 8.0, and a modifier: n-acryloyloxy succinimide (NAS), uniformly mixing, placing the solution at 4 ℃ for reacting for 2.5h, and then sequentially adding monomers into the enzyme solution: acrylamide (AAM) and N- (3-aminopropyl) -methacrylamide hydrochloride (APM), crosslinker: after 3min of nitrogen gas introduction into N-methylene Bisacrylamide (BIS), initiator was added: ammonium Persulfate (APS) and catalyst: tetramethylethylenediamine (TEMED) initiates an aqueous phase in-situ free radical polymerization reaction, and the adding proportion (mass ratio) of the components is HRP: NAS: AAM: APM: BIS: APS: TEMED ═ 1: 0.01: 2:2:0.2:0.5: and 2, the reaction time is 2h, a 1.0kD dialysis bag is adopted for dialysis and purification to obtain an enzyme nanocapsule (nOPH), and the BCA method is adopted to determine the concentration of the final enzyme nanocapsule.

(3) For the graphene oxide dispersion prepared in step (1), 0.25mL was transferred and placed in a 10mL centrifuge tube, and the enzyme solution of step (2), i.e., 0.5mL was added according to nHPR: GO 2:1 (mass ratio), and the total volume was made up to 1mL with a phosphate buffer solution of 50mM and pH 9.0. Vortex mixing, transferring to a refrigerator at 4 deg.C, standing for 24h, centrifuging at 10000r/min, washing with phosphate buffer solution for 5 times, and collecting precipitate as dual immobilized enzyme nHRP (2) @ GO.

And (3) carrying out enzyme activity test on the obtained immobilized enzyme samples nHRP (2) @ GO and free HRP. The enzyme activity is determined by adopting a colorimetric method for detecting the activity of GB/T32131-.

And (3) measuring the protein content in the supernatant by using a Brandford method, and calculating the enzyme immobilization amount and the immobilization rate when the enzyme/material ratio is 2, wherein the immobilization amount of the nOPH reaches 1.64mg/mg, and the immobilization amount of the OPH is 0.86 mg/mg. The immobilization amount of the enzyme nanocapsule on the carbon nanomaterial is far higher than that of the unmodified enzyme molecule.

Example 6

(1) Accurately weighing a certain amount of Graphene Oxide (GO), preparing a dispersion liquid with the concentration of 5mg/mL by using a phosphate buffer solution with the pH value of 9.0 and 50mmol/L, and treating for 2.5h by combining mechanical stirring (100r/min) with ultrasound (200W);

(2) a Glucose Oxidase (GOD) solution having a concentration of 5mg/mL was prepared using a phosphate buffer solution having a pH of 9.0, and a modifier: sodium acrylate (AAS), mixed well, the solution was reacted at 4 ℃ for 5h, and then the monomers were added to the enzyme solution in order: acrylamide (AAM) and N- (3-aminopropyl) -methacrylamide hydrochloride (APM), crosslinker: after 3min of nitrogen gas introduction into N-methylene Bisacrylamide (BIS), initiator was added: ammonium Persulfate (APS) and catalyst: tetramethylethylenediamine (TEMED) initiates the aqueous phase in-situ free radical polymerization reaction, and the addition ratio (mass ratio) of the components is OPH: AAS: AAM: APM: BIS: APS: TEMED ═ 1: 0.08: 2:2:0.4:1: and 2, the reaction time is 2.5h, a 1.0kD dialysis bag is adopted for dialysis and purification to obtain an enzyme nanocapsule (nGOD), and the final concentration of the enzyme nanocapsule is determined by a BCA method.

(3) For the graphene oxide dispersion prepared in step (1), 0.25mL is transferred and placed in a 10mL centrifuge tube, the enzyme solution in step (2) is added according to the ratio of nGOD: GO 2:1 (mass ratio), that is, 0.5mL is added, and the total volume is made up to 1mL with a phosphate buffer solution of 50mmol/L and pH 9.0. Mixing by vortex, transferring to refrigerator at 4 deg.C, standing for 12h, centrifuging at 10000r/min, washing with phosphate buffer solution for 5 times, and collecting precipitate as dual immobilized enzyme nGOD (2) @ GO.

And (3) carrying out enzyme activity test on the obtained immobilized enzyme samples nGOD (2) @ GO and free GOD. A spectrophotometric method is adopted, the result is expressed by relative enzyme activity, the relative enzyme activity of free GOD is set to be 100%, and the relative enzyme activity of nGOD (2) @ GO is 126.09%.

And (3) measuring the protein content in the supernatant by using a Brandford method, and calculating the enzyme immobilization amount and the immobilization rate when the enzyme/material ratio is 2, wherein the immobilization amount of the nOPH reaches 1.51mg/mg, and the immobilization amount of the OPH is 1.03 mg/mg. The immobilization amount of the enzyme nanocapsule on the carbon nanomaterial is far higher than that of the unmodified enzyme molecule.

Example 7

(1) Accurately weighing a certain amount of Graphene Oxide (GO), preparing a dispersion liquid with the concentration of 4mg/mL by using a phosphate buffer solution with the pH value of 8.5 and the concentration of 50mmol/L, and treating for 2.5h by combining mechanical stirring (500r/min) with ultrasound (200W);

(2) a Lipase (LPS) solution at a concentration of 5mg/mL was prepared with a phosphate buffer at pH 8.5, and a modifier: acrylic Acid (AA) is mixed evenly, the solution is put to react for 5 hours at 4 ℃, and then the monomers are added into the enzyme solution in sequence: acrylamide (AAM) and N- (3-aminopropyl) -methacrylamide hydrochloride (APM), crosslinker: after 3min of nitrogen gas introduction into N-methylene Bisacrylamide (BIS), initiator was added: ammonium Persulfate (APS) and catalyst: tetramethylethylenediamine (TEMED) initiates the aqueous phase in-situ free radical polymerization reaction, and the addition ratio (mass ratio) of the components is OPH: AA: AAM: APM: BIS: APS: TEMED ═ 1: 0.05: 2:2:0.4:1: and 2, the reaction time is 2.5h, a 1.0kD dialysis bag is adopted for dialysis and purification to obtain an enzyme nanocapsule (nLPS), and the final concentration of the enzyme nanocapsule is determined by a BCA method.

(3) For the graphene oxide dispersion prepared in step (1), 0.25mL is transferred and placed in a 10mL centrifuge tube, the enzyme solution in step (2) is added according to the nLPS: GO 2:1 (mass ratio), that is, 0.5mL is added, and the total volume is made up to 1mL with a phosphate buffer solution of 50mmol/L and pH 8.5. Vortex mixing, transferring to 4 deg.C refrigerator, standing for 6h, centrifuging at 10000r/min, washing with phosphate buffer solution for 5 times, and collecting precipitate as dual immobilized enzyme nLPS (2) @ GO.

And (3) carrying out enzyme activity test on the obtained immobilized enzyme sample nLPS (2) @ GO and free LPS. By adopting a p-nitrophenol method, the result is expressed by relative enzyme activity, the relative enzyme activity of free LPS is set to be 100%, and the relative enzyme activity of nLPS (2) @ GO is 109.09%.

And (3) measuring the protein content in the supernatant by using a Brandford method, and calculating the enzyme immobilization amount and the immobilization rate when the enzyme/material ratio is 2, wherein the immobilization amount of the nOPH reaches 1.49mg/mg, and the immobilization amount of the OPH is 0.75 mg/mg. The immobilization amount of the enzyme nanocapsule on the carbon nanomaterial is far higher than that of the unmodified enzyme molecule.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A preparation method of a dual immobilized enzyme is characterized by comprising the following steps:

(1) uniformly dispersing a carbon nano material in a phosphate buffer solution with the pH = 6-9 to obtain a carbon nano material dispersion liquid;

(2) adding an enzyme into a phosphate buffer solution with the pH = 6-9 to form an enzyme solution, adding a modifier into the enzyme solution, uniformly mixing, carrying out an enzyme modification reaction, adding a monomer and a cross-linking agent into the enzyme solution after the reaction is finished, then adding an initiator and a catalyst into the enzyme solution under an anaerobic condition to initiate an in-situ polymerization reaction, and purifying the obtained reaction solution after the reaction is finished to obtain an enzyme nanocapsule solution;

(3) adding the enzyme nanocapsule solution prepared in the step (2) into the carbon nanomaterial dispersion liquid in the step (1), uniformly mixing, placing into a refrigerator for standing, and finally separating and washing the mixed liquid to obtain a precipitate, namely the dual immobilized enzyme;

the modifier in the step (2) is sodium acrylate;

the monomer in the step (2) is at least one of styrene, acrylamide, acrylate compounds, methacrylate compounds, pyrrole, thiophene and pyridine;

the cross-linking agent in the step (2) is N-methylene bisacrylamide;

the initiator in the step (2) is ammonium persulfate;

the catalyst in the step (2) is tetramethylethylenediamine.

2. The method for preparing a dual immobilized enzyme according to claim 1, wherein:

the carbon nano material in the step (1) is one or a mixture of several of graphene, graphene oxide, reduced graphene oxide, single-walled carbon nanotubes, multi-walled carbon nanotubes, carbon fibers and carbon nanospheres;

the dispersion in the step (1) is to disperse by at least one of stirring or ultrasound,

the concentration of the carbon nano-material dispersion liquid in the step (1) is 1-10 mg/mL.

3. The method for preparing a dual immobilized enzyme according to claim 2, wherein:

the carbon nano material in the step (1) is one or a mixture of several of graphene oxide, reduced graphene oxide and single-walled carbon nano tubes;

the dispersing in the step (1) is performed by mechanical stirring and ultrasonic treatment for 1-5 hours, wherein the speed of the mechanical stirring is 100-1000 r/min, and the ultrasonic power is 100-200W.

4. The method for preparing a dual immobilized enzyme according to claim 1, wherein:

the enzymes in the step (2) include, but are not limited to, organophosphorus hydrolase, lipase, horseradish peroxidase, laccase, formate dehydrogenase, formaldehyde dehydrogenase, methanol dehydrogenase, glucose oxidase, catalase, glucose isomerase, malic enzyme and alanine dehydrogenase;

the enzyme solution in the step (2) is an enzyme solution with the concentration of 1-10 mg/mL.

5. The method for preparing a dual immobilized enzyme according to claim 1, wherein:

the mass ratio of the enzyme, the modifier, the monomer, the cross-linking agent, the initiator and the catalyst in the step (2) is 1: (0.01-0.1): (1-10): (0.1-1): (0.5-1): (1-2);

the enzyme modification reaction in the step (2) is carried out at 4-30 ℃ for 2-10 h;

6. The method for preparing a dual immobilized enzyme according to claim 1, wherein:

the using amount of the carbon nano material dispersion liquid and the enzyme nano capsule solution in the step (3) meets the requirement that the mass ratio of the enzyme nano capsule to the carbon nano material is 0.5-3: 1;

the step (3) of placing in a refrigerator for standing refers to standing in a refrigerator at 4 ℃ for 0.5-24 hours;

the separation in the step (3) is separation through one of centrifugation, filtration and suction filtration; the washing is to wash for 3-5 times by using a phosphate buffer solution.

7. A dual immobilized enzyme prepared according to the method of any one of claims 1 to 6.

8. Use of the dual immobilized enzyme of claim 7 for catalysis.