CN115354038A - A carbonic anhydrase immobilization method based on visible light-induced graft polymerization - Google Patents

Abstract

The invention discloses a carbonic anhydrase immobilization method based on visible light induced graft polymerization, which comprises the steps of firstly, dropwise adding ITX acetone saturated liquid on two sides of an LDPE (low-density polyethylene) film, clamping the LDPE film between two quartz plates, irradiating the LDPE film under an ultraviolet mercury lamp, washing and drying to obtain an LDPE-ITXSP film; mixing PEGDA, epsilon-PLL and deionized water, casting the mixture on two sides of an LDPE-ITXSP membrane, placing the LDPE-ITXSP membrane between two quartz plates, irradiating the LDPE-g-PEGDA-epsilon-PLL membrane for polymerization under visible light, washing and drying the LDPE-g-PEGDA-epsilon-PLL membrane, immersing the LDPE-g-PEGDA-epsilon-PLL membrane in a glutaraldehyde solution, oscillating the LDPE-g-PEGDA-epsilon-PLL membrane, immersing the LDPE-g-PEGDA-epsilon-PLL membrane in a carbonic anhydrase/PBS buffer solution, and oscillating the LDPE-g-PEGDA-epsilon-PLL membrane. The method can fix the carbonic anhydrase CA on the polyethylene film through the flexible polymer brush with the long molecular chain structure, and the flexibility of the polymer brush is utilized to ensure that the fixed CA still has the flexible displacement capacity, thereby improving the CO displacement capacity of the fixed CA ₂ The capture efficiency of (a).

Description

A carbonic anhydrase immobilization method based on visible light-induced graft polymerization

technical field

The invention belongs to the technical field of enzyme immobilization technology, in particular to a carbonic anhydrase immobilization method based on visible light-induced graft polymerization.

Background technique

At present, there are many technologies for capturing carbon dioxide from industrial flue gas, including the use of photoelectric catalysis, chemical/physical solvent absorption, solid adsorption, membrane separation, low-temperature separation and other technologies. Although these technologies have high carbon capture capacity, but In the actual application process, high energy and pressure are often required, which will cause additional energy consumption and environmental pollution problems, and increase the cost of industrial carbon capture. And nature has evolved a variety of biological enzymes used to transport and convert carbon dioxide. Among them, carbonic anhydrase (CA) can efficiently catalyze the hydration of carbon dioxide, high selectivity (regio/stereo/enantiomer specificity) towards _CO2 , high conversion activity under mild reaction conditions, and biodegradable and Non-toxic and other characteristics provide a broad development for the efficient utilization of _CO2 .

However, the cost of free CA is high and its stability is weak. In practical application, it is necessary to improve its stability by immobilizing CA, and realize the recycling of enzymes to save the cost of biocatalysis. At present, researchers have immobilized CA on the surface or inside of various organic polymer substrates, inorganic materials, and organic-inorganic composite materials through adsorption, encapsulation, cross-linking, and covalent linkage strategies. The stability of immobilized CA is greatly improved, but due to the interaction between the immobilized substrate and CA, the active center of CA is greatly restricted by mass transfer, and cannot fully contact with _CO2 , nor can it fully develop Catalytic activity of CA.

Contents of the invention

The object of the present invention is to provide a carbonic anhydrase immobilization method based on visible light-induced graft polymerization, which improves the storage stability and cycle stability of carbonic anhydrase.

The technical scheme adopted in the present invention is a method for immobilizing carbonic anhydrase based on visible light-induced graft polymerization, specifically implemented according to the following steps:

Step 1. Evenly add ITX acetone saturated solution on both sides of the LDPE film, sandwich the LDPE film between two quartz plates, place it under a UV mercury lamp and irradiate it at room temperature, then soak it in acetone, wash the surface with acetone, and dry it in vacuum , to obtain LDPE-ITXSP film;

Step 2, mix PEGDA, ε-PLL and deionized water evenly to form a mixed solution, then cast it on both sides of the LDPE-ITXSP film, place it between two quartz plates, and fix it with clips, irradiate polymerization under visible light, and then place Soak in deionized water, wash with deionized water, and dry in vacuum to obtain LDPE-g-PEGDA/ε-PLL membrane;

Step 3, immerse the LDPE-g-PEGDA-ε-PLL membrane completely in the glutaraldehyde aqueous solution, vibrate in a constant temperature shaking box, wash, and remove excess glutaraldehyde on the surface of the membrane;

Step 4, mix the PBS buffer solution and carbonic anhydrase evenly to obtain carbonic anhydrase/PBS buffer solution, then immerse the membrane in step 3 in the solution, and shake at a constant temperature to make the aldehyde group at the other end of the glutaraldehyde and The amino group on the CA undergoes a condensation reaction, and the CA is covalently immobilized on the polylysine brush to complete the immobilization of carbonic anhydrase.

The present invention is also characterized in that,

In step 1, the irradiation time is 3-6 minutes, the wavelength of the ultraviolet mercury lamp is 254 nm, and the light intensity is 9 mW/cm ² ; the soaking time is 12-24 hours.

In step 1, the specific preparation process of ITX acetone saturated liquid is as follows: dissolving isopropylthioxanthone ITX in acetone, shaking until a small amount of ITX crystals are precipitated.

In step 2, the irradiation polymerization time is 60-120 min; the wavelength of visible light is 420 nm, and the light intensity is 3 mW/cm ² ; the soaking time is 12-24 h.

In step 2, the mass ratio of PEGDA, ε-PLL and deionized water is 1-2:0.04-0.28:3-4.

In step 3, the oscillation time is 4-6h.

In step 4, the shaking temperature is 35° C., and the shaking time is 6-12 hours.

In step 4, the concentration of PBS buffer solution is 0.05mol/L, and the pH is 8.

The beneficial effect of the present invention is: the present invention method can carbonic anhydrase (CA) be fixed on the polyethylene film by the flexible polymer brush with long molecular chain structure, utilize the flexibility of polymer brush to make the CA after fixing still have The ability of flexible displacement, thereby improving the capture efficiency of _CO2 by immobilized CA. In addition, the immobilization system can realize the recycling and reuse of CA very conveniently, and improve the industrial application value of CA.

Description of drawings

Fig. 1 is the synthetic mechanism figure of the inventive method;

Figure 2 is a comparison chart of the change trend of the pH value of the buffer solution over time when the free enzyme and the immobilized enzyme catalyze the capture of _CO ;

Fig. 3 is a graph showing the relative activity of immobilized enzymes as a function of the number of cycles.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

A carbonic anhydrase immobilization method based on visible light-induced graft polymerization of the present invention is specifically implemented according to the following steps:

Step 1, dissolve isopropylthioxanthone ITX in acetone, shake until ITX has a small amount of crystals to precipitate, and form ITX acetone saturated liquid; evenly drop the ITX acetone saturated liquid on both sides of the low-density polyethylene (LDPE) film , the LDPE film is sandwiched between two quartz plates to form a "sandwich" structure, placed under a UV mercury lamp at room temperature, then soaked in acetone for 12-24h, and washed the surface with acetone 3 times to remove residual ITX, vacuum Dried to obtain the LDPE (LDPE-ITXSP) film grafted with ITX semipinacol free radical dormant species;

The irradiation time is 3-6min, the wavelength of the ultraviolet mercury lamp is 254nm, and the light intensity is 9mW/cm ² ;

Step 2, mix polyethylene glycol diacrylate (PEGDA), ε-polylysine (ε-PLL) and deionized water evenly to form a mixed solution, and then cast it on both sides of the LDPE film connected with ITX, and place it on Between two quartz plates, fix them with clips, place them under visible light to irradiate and polymerize, then soak them in deionized water for 12-24 hours, wash them with deionized water to remove unimmobilized substances, and finally dry them in vacuum to get LDPE - g-PEGDA/ε-PLL film;

The irradiation polymerization time is 60-120min;

The wavelength of visible light is 420nm, and the light intensity is 3mW/cm ² ;

The mass ratio of PEGDA, ε-PLL and deionized water is 1-2g: 0.04-0.28g: 3-4g;

Step 3, prepare a glutaraldehyde aqueous solution with a volume fraction of 3%-5%, and then completely immerse the LDPE-g-PEGDA-ε-PLL membrane in the above solution, shake it in a constant temperature shaking box for 4-6h, and then use it to Wash 3 times with deionized water to remove excess glutaraldehyde on the membrane surface.

Step 4, configure PBS buffer solution, put PBS buffer solution in a centrifuge tube, add carbonic anhydrase to obtain carbonic anhydrase/PBS buffer solution, then immerse the above membrane in the solution, and shake at constant temperature (35°C, 270rpm) 6-12h, the aldehyde group at the other end of glutaraldehyde and the amino group on CA undergo a condensation reaction, and CA is covalently immobilized on the polylysine brush to complete the immobilization of carbonic anhydrase;

The concentration of PBS buffer solution is 0.05mol/L, pH is 8; The concentration of carbonic anhydrase/PBS buffer solution is 1mg/ml;

The polyethylene film system grafted with a semi-embedded polymer brush structure prepared by the present invention immobilizes CA through the organic polymer polymer chain structure, has good enzyme loading efficiency for CA, and the immobilized CA also has Better storage stability and cycle stability.

Example 1

Take 6mL of acetone solution, slowly add ITX and oscillate until ITX is almost completely dissolved (fine grains are precipitated), and ITX-acetone saturated solution is obtained. Take the above solution with a pipette and add it dropwise on both sides of the LDPE film, then add it between two quartz plates to build a sandwich structure, press the quartz plate until there are no air bubbles, and place it in an ultraviolet mercury lamp (wavelength 254nm, light intensity 9mW/cm ² ) at room temperature for 6 minutes to obtain an LDPE film (LDPE-ITXSP) grafted with ITX semipinacol free radical dormant species. Soak and wash the membrane in acetone solution to remove residual ITX;

Mix 1.2g of PEGDA, 0.28g of ε-PLL and 3.32g of deionized water, oscillate for 10 minutes and let it stand still, use a pipette gun to evenly coat both sides of LDPE-ITXSP, and place it in the middle of two quartz plates to construct With a sandwich structure, the sandwich structure was irradiated with a visible light source (xenon lamp plus filter, light-passing band 420nm, light intensity 3mW/cm ² , λ=420nm) for 2h, and then the film was extracted with deionized water for 12h to remove residual PEGDA: ε-PLL, to prepare LDPE-g-PEGDA/ε-PLL film;

Prepare a glutaraldehyde aqueous solution with a volume ratio of 5%, take 30ml and put it in a centrifuge tube, place the LDPE-g-PEGDA/ε-PLL membrane in the glutaraldehyde aqueous solution at 35°C, shake at 270rpm for 6h, and make the glutaraldehyde Fully react with ε-PLL; prepare 1mg/ml free CA enzyme solution, add 30ml buffer and 1.2ml free CA enzyme solution to the centrifuge tube respectively, and place in a constant temperature shaking box (270rpm, 35°C) to react for 6h , to complete the immobilization of carbonic anhydrase.

Example 2

Dissolve isopropylthioxanthone ITX in acetone, and shake until the ITX is completely dissolved to form a saturated solution of ITX acetone; evenly drop the saturated solution of ITX acetone on both sides of the low-density polyethylene (LDPE) film, clamp the LDPE film In the middle of two quartz plates, place them under an ultraviolet mercury lamp for room temperature irradiation, then soak in acetone for 20 hours, wash the surface with acetone for 3 times, and dry in vacuum to obtain an LDPE-ITXSP film;

The irradiation time is 5 minutes, the wavelength of the ultraviolet mercury lamp is 254nm, and the light intensity is 9mW/cm ² ;

Mix polyethylene glycol diacrylate (PEGDA), ε-polylysine (ε-PLL) and deionized water evenly to form a mixed solution, and then cast it on both sides of the LDPE film connected with ITX, and place it on two pieces of quartz between the plates, and fixed with clips, placed under visible light to irradiate polymerization, then soaked in deionized water for 15 hours, washed with deionized water, and dried in vacuum to obtain LDPE-g-PEGDA/ε-PLL film;

The irradiation polymerization time is 100min; the wavelength of visible light is 420nm, and the light intensity is 3mW/cm ² ; the mass ratio of PEGDA, ε-PLL and deionized water is 1.5:0.01:3g;

Prepare a glutaraldehyde aqueous solution with a volume fraction of 4%, and then completely immerse the LDPE-g-PEGDA-ε-PLL membrane in the above solution, shake it in a constant temperature shaking box for 5 hours, and then wash it with deionized water for 3 times;

Prepare PBS buffer solution, put PBS buffer solution in a centrifuge tube, add carbonic anhydrase to obtain carbonic anhydrase/PBS buffer solution, then immerse the above-mentioned membrane in the solution, shake at constant temperature (35°C, 270rpm) for 10h, and make The aldehyde group at the other end of glutaraldehyde undergoes a condensation reaction with the amino group on CA, and the CA is covalently immobilized on the polylysine brush to complete the immobilization of carbonic anhydrase.

Example 3

Dissolve isopropylthioxanthone ITX in acetone, and shake until the ITX is completely dissolved to form a saturated solution of ITX acetone; evenly drop the saturated solution of ITX acetone on both sides of the low-density polyethylene (LDPE) film, clamp the LDPE film In the middle of two quartz plates, a "sandwich" structure was formed, placed under a UV mercury lamp at room temperature, then soaked in acetone for 20 hours, and washed the surface with acetone for 3 times to remove residual ITX, and dried in vacuum to obtain LDPE-ITXSP film ;

The irradiation time is 3min, the wavelength of the ultraviolet mercury lamp is 254nm, and the light intensity is 9mW/cm ² ;

Mix polyethylene glycol diacrylate (PEGDA), ε-polylysine (ε-PLL) and deionized water evenly to form a mixed solution, and then cast it on both sides of the LDPE film connected with ITX, and place it on two pieces of quartz between the plates, and fixed with clips, placed under visible light to irradiate polymerization, then soaked in deionized water for 18 hours, washed with deionized water, and dried in vacuum to obtain LDPE-g-PEGDA/ε-PLL film;

The irradiation polymerization time is 110min; the wavelength of visible light is 420nm, and the light intensity is 3mW/cm ² ; the mass ratio of PEGDA, ε-PLL and deionized water is 2:0.28:3;

Prepare a glutaraldehyde aqueous solution with a volume fraction of 3%, and then completely immerse the LDPE-g-PEGDA-ε-PLL membrane in the above solution, shake it in a constant temperature shaking box for 4 hours, and then wash it with deionized water 3 times to remove the membrane Surface excess glutaraldehyde.

Prepare PBS buffer solution, put PBS buffer solution in a centrifuge tube, add carbonic anhydrase to obtain carbonic anhydrase/PBS buffer solution, then immerse the above-mentioned membrane in the solution, shake at constant temperature (35°C, 270rpm) for 12h, and make The aldehyde group at the other end of glutaraldehyde undergoes a condensation reaction with the amino group on CA, and the CA is covalently immobilized on the polylysine brush to complete the immobilization of carbonic anhydrase;

Fig. 1 is the synthesizing mechanism diagram of polymer brush immobilized CA enzyme membrane of the present invention, starts from visible light active graft polymerization technology, immobilizes carbonic anhydrase with a kind of reaction condition simple and gentle method. First, ITX free radical dormant species were planted on the surface of LDPE by ultraviolet light, and then the foam board planted with ITXSP could initiate the active graft polymerization of PEGDA/ε-PLL under visible light, and finally CA was fixed on the ε-PLL by covalent action of glutaraldehyde. Brush on the PLL to get a complete _CO2 enzyme membrane catalytic system.

Fig. 2 is a comparison chart of the pH value of the buffer solution over time when the free enzyme and the immobilized enzyme catalyze the capture of CO ₂ . Figure 2 shows that the capture rate of free CA for _CO2 is slightly higher than that of immobilized CA at 0-10s, and the capture efficiency of immobilized enzyme is slightly higher than that of free enzyme at 10-20s as the pH value of the buffer solution changes. Subsequently, the two have almost the same _CO2 capture ability, which shows that the CA immobilized by polymer brushes has a _CO2 capture efficiency comparable to that of free CA.

Fig. 3 is the result graph of the cycle stability of the immobilized CA enzyme membrane. It can be seen from the graph that after 10 batches of catalysis cycles, the CA enzyme activity still remains above 85%. It can be seen that the enzyme membrane has good cycle stability and can greatly reduce the carbon capture cost of CA in practical industrial applications.

Claims (8)

1. A carbonic anhydrase immobilization method based on visible light induced graft polymerization is characterized by specifically comprising the following steps:

step 1, uniformly dropwise adding ITX acetone saturated liquid on two sides of an LDPE film, clamping the LDPE film between two quartz plates, placing the LDPE film under an ultraviolet mercury lamp for room-temperature irradiation, then soaking the LDPE film in acetone, washing the surface of the LDPE film with the acetone, and performing vacuum drying to obtain an LDPE-ITXSP film;

step 2, uniformly mixing PEGDA, epsilon-PLL and deionized water to form a mixed solution, then casting the mixed solution on two sides of an LDPE-ITXSP membrane, placing the mixed solution between two quartz plates, fixing the mixed solution by using a clamp, irradiating and polymerizing the mixed solution under visible light, then soaking the mixed solution in the deionized water, washing the soaked solution with the deionized water, and drying the soaked solution in vacuum to obtain the LDPE-g-PEGDA/epsilon-PLL membrane;

step 3, completely immersing the LDPE-g-PEGDA-epsilon-PLL membrane in a glutaraldehyde aqueous solution, oscillating in a constant-temperature oscillation box, washing, and removing redundant glutaraldehyde on the surface of the membrane;

and 4, uniformly mixing the PBS buffer solution and the carbonic anhydrase to obtain carbonic anhydrase/PBS buffer solution, then immersing the membrane in the solution in the step 3, oscillating at constant temperature to enable aldehyde group at the other end of the glutaraldehyde to perform condensation reaction with amino group on the CA, and covalently fixing the CA on a polylysine brush to complete the fixation of the carbonic anhydrase.

2. The method for immobilizing carbonic anhydrase based on visible light-induced graft polymerization as claimed in claim 1, wherein in step 1, the irradiation time is 3-6min, the wavelength of the ultraviolet mercury lamp is 254nm, and the light intensity is 9mW/cm ² (ii) a The soaking time is 12-24h.

3. The method for immobilizing carbonic anhydrase based on visible light-induced graft polymerization as claimed in claim 1, wherein in step 1, the specific preparation process of the ITX acetone saturated solution is as follows: dissolving the ITX in acetone, and oscillating until the ITX has micro crystal separated out.

4. The method for immobilizing carbonic anhydrase based on visible light-induced graft polymerization as claimed in claim 1, wherein in the step 2, the irradiation polymerization time is 60-120min; the wavelength of visible light is 420nm, and the light intensity is 3mW/cm ² (ii) a The soaking time is 12-24h.

5. The method for immobilizing carbonic anhydrase based on visible light-induced graft polymerization as claimed in claim 1, wherein in step 2, the mass ratio of PEGDA, epsilon-PLL and deionized water is 1-2g:0.04-0.28g:3-4g.

6. The method for immobilizing carbonic anhydrase based on visible light-induced graft polymerization as claimed in claim 1, wherein the shaking time in step 3 is 4-6h.

7. The method for immobilizing carbonic anhydrase based on visible light-induced graft polymerization as claimed in claim 1, wherein in step 4, the shaking temperature is 35 ℃ and the shaking time is 6-12h.

8. The method for immobilizing carbonic anhydrase based on visible light-induced graft polymerization as claimed in claim 1, wherein in step 4, the concentration of PBS buffer is 0.05mol/L and the pH is 8.

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