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

Abstract

The application discloses a method for carbonic anhydrase immobilization based on visible light induced graft polymerization, first, ITX acetone saturated liquid drops are added on both sides of an LDPE film, and the LDPE film is clamped between two quartz plates, and then the LDPE film is irradiated under a UV mercury lamp, and then the LDPE film is washed, dried and obtained as an LDPE-ITXSP film; PEGDA, epsilon-PLL and deionized water are mixed, and then cast on both sides of the LDPE-ITXSP film, and then placed between two quartz plates, and then irradiated and polymerized under visible light, and then washed, dried, immersed in a glutaraldehyde solution, shaken, then immersed in a carbonic anhydrase / PBS buffer solution, and shaken, and the method is completed. The method can immobilize the carbonic anhydrase CA on the polyethylene film through the flexible polymer brush with a long molecular chain structure, and the flexibility of the polymer brush enables the immobilized CA to still have the ability of flexible displacement, so that the immobilized CA can improve the CO2 capture efficiency.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of enzyme immobilization process, and particularly relates to a carbonic anhydrase immobilization method based on visible light induced graft polymerization. BACKGROUND

[0002] At present, there are many technologies for capturing carbon dioxide from industrial flue gas, including the use of photoelectrocatalysis, chemical / physical solvent absorption, solid adsorption, membrane separation, low-temperature separation and the like. Although these technologies have high carbon capture capacity, they often require high energy and pressure in actual application, thereby causing additional energy consumption and environmental pollution problems, and increasing the cost of industrial carbon capture. The nature has evolved a variety of biological enzymes for transporting and transforming carbon dioxide. Among them, carbonic anhydrase (CA) can efficiently catalyze the hydration of carbon dioxide, has high selectivity (regio- / stereo- / enantio-specificity) for CO2, high conversion activity under mild reaction conditions, and biodegradability and non-toxicity, which provides a broad development for the efficient utilization of CO2.

[0003] However, free CA has high cost and weak stability, and in actual application, the stability of CA needs to be improved through CA immobilization to realize the recycling of the enzyme and save the cost of biological catalysis. 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 connection strategies. The stability of the immobilized CA is greatly improved, but due to the interaction between the immobilized substrate and CA, the active center of CA is greatly limited by mass transfer, which cannot fully contact with CO2 and cannot fully exert the catalytic activity of CA. SUMMARY

[0004] The purpose of the present application is to provide a carbonic anhydrase immobilization method based on visible light induced graft polymerization, which improves the storage stability and recycling stability of carbonic anhydrase.

[0005] The technical solution adopted by the present application is a carbonic anhydrase immobilization method based on visible light induced graft polymerization, which is implemented according to the following steps:

[0006] Step 1, uniformly drop ITX acetone saturated liquid on both sides of the LDPE film, clamp the LDPE film between two pieces of quartz plate, place it under the ultraviolet mercury lamp at room temperature, then soak it in acetone, wash the surface with acetone, and vacuum dry to obtain LDPE-ITXSP film;

[0007] Step 2, uniformly mix PEGDA, epsilon-PLL and deionized water to form a mixed solution, then cast on both sides of the LDPE-ITXSP film, place between two quartz plates, and fix with a clamp, irradiate polymerization under visible light, then immerse in deionized water, wash with deionized water, and vacuum dry to obtain the LDPE-g-PEGDA / epsilon-PLL film;

[0008] Step 3, immerse the LDPE-g-PEGDA-epsilon-PLL film in the aqueous glutaraldehyde solution, shake in a constant temperature shaking box, wash, and remove excess glutaraldehyde on the surface of the film;

[0009] Step 4, uniformly mix PBS buffer and carbonic anhydrase to obtain carbonic anhydrase / PBS buffer, then immerse the film in step 3 in the solution, constant temperature oscillation, so that the aldehyde group at the other end of glutaraldehyde reacts with the amino group on CA, and CA is covalently fixed on the polylysine brush, so that the carbonic anhydrase is fixed.

[0010] The application also has the characteristics that,

[0011] In step 1, the irradiation time is 3-6 min, the wavelength of the ultraviolet mercury lamp is 254 nm, and the light intensity is 9 mW / cm 2 ; the immersion time is 12-24 h.

[0012] In step 1, the specific preparation process of the ITX acetone saturated solution is as follows: dissolve isopropyl thioxanthone ITX in acetone, and shake until a small amount of ITX crystals are precipitated.

[0013] In step 2, the irradiation polymerization time is 60-120 min; the wavelength of the visible light is 420 nm, the light intensity is 3 mW / cm 2 ; the immersion time is 12-24 h.

[0014] In step 2, the mass ratio of PEGDA, epsilon-PLL and deionized water is 1-2:0.04-0.28:3-4.

[0015] In step 3, the oscillation time is 4-6 h.

[0016] In step 4, the oscillation temperature is 35℃, and the time is 6-12 h.

[0017] In step 4, the concentration of the PBS buffer is 0.05 mol / L, and the pH is 8.

[0018] The method can fix carbonic anhydrase (CA) on a polyethylene film through a flexible polymer brush with a long molecular chain structure, and the flexibility of the polymer brush enables the fixed CA to still have the ability of flexible displacement, thereby improving the capture efficiency of the fixed CA on CO2. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a synthesis mechanism diagram of the method of the application;

[0020] Figure 2 is a comparison diagram of the change trend of the pH value of the buffer solution with time when the free enzyme and the immobilized enzyme catalyze to capture CO2;

[0021] Figure 3 is a curve diagram of the change of the relative activity of the immobilized enzyme with the recycling number. DETAILED DESCRIPTION

[0022] The application will be described in detail below in combination with the drawings and specific embodiments.

[0023] The application is a carbonic anhydrase immobilization method based on visible light-induced graft polymerization, which is specifically implemented according to the following steps:

[0024] Step 1, dissolve isopropyl thioxanthone ITX in acetone, shake until a small amount of ITX crystals are precipitated to form ITX acetone saturated solution; uniformly drop the ITX acetone saturated solution on both sides of a low-density polyethylene (LDPE) film, clamp the LDPE film between two quartz plates to form a “sandwich” structure, and place it under a UV mercury lamp for irradiation at room temperature, then immerse it in acetone for 12-24h, and wash the surface with acetone for 3 times to remove residual ITX, and vacuum dry to obtain an LDPE (LDPE-ITXSP) film grafted with ITX semipinacol free radical dormant species;

[0025] The irradiation time is 3-6min, the wavelength of the UV mercury lamp is 254nm, and the light intensity is 9mW / cm 2 ;

[0026] Step 2, uniformly mix polyethylene glycol diacrylate (PEGDA), ε-polylysine (ε-PLL) and deionized water to form a mixed solution, then cast on both sides of the LDPE film grafted with ITX, place it between two quartz plates and fix it with a clamp, irradiate polymerization under visible light, then immerse it in deionized water for 12-24h, and wash it with deionized water to remove the unimmobilized substances, and finally vacuum dry to obtain an LDPE-g-PEGDA / ε-PLL film;

[0027] The irradiation polymerization time is 60-120 min;

[0028] The wavelength of visible light is 420 nm, and the light intensity is 3 mW / cm 2 ;

[0029] The mass ratio of PEGDA, epsilon-PLL and deionized water is 1-2 g: 0.04-0.28 g: 3-4 g;

[0030] Step 3, configure a 3%-5% volume fraction of glutaraldehyde aqueous solution, then immerse the LDPE-g-PEGDA-epsilon-PLL film in the above solution, shake in a constant temperature shaking box for 4-6 h, and then wash with deionized water for 3 times to remove the excess glutaraldehyde on the surface of the film.

[0031] Step 4, configure PBS buffer, take the PBS buffer in a centrifugal tube, add carbonic anhydrase to obtain carbonic anhydrase / PBS buffer, then immerse the above film in the solution, constant temperature oscillation (35℃, 270 rpm) for 6-12 h, so that the aldehyde group at the other end of glutaraldehyde reacts with the amino group on CA to covalently fix CA on the polylysine brush, and the fixation of carbonic anhydrase is completed;

[0032] The concentration of the PBS buffer is 0.05 mol / L, and the pH is 8; the concentration of the carbonic anhydrase / PBS buffer is 1 mg / ml;

[0033] The polyethylene film system grafted with the semi-embedded polymer brush structure prepared by the application has good enzyme loading efficiency for CA by fixing CA through the organic polymer macromolecular chain structure, and the immobilized CA also has better storage stability and cycle stability.

[0034] Example 1

[0035] Take 6 mL of acetone solution, slowly add ITX and oscillate until ITX is almost completely dissolved (with a small amount of crystal grains precipitated), to obtain ITX-acetone saturated solution. Use a pipette to add the above solution to both sides of the LDPE film, then add it between two quartz plates to form a sandwich structure, press the quartz plates to remove air bubbles, and place it under a UV mercury lamp (wavelength 254 nm, light intensity 9 mW / cm 2 ) for 6 minutes at room temperature to obtain LDPE film grafted with ITX semi-pinacol free radical dormant species (LDPE-ITXSP). Soak and wash the film in acetone solution to remove residual ITX;

[0036] 1.2 g of PEGDA, 0.28 g of ε-PLL and 3.32 g of deionized water were mixed, shaken for 10 min and left to stand, and then uniformly coated on both sides of the LDPE-ITXSP using a pipette, and placed in the middle of two quartz plates to form a sandwich structure, and irradiated with a visible light source (xenon lamp with a filter, light wavelength of 420 nm, light intensity of 3 mW / cm 2 , λ = 420 nm) for 2 h, and then extracted with deionized water for 12 h to remove residual PEGDA: ε-PLL, to prepare an LDPE-g-PEGDA / ε-PLL film;

[0037] A 5% glutaraldehyde aqueous solution was prepared, 30 ml of which was placed in a centrifuge tube, and the LDPE-g-PEGDA / ε-PLL film was placed in the glutaraldehyde aqueous solution and shaken at 270 rpm at 35°C for 6 h to allow the glutaraldehyde to fully react with the ε-PLL; a 1 mg / ml free CA enzyme solution was prepared, and 30 ml of buffer and 1.2 ml of the free CA enzyme solution were added to the centrifuge tube, which was placed in a constant-temperature shaking incubator (270 rpm, 35°C) for 6 h to complete the immobilization of the carbonic anhydrase.

[0038] Example 2

[0039] Isopropyl thioxanthone ITX was dissolved in acetone and shaken until the ITX was completely dissolved to form an ITX acetone saturated solution; the ITX acetone saturated solution was uniformly added to both sides of a low-density polyethylene (LDPE) film, and the LDPE film was clamped between two quartz plates and irradiated under a UV mercury lamp at room temperature, and then soaked in acetone for 20 h and washed with acetone three times, and vacuum dried to obtain an LDPE-ITXSP film;

[0040] The irradiation time was 5 min, the wavelength of the UV mercury lamp was 254 nm, and the light intensity was 9 mW / cm 2 ;

[0041] Polyethylene glycol diacrylate (PEGDA), ε-polylysine (ε-PLL) and deionized water were mixed uniformly to form a mixed solution, which was then cast on both sides of the LDPE film with ITX, placed between two quartz plates and fixed with a clamp, and irradiated with visible light to polymerize, and then soaked in deionized water for 15 h, washed with deionized water and vacuum dried to obtain an LDPE-g-PEGDA / ε-PLL film;

[0042] The irradiation polymerization time was 100 min; the wavelength of the visible light was 420 nm, and the light intensity was 3 mW / cm 2 ; the mass ratio of PEGDA, ε-PLL and deionized water was 1.5:0.01:3 g;

[0043] A 4% glutaraldehyde aqueous solution was prepared, and then the LDPE-g-PEGDA-ε-PLL film was completely immersed in the above solution, shaken in a constant temperature shaking box for 5 h, and then washed with deionized water for 3 times;

[0044] A PBS buffer was prepared, and then the carbonic anhydrase was added into the PBS buffer in a centrifuge tube to obtain a carbonic anhydrase / PBS buffer. Then the above film was immersed in the solution, and shaken at a constant temperature (35℃) and a constant speed (270 rpm) for 10 h, so that the aldehyde group at the other end of glutaraldehyde reacted with the amino group on the CA to covalently fix the CA on the polylysine brush, and the fixation of the carbonic anhydrase was completed.

[0045] Example 3

[0046] Isopropyl thioxanthone ITX was dissolved in acetone, and shaken until the ITX was completely dissolved to form an ITX acetone saturated solution. The ITX acetone saturated solution was uniformly dropped on both sides of a low-density polyethylene (LDPE) film, the LDPE film was clamped between two quartz plates to form a “sandwich” structure, and then placed under a UV mercury lamp for irradiation at room temperature. After that, the LDPE film was immersed in acetone for 20 h, and the surface was washed with acetone for 3 times to remove the residual ITX, and then vacuum dried to obtain an LDPE-ITXSP film.

[0047] The irradiation time was 3 min, the wavelength of the UV mercury lamp was 254 nm, and the light intensity was 9 mW / cm 2 ;

[0048] Polyethylene glycol diacrylate (PEGDA), ε-polylysine (ε-PLL) and deionized water were uniformly mixed to form a mixed solution, and then cast on both sides of the LDPE film connected with ITX, and placed between two quartz plates and fixed with a clamp. The mixed solution was irradiated under visible light, and then immersed in deionized water for 18 h, and washed with deionized water. After vacuum drying, an LDPE-g-PEGDA / ε-PLL film was obtained.

[0049] The irradiation polymerization time was 110 min, the wavelength of the visible light was 420 nm, and the light intensity was 3 mW / cm 2 ; the mass ratio of PEGDA, ε-PLL and deionized water was 2:0.28:3;

[0050] A 3% glutaraldehyde aqueous solution was prepared, and then the LDPE-g-PEGDA-ε-PLL film was completely immersed in the above solution, shaken in a constant temperature shaking box for 4 h, and then washed with deionized water for 3 times to remove the excess glutaraldehyde on the surface of the film.

[0051] The PBS buffer solution is configured, the PBS buffer solution is taken into a centrifugal tube, carbonic anhydrase is added to obtain carbonic anhydrase / PBS buffer solution, then the above-mentioned membrane is immersed in the solution, constant temperature oscillation (35℃, 270rpm) is carried out for 12h, condensation reaction of aldehyde groups at the other end of glutaraldehyde and amino groups on CA is carried out, CA is covalently fixed on the polylysine brush, and the fixation of carbonic anhydrase is completed;

[0052] Figure 1 is a synthesis mechanism diagram of the CA enzyme membrane of the polymer brush of the application, and the CA enzyme is immobilized by a simple and mild method starting from visible light active graft polymerization technology. First, ITX free radical dormant seeds are planted on the surface of LDPE by ultraviolet light, then the foam board planted with ITXSP can initiate active graft polymerization of PEGDA / ε-PLL under visible light, finally CA is covalently fixed on the ε-PLL brush through glutaraldehyde, and a complete CO2 enzyme membrane catalytic system is obtained.

[0053] Figure 2 is a comparison diagram of the change trend of the pH value of the buffer solution with time when free enzymes and immobilized enzymes catalyze the capture of CO2. Figure 2 It is shown that, in 0-10s, the capture rate of CO2 of free CA is slightly higher than that of immobilized CA, in 10-20s, with the change of the pH value of the buffer solution, the capture efficiency of immobilized enzymes is slightly higher than that of free enzymes, and then the capture ability of CO2 of the two is almost the same, so it can be seen that the immobilized CA by the polymer brush has the same CO2 capture efficiency as free CA.

[0054] Figure 3 is a result diagram of the cycle stability of the immobilized CA enzyme membrane, and it can be seen from the diagram that after 10 batches of cycle catalysis, the CA enzyme activity is still retained 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 actual industrial application.

Claims

1. A method for immobilizing carbonic anhydrase based on visible light-induced graft polymerization, characterized in that, The specific steps are as follows: Step 1: ITX saturated acetone solution is evenly dropped onto both sides of the LDPE membrane. The LDPE membrane is sandwiched between two quartz plates and irradiated at room temperature under a UV mercury lamp. Then it is immersed in acetone, the surface is washed with acetone, and vacuum dried to obtain the LDPE-ITXSP membrane. The specific preparation process of ITX acetone saturated solution is as follows: Dissolve isopropylthioxanthone (ITX) in acetone and shake until trace amounts of ITX crystals precipitate out; Step 2: Mix PEGDA, ε-PLL and deionized water evenly to form a mixture, then cast it onto both sides of the LDPE-ITXSP membrane, place it between two quartz plates and fix it with clips, irradiate it under visible light to polymerize, then immerse it in deionized water, wash it with deionized water, and vacuum dry it to obtain the LDPE-g-PEGDA / ε-PLL membrane. The polymerization time was 60-120 min; the wavelength of visible light was 420 nm, and the light intensity was 3 mW / cm². 2 Soaking time is 12-24 hours; the mass ratio of PEGDA, ε-PLL and deionized water is 1-2g: 0.04-0.28g: 3-4g. Step 3: Completely immerse the LDPE-g-PEGDA-ε-PLL membrane in a glutaraldehyde aqueous solution, shake it in a constant temperature shaking box, wash it, and remove excess glutaraldehyde from the membrane surface. Step 4: Mix PBS buffer and carbonic anhydrase evenly to obtain carbonic anhydrase / PBS buffer. Then immerse the membrane from step 3 in this solution and shake at a constant temperature to allow the aldehyde group at the other end of glutaraldehyde to undergo a condensation reaction with the amino group on CA, and covalently fix CA on the polylysine brush to complete the fixation of carbonic anhydrase. The concentration of the PBS buffer was 0.05 mol / L, and the pH was 8.

2. The method for immobilizing carbonic anhydrase based on visible light-induced graft polymerization according to claim 1, 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 / cm². 2 Soaking time is 12-24 hours.

3. The carbonic anhydrase immobilization method based on visible light-induced graft polymerization according to claim 1, characterized in that, In step 3, the oscillation time is 4-6 hours.

4. The method for immobilizing carbonic anhydrase based on visible light-induced graft polymerization according to claim 1, characterized in that, In step 4, the oscillation temperature is 35℃ and the time is 6-12h.

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