Near-infrared light enzyme catalyst as well as preparation method and application thereof

Abstract

The invention discloses a near-infrared light enzyme catalyst as well as a preparation method and application thereof. The near-infrared light enzyme catalyst takes a polydopamine modified copper porphyrin metal organic framework as a substrate and lipase as an active component. The preparation method comprises the following steps: dissolving 5, 10, 15, 20-tetra (4-carboxyl phenyl) porphyrin TCPP in dimethylformamide DMF, uniformly mixing, adding a copper nitrate solution and benzoic acid, heating and stirring, after the reaction is finished, centrifugally separating and precipitating, and drying in vacuum to obtain the copper porphyrin metal organic framework Cu-TCPP; the preparation method comprises the following steps: dispersing Cu-TCPP in a phosphoric acid buffer solution, adding dopamine hydrochloride and lipase, stirring, centrifuging, separating solids, and freeze-drying to obtain the polydopamine-modified copper porphyrin metal organic framework-loaded lipase photo-enzyme catalyst. The near-infrared light enzyme catalyst can play a photo-enzyme synergistic catalysis role under near-infrared irradiation, and phthalate compounds can be effectively degraded.

Description

Technical Field

[0001] The present invention relates to the field of photoenzyme catalysts, and in particular to a near-infrared photoenzyme catalyst and a preparation method and application thereof. Background Art

[0002] Phthalates (PAEs) are considered endocrine disrupting chemicals that can cause cancer, teratogenesis, and mutagenesis even at very low concentrations. Their large-scale production and use as plasticizers increase the risk of harming human health.

[0003] Under natural conditions, PAEs are degraded only by photolysis and hydrolysis, but these two methods are extremely slow. Currently, the artificial degradation technologies for PAEs mainly include biodegradation, physical adsorption and advanced oxidation. The biodegradation method is mainly achieved through bacteria, esterases or lipases produced by bacteria. However, there are problems such as difficulty in screening strains and enzymes, a single degradation target, difficulty in degrading large molecules or complex PAEs, and the easy production of highly toxic intermediates during the degradation process. The physical adsorption method transfers pollutants but does not completely degrade them, which may cause secondary pollution. Advanced oxidation technologies include chemical oxidation, photochemical oxidation and photocatalytic oxidation. Among them, the light sources used in photochemical oxidation and photocatalytic oxidation require strong light, consume a lot of energy and have poor effects.

[0004] Free lipase is easily inactivated and has poor stability, so it is usually necessary to immobilize it for use. Using materials with good adsorption properties can not only immobilize the lipase, but also effectively degrade PAEs by synergistic effects of enzyme catalysis and adsorption. Chinese patent CN112845566B discloses a biochar-immobilized Candida lipase, which achieves the degradation of most types of PAEs. However, its degradation rate of di(2-ethylhexyl) phthalate (DEHP) is less than 30% in 120 hours at room temperature. Chinese patent CN119345901A discloses the application of a nano-titanium dioxide in the degradation of volatile phthalates in air. The material's adsorption, its own catalytic performance, and photocatalytic performance achieve almost complete degradation of phthalates, but DEHP cannot be effectively degraded and the xenon lamp used has disadvantages such as high temperature and large energy consumption. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a polydopamine (PDA)-modified copper porphyrin metal organic framework (Cu-TCPP) loaded with lipase near-infrared photoenzyme catalyst. The present invention uses a polydopamine (PDA)-modified copper porphyrin metal organic framework (Cu-TCPP) as a substrate, utilizing its good absorption and good photothermal conversion effect in the near-infrared region to load lipase to form a novel near-infrared photoenzyme catalyst. This catalyst can achieve the three-in-one effect of biodegradation, physical adsorption and photocatalytic degradation of PAEs, and can also replace traditional heating by near-infrared light irradiation, reducing energy consumption and regulating enzyme activity in real time.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned near-infrared photoenzyme catalyst.

[0007] Another object of the present invention is to provide applications of the above-mentioned near-infrared photoenzyme catalyst.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A near-infrared photoenzyme catalyst is based on a polydopamine-modified copper porphyrin metal organic framework (PDA / Cu-TCPP) and lipase as an active ingredient, with a mass ratio of the substrate to the lipase being 10:1 to 15.

[0010] The preparation method of the above-mentioned near-infrared light enzyme catalyst comprises the following steps:

[0011] (1) 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin TCPP was dissolved in dimethylformamide (DMF) and mixed well. Then, copper nitrate solution and benzoic acid were added. The mixture was heated and stirred. After the reaction was completed, the precipitate was separated by centrifugation and vacuum dried to obtain a copper porphyrin metal organic framework, named Cu-TCPP.

[0012] (2) The Cu-TCPP obtained in step (1) is dispersed in a phosphate buffer solution, dopamine hydrochloride and lipase are added, stirred, centrifuged, the solid is separated, and freeze-dried to obtain a polydopamine-modified copper porphyrin metal organic framework (PDA / Cu-TCPP)-loaded lipase photoenzyme catalyst.

[0013] Preferably, the amount of benzoic acid used in step (1) is 80 to 100 times the mass of TCPP.

[0014] Preferably, the volume mass ratio of DMF to TCPP in step (1) is 1 mL: 1-10 mg.

[0015] Preferably, the volume ratio of the copper nitrate solution to DMF in step (1) is 1:1 to 10.

[0016] Preferably, the concentration of the copper nitrate solution in step (1) is 1-5 mg / mL.

[0017] Preferably, the reaction in step (1) is carried out at 100-500 rpm and 80-100° C. for 2-6 hours.

[0018] Preferably, the centrifugation in step (1) is performed at 5000-10000 rpm for 1-10 min.

[0019] Preferably, the vacuum drying in step (1) is carried out at 50-70° C. for 3-6 hours.

[0020] Preferably, the mass volume ratio of Cu-TCPP to phosphate buffer in step (2) is 1 mg: 1-5 mL.

[0021] Preferably, the pH of the phosphate buffer solution in step (2) is 7.5 to 8.5.

[0022] Preferably, the amount of dopamine hydrochloride added in step (2) is 50% to 150% of the mass of Cu-TCPP.

[0023] Preferably, the amount of lipase added in step (2) is 10% to 150% of the mass of Cu-TCPP.

[0024] Preferably, the reaction in step (2) is carried out at room temperature and stirred at 300-500 rpm for 1-3 hours.

[0025] Preferably, the centrifugation in step (2) is performed at 8000-10000 rpm for 15-30 min.

[0026] Preferably, the lipase in step (2) is one or more of Burkholderia cepacia lipase and Candida lipase.

[0027] Application of the above-mentioned near-infrared photoenzyme catalyst in the degradation of phthalate compounds.

[0028] Preferably, the phthalate compound is one or more of di(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), and butyl benzyl phthalate (BBP).

[0029] Preferably, the application method comprises the following steps: adding 50% to 200% of the substrate mass of the photoenzyme catalyst, using 0.6 to 2 W / cm 2 Irradiation with a near-infrared laser for 1 to 3 hours can effectively degrade PAEs by exerting a photoenzyme-mediated catalytic effect.

[0030] The principle of the present invention is that Cu-TCPP is a metal organic framework with porphyrin units as organic ligands. PDA modification of Cu-TCPP is achieved by oxidative self-polymerization of dopamine in an alkaline oxygen-containing environment. The stability and rich chemical functional groups of PDA not only enhance the stability of Cu-TCPP but also increase the enzyme loading capacity of Cu-TCPP. 2+ Due to the dd band transition and the ultra-thin morphology of Cu-TCPP, Cu-TCPP has a certain absorption in the near-infrared region and has photothermal conversion ability under near-infrared irradiation. At the same time, the ultra-thin 2D morphology of Cu-TCPP means that Cu-TCPP has a larger specific surface area and more active sites, thereby being able to adsorb more PAEs. Under near-infrared irradiation, PDA / Cu-TCPP can convert light energy into heat energy to remotely locally heat lipase to enhance the catalytic activity of the enzyme, and can also generate electron-hole pairs (h + ,e - ), light stimulates electron migration, and produces a synergistic effect with lipase, thereby increasing the degradation rate of PAEs and reducing secondary pollution.

[0031] The present invention has the following advantages and effects compared to the prior art:

[0032] (1) The Cu-TCPP substrate material of the present invention has good near-infrared light absorption ability and corresponding photothermal conversion ability, which can not only realize real-time regulation of enzyme activity, thereby enhancing the degradation ability of lipase on PAEs; but also can replace the traditional heating method and realize the reaction at room temperature, thereby simplifying the entire reaction process and reducing energy consumption.

[0033] (2) The PDA / Cu-TCPP of the present invention has a porous structure and an ultrathin 2D morphology as a substrate material for immobilizing lipase, which provides a large number of adsorption sites for PAEs; PDA / Cu-TCPP can generate electron-hole pairs (h + ,e - ), which enables photocatalytic degradation of PAEs and secondary pollutants produced by enzymatic degradation of PAEs. This means that the photoenzymatic catalyst can achieve a three-in-one biodegradation, physical adsorption, and photocatalytic degradation of PAEs, thereby achieving a faster, gentler, and greener degradation of PAEs. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the infrared spectrum of PDA / Cu-TCPP loaded lipase;

[0035] Figure 2 is the UV-visible diffuse reflectance spectrum of PDA / Cu-TCPP loaded lipase;

[0036] Figure 3 is the Tauc plot of PDA / Cu-TCPP loaded lipase;

[0037] Figure 4 is the SEM image of PDA / Cu-TCPP loaded lipase;

[0038] Figure 5 This is the photothermal temperature rise curve of PDA / Cu-TCPP loaded lipase;

[0039] Figure 6 This is a diagram of the degradation of DEHP catalyzed by PDA / Cu-TCPP-loaded lipase. DETAILED DESCRIPTION

[0040] The present invention will be further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0041] Example 1

[0042] (1) Dissolve 100 mg of TCPP in 100 mL of DMF and mix well. Then, add 10 mL of copper nitrate solution (2 mg / mL) to DMF. At the same time, add 9 g of benzoic acid. React at 400 rpm and 90 °C for 4 h. After the reaction, centrifuge at 10,000 rpm for 5 min to separate the precipitate. Dry under vacuum at 50 °C for 6 h to obtain 120 mg of Cu-TCPP.

[0043] (2) Disperse 100 mg of Cu-TCPP in 200 mL of phosphate buffer (pH = 8), add 100 mg of dopamine hydrochloride and 120 mg of Burkholderia cepacia lipase, stir at 400 rpm for 2 h at room temperature, centrifuge at 8000 rpm for 30 min, separate the solid, and freeze-dry to obtain 220 mg of PDA / Cu-TCPP-loaded lipase photoenzyme catalyst.

[0044] (3) Take 50mL of 5mg / L DEHP solution, add 0.5mg of photoenzyme catalyst, and use 1W / cm 2 DEHP was degraded by near-infrared laser irradiation for 1 hour, and the degradation rate of DEHP was 65.08%.

[0045] Example 2

[0046] (1) Dissolve 100 mg of TCPP in 500 mL of DMF and mix well. Then, add 100 mL of copper nitrate solution (3 mg / mL) to DMF. At the same time, add 8 g of benzoic acid. The mixture is reacted at 300 rpm and 100 °C for 2 h. After the reaction, centrifuge at 8000 rpm for 10 min to separate the precipitate. Dry under vacuum at 70 °C for 3 h to obtain 300 mg of Cu-TCPP.

[0047] (2) Disperse 100 mg of Cu-TCPP in 500 mL of phosphate buffer (pH = 8.5), add 70 mg of dopamine hydrochloride and 100 mg of Candida lipase, stir at 500 rpm for 1 h at room temperature, centrifuge at 10,000 rpm for 20 min, separate the solid, and freeze-dry to obtain 260 mg of PDA / Cu-TCPP-loaded lipase photoenzyme catalyst.

[0048] (3) Take 50 mL of 10 mg / L dibutyl phthalate (DBP) solution, add 0.9 mg of photoenzyme catalyst, and use 1.2 W / cm 2 DBP was degraded by near-infrared laser irradiation for 2 h, and the degradation rate of DBP was 88.6%.

[0049] Example 3

[0050] (1) Dissolve 100 mg of TCPP in 1 L of DMF and mix well. Then, add 100 mL of copper nitrate solution (5 mg / mL) to DMF. At the same time, add 10 g of benzoic acid. React at 300 rpm and 80 °C for 6 h. After the reaction, centrifuge at 5000 rpm for 10 min to separate the precipitate. Dry under vacuum at 60 °C for 5 h to obtain 400 mg of Cu-TCPP.

[0051] (2) Disperse 100 mg of Cu-TCPP in 300 mL of phosphate buffer (pH = 7.5), add 50 mg of dopamine hydrochloride and 80 mg of Burkholderia cepacia lipase, stir at 300 rpm for 3 h at room temperature, centrifuge at 8000 rpm for 30 min, separate the solid, and freeze-dry to obtain 220 mg of PDA / Cu-TCPP-loaded lipase photoenzyme catalyst.

[0052] (3) Take 50 mL of 20 mg / L butyl benzyl phthalate (BBP) solution, add 1.6 mg of photoenzyme catalyst, and use 0.8 W / cm 2 Near-infrared laser irradiation for 3 hours was used to degrade BBP, and the degradation rate of BBP was 95.7%.

[0053] Effect Example 1

[0054] Test 1

[0055] Structural characterization of the near-infrared photoenzyme catalyst prepared in Example 1

[0056] Method: An appropriate amount of the near-infrared photoenzyme catalyst prepared in Experimental Example 1 was mixed with potassium bromide and pressed into a tablet, and infrared spectroscopy and ultraviolet-visible diffuse reflectance spectroscopy were performed; another sample was taken for electron microscopy observation.

[0057] result: Figure 1The infrared spectrum of PDA / Cu-TCPP loaded lipase, 515 cm -1 The group at 1614cm belongs to the carboxylic acid stretching band, and its intensity is higher than that of 1405cm -1 The metal coordination strength of the carboxylic acid group at 2+ There is positive coordination between the porphyrin molecules. Figure 2 This is the UV-visible diffuse reflectance spectrum of PDA / Cu-TCPP loaded lipase. Figure 3 Based on Figure 2 The Tauc plot was made, and based on the plot, it was calculated that the PDA / Cu-TCPP loaded lipase belongs to the direct band gap, and the band gap is 1.52 eV. Figure 4 is the SEM image of Cu-TCPP. The formed Cu-TCPP exhibits a layered nanosheet structure.

[0058] Test 2

[0059] Determination of the photothermal temperature rise curve of the near-infrared photoenzyme catalyst prepared in Example 1

[0060] Method: An appropriate amount of the near-infrared photocatalyst prepared in Example 1 was dispersed in a solution, and the photothermal temperature rise curve of the catalyst was measured using a near-infrared laser and a digital thermocouple thermometer.

[0061] result: Figure 5 The photothermal temperature rise curve of PDA / Cu-TCPP-loaded lipase shows that after 10 minutes of laser irradiation, the maximum temperature of lipase / PDA / Cu-TCPP reaches 48.3°C.

[0062] Test 3

[0063] Method: 50 mL of 5 mg / L DEHP solution was added with 0.5 mg free lipase, PDA / Cu-TCPP prepared in Example 1, lipase / PDA / Cu-TCPP (without NIR), and lipase / PDA / Cu-TCPP (NIR). The near-infrared light was irradiated at 0.8 W / cm 2 After irradiation with near-infrared laser for 1 h, the product was detected by high performance liquid chromatography to calculate the DEHP degradation rate.

[0064] result: Figure 6From left to right, columns 1-4 show a comparison of the degradation of DEHP catalyzed by free lipase, PDA / Cu-TCPP, lipase / PDA / Cu-TCPP (without NIR), and lipase / PDA / Cu-TCPP (with NIR) at room temperature. The lower degradation rate of PAEs by lipase / PDA / Cu-TCPP at room temperature without NIR irradiation is due to the low enzyme activity at 20°C and the fact that the enzyme molecules occupy some adsorption sites on the PDA / Cu-TCPP surface. The results demonstrate that the catalytic activity of the lipase is significantly enhanced after immobilization with Cu-TCPP. Under NIR irradiation, NIR not only replaces traditional heating methods for real-time regulation of enzyme activity but also produces a synergistic effect with enzyme catalysis.

[0065] Comparative Example 1 (Increasing NIR irradiation power density)

[0066] The difference between this comparative example and Example 1 is that the NIR irradiation power in step (3) is 0.8 W / cm 2 Change to 5W / cm 2 The degradation rate of DEHP was determined according to Test 3.

[0067] Results The degradation rate of DEHP was 33.78%. The reason for the lower degradation rate was that the NIR irradiation power was too high, which caused the material to heat up too high and the enzyme was partially inactivated.

[0068] Comparative Example 2 (Changing the Light Source)

[0069] The difference between this comparative example and Example 1 is that in step (3), the NIR lamp is replaced by a xenon lamp.

[0070] Results showed that the degradation rate of DEHP was 19.61%.The decrease in the degradation rate was due to the increase in light intensity and the excessively high temperature of the system, which would partially inactivate the enzyme.

[0071] The above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications may be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the claims of the present invention.

Claims

1. A near-infrared photoenzyme catalyst, characterized in that: The polydopamine-modified copper porphyrin metal organic framework is used as the substrate, lipase is used as the active ingredient, and the mass ratio of the substrate to the lipase is 10:1-15.

2. The method for preparing the near-infrared light enzyme catalyst according to claim 1, characterized in that: The steps include: (1) 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin TCPP was dissolved in dimethylformamide (DMF) and mixed well. Then, copper nitrate solution and benzoic acid were added. The mixture was heated and stirred. After the reaction was completed, the precipitate was separated by centrifugation and vacuum dried to obtain a copper porphyrin metal organic framework, named Cu-TCPP. (2) dispersing the Cu-TCPP obtained in step (1) in a phosphate buffer solution, adding dopamine hydrochloride and lipase, stirring, centrifuging, separating the solid, and freeze-drying to obtain a polydopamine-modified copper porphyrin metal organic framework-loaded lipase photoenzyme catalyst.

3. The method for preparing the near-infrared light enzyme catalyst according to claim 2, wherein: The amount of benzoic acid used in step (1) is 80 to 100 times the mass of TCPP; The volume mass ratio of DMF to TCPP in step (1) is 1 mL: 1-10 mg; The volume ratio of the copper nitrate solution to DMF in step (1) is 1:1 to 10; The concentration of the copper nitrate solution described in step (1) is 1-5 mg / mL.

4. The method for preparing the near-infrared light enzyme catalyst according to claim 2, wherein: The reaction in step (1) is carried out at 100-500 rpm and 80-100° C. for 2-6 hours; The centrifugation in step (1) is performed at 5000-10000 rpm for 1-10 min; The vacuum drying in step (1) is performed at 50-70° C. for 3-6 hours.

5. The method for preparing the near-infrared light enzyme catalyst according to claim 2, wherein: The mass volume ratio of Cu-TCPP to phosphate buffer described in step (2) is 1 mg: 1-5 mL; The pH of the phosphate buffer solution in step (2) is 7.5 to 8.5; The amount of dopamine hydrochloride added in step (2) is 50% to 150% of the mass of Cu-TCPP; The amount of lipase added in step (2) is 10% to 150% of the mass of Cu-TCPP.

6. The method for preparing the near-infrared light enzyme catalyst according to claim 2, characterized in that: The reaction in step (2) is carried out at room temperature and stirred at 300-500 rpm for 1-3 hours; The centrifugation in step (2) is performed at 8000-10000 rpm for 15-30 min.

7. The method for preparing the near-infrared light enzyme catalyst according to any one of claims 2 to 6, characterized in that: The lipase in step (2) is one or more of Burkholderia cepacia lipase and Candida lipase.

8. Use of the near-infrared photoenzyme catalyst obtained by the preparation method according to any one of claims 2 to 6 in the degradation of phthalate compounds.

9. The use according to claim 8, characterized in that: The phthalate compound is one or more of di(2-ethylhexyl) phthalate DEHP, dibutyl phthalate DBP, and butyl benzyl phthalate BBP.

10. The use according to claim 8, characterized in that: The application method comprises the following steps: adding 50% to 200% of the substrate mass of the photoenzyme catalyst, using 0.6 to 2W / cm 2 Near-infrared laser irradiation for 1 to 3 hours.

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