An immobilized enzyme carrier and preparation method thereof

Abstract

The present invention provides an immobilized enzyme carrier and a preparation method thereof, belonging to the technical field of immobilized enzymes. The present invention disperses nano antimony-doped tin dioxide, nano aluminum oxide-doped zinc oxide, iodine and carboxylic acid in a polar organic solvent to obtain a nano powder dispersion; the mass of the nano antimony-doped tin dioxide is 70-100% of the total mass of the nano antimony-doped tin dioxide and the nano aluminum oxide-doped zinc oxide; the particle sizes of the nano antimony-doped tin dioxide and the nano zinc oxide are independently 10-100 nm; a conductive metal substrate is placed in the nano powder dispersion, and a metal oxide film is deposited on the surface of the conductive metal substrate by electrophoresis to obtain the immobilized enzyme carrier. The immobilized enzyme carrier of the present invention has the advantages of reducing reduction voltage, accelerating reaction rate, reducing energy consumption, being renewable, being covalently linked to the enzyme, and being inexpensive, and is an ideal immobilized enzyme carrier.

Description

Technical Field

[0001] The invention relates to the technical field of immobilized enzymes, and in particular to an immobilized enzyme carrier and a preparation method thereof. Background Art

[0002] Enzymes are a type of biological catalyst, and the chemical nature of most enzymes is protein. Compared with chemical catalysts, enzymes have the advantages of strong specificity, high catalytic efficiency, mild reaction conditions, and controllable activity. However, due to the instability of enzymes, they are easily affected by the external environment and lose their catalytic activity. In addition, most enzymes are water-soluble, which makes it difficult to separate them from the substrate and product after the catalytic reaction, which not only affects the purity of the product but also makes the enzyme unable to be reused, resulting in enzyme catalysis in industrial applications. The high cost constraint makes it impossible to achieve ideal economic benefits.

[0003] The emergence of enzyme immobilization technology provides an effective way to overcome the above shortcomings and carry out large-scale applications. As an important branch of enzyme engineering, immobilized enzyme technology is a technology that uses physical and chemical methods to immobilize water-soluble enzymes on specific carriers or confine enzymes to a certain spatial range to restrict the movement of enzyme molecules, but still allows the enzymes to play their catalytic functions and the enzymes can be reused after the reaction. Compared with free enzymes, immobilized enzymes retain the advantages of water-soluble enzymes while having the advantages of reusability, increased activity, simple process, and large-scale application.

[0004] Immobilized enzyme carriers and enzyme-carrier immobilization processes are two key technologies for immobilized enzymes. In recent years, as enzyme-catalyzed reactions have become increasingly complex, multi-enzyme catalytic systems, especially multi-enzyme immobilization systems that can provide electrical conductivity, have been proven to be one of the most effective methods on carrier materials. The unique electrical conductivity allows the reaction enzyme to not only be fixed on the carrier material, but also to achieve efficient coenzyme regeneration through new energy sources such as hydrogen and electricity.

[0005] However, the existing immobilized enzyme carriers have low affinity with the enzyme, which easily causes the enzyme to fall off the carrier during the catalytic reaction or cyclic catalytic reaction, thus greatly reducing the number of enzyme recycling. For example, the Kylie A. Vincent laboratory at the University of Oxford used nanocarbon powder as a conductive enzyme carrier. The experiment showed that after the reaction was repeated four times, the average shedding rate was 77% (Reeve HA, Lauterbach L, Lenz O, et al. Inside Back Cover: Enzyme-Modified Particles for Selective Biocatalytic Hydrogenation by Hydrogen-Driven NADH Recycling (ChemCatChem 21 / 2015) [J]. ChemCatChem, 2015.). Summary of the invention

[0006] The purpose of the present invention is to provide an immobilized enzyme carrier and a preparation method thereof. The immobilized enzyme carrier of the present invention has good affinity with the enzyme, and the immobilized enzyme system formed after loading the enzyme has good circulation stability.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing an immobilized enzyme carrier, comprising the following steps:

[0009] Dispersing nano antimony-doped tin dioxide, nano aluminum oxide-doped zinc oxide, iodine and carboxylic acid in a polar organic solvent to obtain a nano powder dispersion; the mass of the nano antimony-doped tin dioxide is 70-100% of the total mass of the nano antimony-doped tin dioxide and the nano aluminum oxide-doped zinc oxide; the particle sizes of the nano antimony-doped tin dioxide and the nano zinc oxide are independently 10-100 nm;

[0010] The conductive metal substrate is placed in the nano powder dispersion, and a metal oxide film is deposited on the surface of the conductive metal substrate by electrophoresis to obtain the immobilized enzyme carrier.

[0011] Preferably, the conditions for depositing the metal oxide film on the surface of the conductive metal substrate by electrophoresis include: a voltage of 5 to 20 V and a time of 10 to 60 minutes.

[0012] Preferably, the resistance of the conductive metal substrate is 1×10 5 ~1×10 12 Ω.

[0013] Preferably, the conductive metal substrate includes aluminum, magnesium alloy, stainless steel, titanium, lead or lead-silver alloy.

[0014] Preferably, the amount of nano antimony doped tin dioxide and nano aluminum oxide doped zinc oxide in the nano powder dispersion is 0.0025-0.01 g / 1 cm 2 Conductive metal substrate.

[0015] Preferably, the carboxylic acid includes stearic acid; and the mass ratio of the total mass of the nano-antimony doped tin dioxide and the nano-aluminum oxide doped zinc oxide to the carboxylic acid is 2:1.

[0016] Preferably, the mass ratio of the total mass of the nano-antimony-doped tin dioxide and the nano-aluminum oxide-doped zinc oxide to iodine is 2:1.

[0017] Preferably, the polar organic solvent includes acetone, toluene, ethanol, butanone, butyl acetate or ethyl acetate.

[0018] The present invention provides an immobilized enzyme carrier prepared by the preparation method described in the above scheme, comprising a conductive metal substrate and a metal oxide film attached to the conductive metal substrate; the metal oxide film comprises antimony-doped tin dioxide and zinc oxide-doped aluminum; the mass content of antimony-doped tin dioxide in the metal oxide film is 70-100%; and the metal oxide film is a three-dimensional network porous structure.

[0019] Preferably, the thickness of the metal oxide film is 1 to 5 μm.

[0020] The invention provides a method for preparing an immobilized enzyme carrier, comprising the following steps: dispersing nano antimony-doped tin dioxide, nano aluminum oxide-doped zinc oxide, iodine and carboxylic acid in a polar organic solvent to obtain a nano powder dispersion; the mass of the nano antimony-doped tin dioxide is 70-100% of the total mass of the nano antimony-doped tin dioxide and the nano aluminum oxide-doped zinc oxide; the particle sizes of the nano antimony-doped tin dioxide and the nano zinc oxide are independently 10-100 nm; placing a conductive metal substrate in the nano powder dispersion, and depositing a metal oxide film on the surface of the conductive metal substrate by electrophoresis to obtain the immobilized enzyme carrier.

[0021] Nano antimony doped tin dioxide (ATO) and nano aluminum oxide doped zinc oxide (AZO) both have good electrical conductivity. The carrier obtained by compounding the two in the present invention has good electrical conductivity. When the carrier is energized, the negative charge of the electrode can form an electrostatic interaction with the numerous amino acid groups of the enzyme, thereby improving the affinity for the enzyme. By controlling the particle size of ATO and AZO, it is ensured that the formed metal oxide film has a three-dimensional network porous structure. In addition, ATO itself has a mesh structure, which can well simulate the space-time ratio of the enzyme space in nature, providing a higher specific surface and a more solid carrier environment for the enzyme. The results of the embodiment show that the carrier of the present invention is used for enzyme fixation, and after 4 recovery tests, nearly 100% of the enzyme activity is still retained, with good cyclic stability.

[0022] In addition, ATO and AZO are cheap, highly biocompatible and conductive materials. They are mainly used for the preparation of transparent conductive films. The conductivity of the mixed material is similar to that of the commonly used conductive film material ITO under low voltage conditions, but the cost is only 10% to 20% of ITO. Therefore, the present invention also has the advantage of low cost.

[0023] After the enzyme is fixed by the carrier of the present invention, the coenzyme can be regenerated by electrochemistry, and the conductive fixed electrode can directly activate the coenzyme that has lost its reducing ability through the electrons of the current, so that it can be continuously used in the enzymatic reaction, ultimately reducing the amount of coenzyme added and reducing the overall reaction cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the scanning electron microscope image of ATO powder;

[0025] Figure 2 is the scanning electron microscope image of ATO powder;

[0026] Figure 3 This is a graph showing the results of a cyclic stability test of the immobilized enzyme carrier prepared in Example 1 after loading the enzyme. DETAILED DESCRIPTION

[0027] The present invention provides a method for preparing an immobilized enzyme carrier, comprising the following steps:

[0028] Dispersing nano antimony-doped tin dioxide, nano aluminum oxide-doped zinc oxide, iodine and carboxylic acid in a polar organic solvent to obtain a nano powder dispersion; the mass of the nano antimony-doped tin dioxide is 70-100% of the total mass of the nano antimony-doped tin dioxide and the nano aluminum oxide-doped zinc oxide; the particle sizes of the nano antimony-doped tin dioxide and the nano zinc oxide are independently 10-100 nm;

[0029] The conductive metal substrate is placed in the nano powder dispersion, and a metal oxide film is deposited on the surface of the conductive metal substrate by electrophoresis to obtain the immobilized enzyme carrier.

[0030] In the present invention, unless otherwise specified, the raw materials used are commercially available products well known in the art.

[0031] The invention disperses nano antimony doped with tin dioxide, nano aluminum oxide doped with zinc oxide, iodine and carboxylic acid in a polar organic solvent to obtain a nano powder dispersion.

[0032] In the present invention, the particle size of the nano antimony doped tin dioxide (ATO) and the nano aluminum oxide doped zinc oxide (AZO) is independently 10-100nm, preferably 20-70nm, and more preferably 20-50nm. Those skilled in the art can select a suitable particle size according to the type of enzyme. When the fixed enzyme is coenzyme reductase and L-amino acid enzyme, the particle size of ATO is preferably 20nm, and the particle size of AZO is preferably 40nm. The present invention controls the particle size of ATO and AZO to form a metal oxide film that not only satisfies the deeper adsorption channel of the enzyme, but also has a higher mass transfer capacity. In the present invention, the mass of ATO is 70-100% of the total mass of ATO and AZO; preferably 75-85%, and more preferably 78-82%. The enzyme fixation efficiency of a single AZO powder is very low, and the conductivity of the obtained carrier is not strong enough to meet the requirements of the enzymatic reaction. The present invention uses ATO and AZO at the same time, or uses ATO, and the obtained carrier has good conductivity; and by controlling the mass ratio of the two, a higher enzyme fixation efficiency is ensured.

[0033] In the present invention, the mass ratio of the total mass of the nano antimony-doped tin dioxide and the nano aluminum oxide-doped zinc oxide to the mass of iodine is preferably 2: 1. In the present invention, the iodine helps to generate a nanoparticle coating layer by electrophoresis.

[0034] In the present invention, the carboxylic acid preferably includes stearic acid; the mass ratio of the total mass of the nano antimony doped tin dioxide and the nano aluminum oxide doped zinc oxide to the carboxylic acid is preferably 2: 1. In the present invention, the carboxylic acid functions to remove grease from the surface of ATO and AZO while increasing the dispersibility of the nanoparticles.

[0035] In the present invention, the polar organic solvent preferably includes acetone, toluene, ethanol, butanone, butyl acetate or ethyl acetate, more preferably acetone. In the present invention, the total mass ratio of the nano antimony doped tin dioxide and nano aluminum oxide doped zinc oxide to the polar organic solvent is preferably 1g:500mL.

[0036] In the present invention, nano antimony doped tin dioxide and nano aluminum oxide doped zinc oxide are preferably mixed first, and then the obtained mixed powder is mixed with iodine, stearic acid and acetone under stirring conditions to obtain a nano powder dispersion.

[0037] After obtaining the nano powder dispersion, the present invention places a conductive metal matrix in the nano powder dispersion, and deposits a metal oxide film on the surface of the conductive metal matrix by electrophoresis to obtain the immobilized enzyme carrier.

[0038] Before placing the conductive metal substrate in the nanopowder dispersion, the present invention preferably first cleans the conductive metal substrate; the cleaning preferably includes: rinsing the conductive metal substrate with deionized water for 3 to 5 times, then placing it in an acetone solution for ultrasonic cleaning, taking out the conductive metal substrate and drying it or air-drying it naturally. In the present invention, the ultrasonic cleaning time is preferably 15 minutes.

[0039] In the present invention, the resistance of the conductive metal substrate is preferably 1×10 5 ~1×10 12 Ω; the conductive metal matrix preferably includes aluminum, magnesium alloy, stainless steel, titanium, lead or lead-silver alloy. The present invention can prevent the coenzyme regeneration efficiency from being too low by controlling the resistance of the conductive metal matrix within the above range. The present invention has no special requirements on the size of the conductive metal matrix, which can be selected according to actual needs. In an embodiment of the present invention, the size of the conductive metal matrix is ​​specifically 2cm×5cm. In the present invention, the shape of the conductive metal matrix is ​​preferably a plate, a mesh or a grid.

[0040] In the present invention, the amount of nano antimony doped tin dioxide and nano aluminum oxide doped zinc oxide in the nano powder dispersion is 0.0025-0.01 g / 1 cm 2 Conductive metal substrate, more preferably 0.004 to 0.005 g / 1 cm 2 Conductive metal substrate.

[0041] In the present invention, the conditions for depositing the metal oxide film on the surface of the conductive metal substrate by electrophoresis preferably include: a voltage of 5 to 20 V and a time of 10 to 60 minutes; further, the voltage is preferably 8 to 16 V, more preferably 10 to 14 V; the time is preferably 15 to 50 minutes, more preferably 30 to 40 minutes. Those skilled in the art can adjust the thickness of the metal oxide film by adjusting the electrophoresis time.

[0042] After completing the deposition on one side, the present invention preferably repeats the electrophoresis process on the other side of the metal substrate to obtain a uniform immobilized enzyme carrier with a light blue luster on both sides.

[0043] The present invention provides an immobilized enzyme carrier prepared by the preparation method described in the above scheme, comprising a conductive metal substrate and a metal oxide film attached to the conductive metal substrate; the metal oxide film comprises antimony-doped tin dioxide and zinc oxide-doped aluminum; the mass content of antimony-doped tin dioxide in the metal oxide film is 70-90%; and the metal oxide film is a three-dimensional network porous structure.

[0044] In the present invention, the thickness of the metal oxide film is preferably 1 to 5 μm, more preferably 2 to 4 μm.

[0045] The nano antimony-doped tin dioxide (ATO) and nano aluminum oxide-doped zinc oxide (AZO) both have good electrical conductivity. The carrier obtained by compounding the two in the present invention has good electrical conductivity. When the carrier is energized, the negative charge of the electrode can form an electrostatic interaction with the numerous amino acid groups of the enzyme, thereby improving the affinity for the enzyme. The metal oxide film has a three-dimensional network porous structure, and the ATO itself has a mesh structure, which can well simulate the space-time ratio of the enzyme space in nature, providing the enzyme with a higher specific surface area and a more solid carrier environment.

[0046] The immobilized enzyme carrier of the present invention has the advantages of lowering reduction voltage, accelerating reaction rate, reducing energy consumption, being renewable, being covalently linked to the enzyme, and being inexpensive. It is an ideal immobilized enzyme carrier and has great application potential in the field of immobilized enzymes.

[0047] The immobilized enzyme carrier and the preparation method thereof provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0048] The ATO powder and ATO powder used in the following examples are all from Hengna New Materials. The scanning electron microscope image of ATO powder is as follows Figure 1 The scanning electron microscope image of ATO powder is shown in Figure 2 .Note: Figure 1 and Figure 2 The scale and text in the text box have no practical meaning and can be ignored. Figure 1 It can be seen that AZO is a network structure. Figure 2 It can be seen that the surface of ATO is a granular structure.

[0049] Example 1

[0050] A 2cm×5cm titanium sheet specimen was cut and rinsed with deionized water for 4 times. Then, it was placed in a beaker containing 50 ml of acetone solution. The beaker was transferred to an ultrasonic instrument for 15 minutes of ultrasonic cleaning. After taking out the specimen, it was placed on filter paper to air dry naturally.

[0051] Weigh 0.1g of ATO-AZO mixed powder into a beaker, where the particle size of ATO is 20nm, the particle size of AZO is 40nm, and the mass ratio is 1:9. Add 0.05g of iodine, 0.05g of stearic acid, and 50ml of acetone solution into the beaker at the same time, seal it with plastic wrap, and stir it magnetically until it dissolves to obtain a nanopowder dispersion.

[0052] Step 3: Deposit a metal oxide film on the surface of the conductive metal substrate using an electrophoresis deposition method, control the voltage to be constant at 10V, remove the metal substrate after 20 minutes of electrophoresis, and repeat the electrophoresis process on the other side to obtain a uniform immobilized enzyme carrier with a light blue luster on both sides.

[0053] Performance Test:

[0054] Take a solution of coenzyme reductase and L-amino acid enzyme, in which the concentration of coenzyme reductase is 10 nmol and the concentration ratio of coenzyme reductase to L-amino acid enzyme is 8:1; soak the immobilized enzyme carrier prepared in Example 1 in the enzyme solution and let it stand for 25 minutes to complete the immobilization of the enzyme.

[0055] To prepare the reaction solution, place 10 μM nicotinamide adenine dinucleotide phosphate, 50 mM ammonium chloride, and 0.7 M TAPS buffer in a 50 mL beaker, and then adjust the solution to a pH of 7.5.

[0056] Place the enzyme-fixed carrier in the reaction solution, connect the electrode voltage, adjust the voltage to 1V, turn on the switch, and after the reaction has been going on for ten minutes, draw out all the reaction solution and replace it with new reaction solution. Repeat this four times and record the current changes. Figure 3 As shown, Figure 3 The horizontal axis is the reaction time, in seconds; the vertical axis is the reaction current intensity (which represents the enzyme reaction rate), in microamperes.

[0057] Depend on Figure 3 It can be seen that after four reactions, through each initial current and current rate change (slope), it can be seen that the enzyme content and activity are still close to 100%, indicating that the immobilized enzyme carrier of the present invention has good affinity with the enzyme, can firmly load the enzyme and is not easy to fall off.

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an immobilized enzyme carrier, characterized in that: The following steps are involved: Dispersing nano antimony-doped tin dioxide, nano aluminum oxide-doped zinc oxide, iodine and carboxylic acid in a polar organic solvent to obtain a nano powder dispersion; the mass of the nano antimony-doped tin dioxide is 70-100% of the total mass of the nano antimony-doped tin dioxide and the nano aluminum oxide-doped zinc oxide; the particle sizes of the nano antimony-doped tin dioxide and the nano zinc oxide are independently 10-100 nm; The conductive metal substrate is placed in the nano powder dispersion, and a metal oxide film is deposited on the surface of the conductive metal substrate by electrophoresis to obtain the immobilized enzyme carrier.

2. The preparation method according to claim 1, characterized in that: The conditions for depositing the metal oxide film on the surface of the conductive metal substrate by electrophoresis include: a voltage of 5 to 20 V and a time of 10 to 60 minutes.

3. The preparation method according to claim 1, characterized in that: The resistance of the conductive metal substrate is 1×10 5 ~1×10 12 Ω.

4. The preparation method according to claim 3, characterized in that: The conductive metal substrate includes aluminum, magnesium alloy, stainless steel, titanium, lead or lead-silver alloy.

5. The preparation method according to claim 1, characterized in that: The amount of nano antimony doped tin dioxide and nano aluminum oxide doped zinc oxide in the nano powder dispersion is 0.0025-0.01 g / 1 cm 2 Conductive metal substrate.

6. The preparation method according to claim 1, characterized in that: The carboxylic acid includes stearic acid; the mass ratio of the total mass of the nano-antimony doped tin dioxide and the nano-aluminum oxide doped zinc oxide to the carboxylic acid is 2:

1.

7. The preparation method according to claim 1, characterized in that: The mass ratio of the total mass of the nano antimony-doped tin dioxide and the nano aluminum oxide-doped zinc oxide to the mass of iodine is 2:

1.

8. The preparation method according to claim 1, characterized in that: The polar organic solvent includes acetone, toluene, ethanol, butanone, butyl acetate or ethyl acetate.

9. The immobilized enzyme carrier prepared by the preparation method according to any one of claims 1 to 8, characterized in that: It comprises a conductive metal substrate and a metal oxide film attached to the conductive metal substrate; the metal oxide film comprises antimony-doped tin dioxide and zinc oxide-doped aluminum; the mass content of antimony-doped tin dioxide in the metal oxide film is 70-100%; and the metal oxide film is a three-dimensional network porous structure.

10. The immobilized enzyme carrier according to claim 9, characterized in that: The thickness of the metal oxide film is 1-5 μm.

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