CN115725563B - Immobilized enzyme carrier and preparation method thereof - Google Patents
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- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- XOLBLPGZBRYERU-UHFFFAOYSA-N tin dioxide Chemical compound O=[Sn]=O XOLBLPGZBRYERU-UHFFFAOYSA-N 0.000 claims abstract description 83
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 claims abstract description 68
- 229910052751 metal Inorganic materials 0.000 claims abstract description 45
- 239000002184 metal Substances 0.000 claims abstract description 45
- 229910052787 antimony Inorganic materials 0.000 claims abstract description 35
- 239000011159 matrix material Substances 0.000 claims abstract description 35
- 239000011787 zinc oxide Substances 0.000 claims abstract description 34
- 229910044991 metal oxide Inorganic materials 0.000 claims abstract description 29
- 150000004706 metal oxides Chemical class 0.000 claims abstract description 29
- PNEYBMLMFCGWSK-UHFFFAOYSA-N Alumina Chemical compound [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000006185 dispersion Substances 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 19
- 239000011858 nanopowder Substances 0.000 claims abstract description 19
- 238000001962 electrophoresis Methods 0.000 claims abstract description 15
- 150000001732 carboxylic acid derivatives Chemical class 0.000 claims abstract description 13
- 239000002245 particle Substances 0.000 claims abstract description 13
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229910052740 iodine Inorganic materials 0.000 claims abstract description 10
- 239000011630 iodine Substances 0.000 claims abstract description 10
- 239000003495 polar organic solvent Substances 0.000 claims abstract description 10
- 238000000151 deposition Methods 0.000 claims abstract description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 19
- 229910052782 aluminium Inorganic materials 0.000 claims description 10
- 239000000758 substrate Substances 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 9
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 claims description 6
- 235000021355 Stearic acid Nutrition 0.000 claims description 5
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 claims description 5
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 claims description 5
- 239000008117 stearic acid Substances 0.000 claims description 5
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 claims description 4
- 239000010936 titanium Substances 0.000 claims description 4
- 229910052719 titanium Inorganic materials 0.000 claims description 4
- 229910001316 Ag alloy Inorganic materials 0.000 claims description 3
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 claims description 3
- 229910000861 Mg alloy Inorganic materials 0.000 claims description 3
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 claims description 3
- 239000011133 lead Substances 0.000 claims description 3
- LWUVWAREOOAHDW-UHFFFAOYSA-N lead silver Chemical compound [Ag].[Pb] LWUVWAREOOAHDW-UHFFFAOYSA-N 0.000 claims description 3
- 239000010935 stainless steel Substances 0.000 claims description 3
- 229910001220 stainless steel Inorganic materials 0.000 claims description 3
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims 1
- 229910052725 zinc Inorganic materials 0.000 claims 1
- 239000011701 zinc Substances 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 abstract description 52
- 102000004190 Enzymes Human genes 0.000 abstract description 52
- WATWJIUSRGPENY-UHFFFAOYSA-N antimony atom Chemical compound [Sb] WATWJIUSRGPENY-UHFFFAOYSA-N 0.000 abstract description 34
- 238000006243 chemical reaction Methods 0.000 abstract description 13
- 239000007788 liquid Substances 0.000 abstract description 13
- 230000008901 benefit Effects 0.000 abstract description 7
- 238000005265 energy consumption Methods 0.000 abstract description 2
- 230000009467 reduction Effects 0.000 abstract description 2
- 239000010408 film Substances 0.000 description 23
- 239000005515 coenzyme Substances 0.000 description 9
- 239000000843 powder Substances 0.000 description 6
- 238000006555 catalytic reaction Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000003197 catalytic effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 229910001887 tin oxide Inorganic materials 0.000 description 3
- 238000004506 ultrasonic cleaning Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000012876 carrier material Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000013329 compounding Methods 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000009881 electrostatic interaction Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000002932 luster Substances 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 239000011812 mixed powder Substances 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- XJLXINKUBYWONI-NNYOXOHSSA-N NADP zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-N 0.000 description 1
- 208000034953 Twin anemia-polycythemia sequence Diseases 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 230000002210 biocatalytic effect Effects 0.000 description 1
- 239000000560 biocompatible material Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000004020 conductor Substances 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000010799 enzyme reaction rate Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910021392 nanocarbon Inorganic materials 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
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- 238000001179 sorption measurement Methods 0.000 description 1
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Landscapes
- Catalysts (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention provides an immobilized enzyme carrier and a preparation method thereof, belonging to the technical field of immobilized enzymes. The preparation method comprises the steps of dispersing nano antimony doped tin dioxide, nano aluminum oxide doped zinc oxide, iodine and carboxylic acid in a polar organic solvent to obtain nano powder dispersion liquid, wherein 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 10-100 nm independently, placing a conductive metal matrix in the nano powder dispersion liquid, and depositing a metal oxide film on the surface of the conductive metal matrix by utilizing an electrophoresis method to obtain the immobilized enzyme carrier. The immobilized enzyme carrier has the advantages of reducing the reduction voltage, accelerating the reaction rate, reducing the energy consumption, being capable of being recycled, being covalently connected with the enzyme and being low in cost, and is an ideal immobilized enzyme carrier.
Description
Technical Field
The invention relates to the technical field of immobilized enzymes, in particular to an immobilized enzyme carrier and a preparation method thereof.
Background
Enzymes are a class of biocatalysts, most enzymes being proteins in their chemical nature. Compared with chemical catalyst, the enzyme has the advantages of strong specificity, high catalytic efficiency, mild reaction condition, controllable activity and the like. However, the enzyme is highly susceptible to external environmental influences and loses catalytic activity due to enzymatic instability. In addition, most enzymes have water solubility, so that the enzymes are not easy to separate from substrates and products after catalytic reaction, not only the purity of the products is affected, but also the enzymes cannot be reused, and ideal economic benefits cannot be realized in industrial application of enzyme catalysis due to high cost restriction.
The advent of enzyme immobilization techniques provides an effective way to overcome the above-mentioned disadvantages and to carry out large-scale applications. Immobilized enzyme technology is an important branch of enzyme engineering, namely, a technology that limits the movement of enzyme molecules by immobilizing water-soluble enzymes on specific carriers or restricting enzymes to a certain spatial range by physical and chemical methods, but still allows the enzymes to exert their catalytic functions and the enzymes can be reused after the reaction. Compared with free enzyme, the immobilized enzyme has the advantages of recycling, activity improvement, simple process, large-scale application and the like while retaining the advantages of water-soluble enzyme.
Immobilized enzyme carrier and enzyme and carrier immobilization process are two key technologies of immobilized enzyme. In recent years, as enzyme catalytic reactions are increasingly complicated, a multienzyme catalytic system, particularly a multienzyme immobilization system capable of providing electric conduction performance, has been proven to be one of the most effective methods on a carrier material, and the uniqueness of the electric conduction performance enables the reactive enzyme to be immobilized on the carrier material, and can realize a high-efficiency coenzyme regeneration function through new energy sources such as hydrogen, electric energy and the like.
However, the existing immobilized enzyme carrier has low affinity with the enzyme, which is easy to cause the enzyme to fall off from the carrier in the catalytic reaction or cyclic catalytic reaction process, thus greatly reducing the cyclic utilization times of the enzyme. For example, kylie A. Vincent laboratory of Oxford university, uses nano carbon powder as a carrier for conducting enzyme, and experiments show that after the reaction is repeated four times, the average falling rate is 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.).
Disclosure of Invention
The invention aims to provide an immobilized enzyme carrier and a preparation method thereof, wherein the immobilized enzyme carrier has good affinity with enzyme, and an immobilized enzyme system formed after enzyme loading has good circulation stability.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of an immobilized enzyme carrier, which comprises 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 nano powder dispersion, wherein 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, and the particle sizes of the nano antimony doped tin dioxide and the nano zinc oxide are 10-100 nm independently;
And placing a conductive metal matrix in the nano powder dispersion liquid, and depositing a metal oxide film on the surface of the conductive metal matrix by utilizing an electrophoresis method to obtain the immobilized enzyme carrier.
Preferably, the condition of depositing the metal oxide film on the surface of the conductive metal matrix by utilizing an electrophoresis method comprises the steps of enabling the voltage to be 5-20V and enabling the time to be 10-60 minutes.
Preferably, the resistance of the conductive metal matrix is 1×10 5～1×1012 Ω.
Preferably, the conductive metal matrix comprises aluminum, magnesium alloy, stainless steel, titanium, lead or lead silver alloy.
Preferably, the consumption of the nano antimony doped tin dioxide and the nano aluminum oxide doped zinc oxide in the nano powder dispersion liquid is 0.0025-0.01 g/1cm 2 of the conductive metal matrix.
Preferably, the carboxylic acid comprises stearic acid, and the mass ratio of the total mass of the nano antimony doped tin oxide and the nano aluminum oxide doped zinc oxide to the carboxylic acid is 2:1.
Preferably, the mass ratio of the total mass of the nano antimony doped tin oxide and the nano aluminum oxide doped zinc oxide to iodine is 2:1.
Preferably, the polar organic solvent comprises acetone, toluene, ethanol, butanone, butyl acetate or ethyl acetate.
The invention provides an immobilized enzyme carrier prepared by the preparation method of the scheme, which comprises a conductive metal matrix and a metal oxide film attached to the conductive metal matrix, wherein the metal oxide film comprises antimony doped tin dioxide and zinc oxide doped aluminum, the mass content of the antimony doped tin dioxide in the metal oxide film is 70-100%, and the metal oxide film is of a three-dimensional network porous structure.
Preferably, the thickness of the metal oxide film is 1-5 μm.
The invention provides a preparation method of an immobilized enzyme carrier, which comprises the following steps of dispersing nano antimony doped tin dioxide, nano aluminum oxide doped zinc oxide, iodine and carboxylic acid in a polar organic solvent to obtain nano powder dispersion liquid, wherein 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 10-100 nm independently, and a conductive metal matrix is placed in the nano powder dispersion liquid, and a metal oxide film is deposited on the surface of the conductive metal matrix by utilizing an electrophoresis method to obtain the immobilized enzyme carrier.
The nano antimony doped tin dioxide (ATO) and nano aluminum oxide doped zinc oxide (AZO) have good conductivity, the carrier obtained by compounding the nano antimony doped tin dioxide (ATO) and the nano aluminum oxide doped zinc oxide (AZO) has good conductivity, negative charges of the electrode can form electrostatic interaction with a plurality of amino acid groups of enzyme when the carrier is electrified, affinity to the enzyme is improved, the formed metal oxide film is ensured to have a three-dimensional network porous structure by controlling particle sizes of the ATO and the AZO, and the ATO has a network structure, so that the space-time ratio of an enzyme space in nature can be well simulated, and a higher specific surface and a firmer carrier environment are provided for the enzyme. The results of the examples show that the vector of the invention is used for enzyme immobilization, and almost 100% of enzyme activity is still retained after 4 recovery tests, and good cycling stability is achieved.
In addition, ATO and AZO are cheap, biocompatible and conductive materials. The method is mainly used for preparing the transparent conductive film, the conductivity of the mixed material is similar to that of the common conductive film material ITO in a low-voltage environment, but the cost is only 10% -20% of that of the ITO, and therefore, the method also has the advantage of low cost.
After the enzyme is immobilized by using the carrier of the invention, the coenzyme can be regenerated through electrochemistry, and the conductive immobilized electrode can directly activate the coenzyme losing the reducing capability through the electrons of the current, so that the carrier is continuously used in the enzymatic reaction, and finally, the addition amount of the coenzyme is reduced, and the comprehensive reaction cost is reduced.
Drawings
FIG. 1 is a scanning electron microscope image of ATO powder;
FIG. 2 is a scanning electron microscope image of ATO powder;
FIG. 3 is a graph showing the results of the cycle stability test after enzyme loading of the immobilized enzyme carrier prepared in example 1.
Detailed Description
The invention provides a preparation method of an immobilized enzyme carrier, which comprises 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 nano powder dispersion, wherein 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, and the particle sizes of the nano antimony doped tin dioxide and the nano zinc oxide are 10-100 nm independently;
And placing a conductive metal matrix in the nano powder dispersion liquid, and depositing a metal oxide film on the surface of the conductive metal matrix by utilizing an electrophoresis method to obtain the immobilized enzyme carrier.
In the present invention, the raw materials used are commercially available products well known in the art, unless specifically described otherwise.
According to the invention, nano antimony doped tin dioxide, nano aluminum oxide doped zinc oxide, iodine and carboxylic acid are dispersed in a polar organic solvent to obtain nano powder dispersion liquid.
In the invention, the particle sizes of the nano antimony doped tin dioxide (ATO) and the nano aluminum oxide doped zinc oxide (AZO) are independently 10-100 nm, preferably 20-70 nm, and more preferably 20-50 nm. The particle size of ATO is preferably 20nm and AZO is preferably 40nm when the immobilized enzymes are coenzyme reductase and L-amino acid enzyme. The metal oxide film formed by controlling the particle sizes of ATO and AZO not only meets the deeper adsorption channel of enzyme, but also has higher mass transfer capacity. In the 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 single AZO powdery enzyme has low fixation efficiency, and the conductivity of the obtained carrier is not strong enough, so that the requirement of enzymatic reaction can not be met. The invention adopts ATO and AZO at the same time, or adopts ATO, the obtained carrier has good conductivity, and the higher enzyme immobilization efficiency is ensured by controlling the mass ratio of the ATO and AZO.
In the invention, 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 preferably 2:1. In the present invention, the iodine aids in the creation of nanoparticle coatings by electrophoresis.
In the invention, the carboxylic acid preferably comprises stearic acid, and the mass ratio of the total mass of the nano antimony doped tin oxide 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 ATO and AZO surface grease while increasing the dispersibility of nanoparticles.
In the present invention, the polar organic solvent preferably includes acetone, toluene, ethanol, butanone, butyl acetate or ethyl acetate, more preferably acetone. In the invention, the ratio of the total mass of the nano antimony doped tin dioxide and the nano aluminum oxide doped zinc oxide to the dosage of the polar organic solvent is preferably 1g to 500mL.
The invention preferably mixes the nano antimony doped tin dioxide and the nano aluminum oxide doped zinc oxide, and then mixes the obtained mixed powder with iodine, stearic acid and acetone under the stirring condition to obtain the nano powder dispersion liquid.
After the nano powder dispersion liquid is obtained, the conductive metal matrix is placed in the nano powder dispersion liquid, and a metal oxide film is deposited on the surface of the conductive metal matrix by utilizing an electrophoresis method, so that the immobilized enzyme carrier is obtained.
The invention preferably carries out cleaning treatment on the conductive metal matrix before the conductive metal matrix is placed in the nano powder dispersion liquid, wherein the cleaning treatment preferably comprises the steps of leaching the conductive metal matrix with deionized water for 3-5 times, placing the conductive metal matrix in acetone solution for ultrasonic cleaning, taking out the conductive metal matrix, and drying or naturally air-drying the conductive metal matrix. In the present invention, the time of the ultrasonic cleaning is preferably 15 minutes.
In the present invention, the resistance of the conductive metal matrix is preferably 1×10 5～1×1012 Ω, and the conductive metal matrix preferably includes aluminum, magnesium alloy, stainless steel, titanium, lead, or lead-silver alloy. The present invention can prevent the regeneration efficiency of the coenzyme from being too low by controlling the resistance of the conductive metal substrate in the above range. The invention has no special requirement on the size of the conductive metal matrix, and the conductive metal matrix is selected according to actual requirements. In an embodiment of the present invention, the conductive metal matrix has dimensions of specifically 2cm×5cm. In the present invention, the shape of the conductive metal base is preferably a plate, net or grid.
In the invention, the consumption of nano antimony doped tin dioxide and nano aluminum oxide doped zinc oxide in the nano powder dispersion liquid is 0.0025-0.01 g/1cm 2 conductive metal matrix, more preferably 0.004-0.005 g/1cm 2 conductive metal matrix.
In the invention, the conditions for depositing the metal oxide film on the surface of the conductive metal substrate by using the electrophoresis method preferably comprise a voltage of 5-20V for 10-60 minutes, and further preferably a voltage of 8-16V, more preferably 10-14V, and a time of 15-50 minutes, more preferably 30-40 minutes. The thickness of the metal oxide thin film can be adjusted by those skilled in the art by adjusting the electrophoresis time.
After one side deposition is completed, the electrophoresis process is repeated on the other side of the optimized power-exchange metal matrix, and the immobilized enzyme carrier with light blue luster and uniformity on both sides is obtained.
The invention provides an immobilized enzyme carrier prepared by the preparation method of the scheme, which comprises a conductive metal matrix and a metal oxide film attached to the conductive metal matrix, wherein the metal oxide film comprises antimony doped tin dioxide and zinc oxide doped aluminum, the mass content of the antimony doped tin dioxide in the metal oxide film is 70-90%, and the metal oxide film is of a three-dimensional network porous structure.
In the present invention, the thickness of the metal oxide film is preferably 1 to 5 μm, more preferably 2 to 4 μm.
The nano antimony doped tin dioxide (ATO) and nano aluminum oxide doped zinc oxide (AZO) have good conductivity, the carrier obtained by compounding the nano antimony doped tin dioxide (ATO) and the nano aluminum oxide doped zinc oxide (AZO) has good conductivity, negative charges of the electrode can form electrostatic interaction with a plurality of amino acid groups of enzyme when the carrier is electrified, affinity to the enzyme is improved, the metal oxide film has a three-dimensional network porous structure, and the ATO has a network structure, so that the space-time ratio of enzyme space in nature can be well simulated, and a higher specific surface and a firmer carrier environment are provided for the enzyme.
The immobilized enzyme carrier has the advantages of reducing reduction voltage, accelerating reaction rate, reducing energy consumption, being renewable, being covalently connected with enzyme and being low in cost, is an ideal immobilized enzyme carrier, and has great application potential in the field of immobilized enzymes.
The immobilized enzyme carrier and the method for preparing the same according to the present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
Both the ATO powder and the ATO powder used in the examples below were from Hengna new materials. The scanning electron microscope of ATO powder is shown in FIG. 1, and the scanning electron microscope of ATO powder is shown in FIG. 2. Note that the scales and text in fig. 1 and 2 have no practical significance and can be ignored. As can be seen from fig. 1, AZO is a network structure, and as can be seen from fig. 2, ATO surface is a particle structure.
Example 1
A piece of a titanium sheet test piece of 2cm multiplied by 5cm is cut, rinsed with deionized water for 4 times respectively, then placed into a beaker filled with 50 milliliters of acetone solution, the beaker is transferred into an ultrasonic instrument, ultrasonic cleaning is carried out for 15 minutes, and the test piece is taken out and then placed on filter paper for natural air drying.
Weighing 0.1gATO-AZO mixed powder, and placing into a beaker, wherein the particle size of ATO is 20nm, the particle size of AZO is 40nm, and the mass ratio is 1:9. And simultaneously adding 0.05g of iodine, 0.05g of stearic acid and 50 ml of acetone solution into the beaker, sealing with a preservative film, and magnetically stirring until the mixture is dissolved to obtain nano powder dispersion.
And thirdly, depositing a metal oxide film on the surface of the conductive metal matrix by adopting an electrophoresis deposition method, controlling the voltage to be constant at 10V, taking down the metal matrix after the electrophoresis time is 20 minutes, and replacing the other surface to repeat the electrophoresis process, thereby obtaining the immobilized enzyme carrier with light blue luster and uniformity on both surfaces.
Performance test:
And (3) taking a coenzyme reductase and L amino acid enzyme solution, wherein the concentration of the coenzyme reductase in the solution is 10nmol, the concentration ratio of the coenzyme reductase to the L amino acid enzyme is 8:1, soaking the immobilized enzyme carrier prepared in the example 1 in the enzyme solution, and standing for 25 minutes to finish enzyme immobilization.
The reaction solution was prepared, and 10. Mu.M nicotinamide adenine dinucleotide phosphate, 50mM ammonium chloride, and 0.7M TAPS buffer were placed in a 50mL beaker, and then the pH of the solution was adjusted to 7.5.
Placing the enzyme-immobilized carrier into the reaction solution, switching on the electrode voltage, adjusting the voltage to 1V, switching on the switch, sucking all the reaction solution after ten minutes of reaction, changing into new reaction solution, repeating the steps four times, and recording the current change. The results are shown in FIG. 3, wherein the abscissa in FIG. 3 represents the reaction time in seconds, and the ordinate represents the reaction current intensity (i.e., representing the enzyme reaction rate) in microamps.
As can be seen from FIG. 3, after four reactions, the enzyme content and activity were still close to 100% by each initial current and current rate change (slope), indicating that the immobilized enzyme carrier of the present invention has good affinity with enzyme, can firmly support enzyme and is not easy to fall off.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
Priority Applications (1)
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