CN108624583B - Cuprous oxide mesoscopic crystal-biological enzyme hybrid material and preparation method thereof - Google Patents
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Abstract
The invention discloses a cuprous oxide mesoscopic crystal-biological enzyme hybrid material and a preparation method thereof. In the invention, biological enzyme is fixed on the surface of the cuprous oxide mesoscopic crystal nanowire through a covalent bond; the cuprous oxide mesoscopic crystal is obtained by taking cuprous oxide nanowires as construction units and performing mutual penetration and self-assembly growth in an orthogonal mode. The catalytic activity of the immobilized enzyme is improved by more than 10 times compared with that of the biological enzyme, the specific activity is greatly improved, the immobilized enzyme can be repeatedly used, the storage stability is high, the preparation method is simple, the cost is low, the large-scale production is easy, the immobilized enzyme can be used as an efficient wastewater treatment catalytic preparation, and the application prospect in biological environment restoration is wide.
Description
Technical Field
The invention relates to a cuprous oxide mesoscopic crystal immobilized bio-enzyme hybrid material and a preparation method thereof, belonging to the field of enzyme immobilization.
Background
The biological enzyme is protein in nature, can efficiently catalyze specific chemical reactions, and has mild reaction conditions. However, the stability of the biological enzyme to heat, strong acid, strong base, organic solvent and the like is poor, the biological enzyme is easily inactivated and denatured under the influence of extreme conditions, is difficult to separate and recycle, and cannot be reused. In order to overcome these problems, immobilized enzymes were produced and developed rapidly in the 20 th century 60 s. The immobilized enzyme is to bind or limit biological enzyme in a certain area by a physical or chemical method, so that enzyme molecules can perform catalytic reaction in the area, and can be recovered and reused for many times. The immobilized enzyme technology can be widely applied to the fields of environmental protection, medicine, agriculture, food, chemistry and the like. Immobilized enzymes have been the enthusiasm for people to widely research, and a plurality of immobilized carrier materials are emerged. Most of traditional carrier materials are bulk phase materials, have obvious mass transfer limiting effect and are often accompanied with activity loss of immobilized enzymes; the emerging nano material carrier immobilized enzyme overcomes mass transfer limitation, but is difficult to separate and recover from a reaction system. The novel biological activity micro-nano material is used as an immobilized enzyme carrier, and has shown unique advantages in immobilized enzyme research, such as obviously enhancing the activity of the immobilized enzyme. However, in the existing immobilized enzyme system with a bioactive carrier, the biological enzyme load is low, mass transfer limitation still exists, the stability needs to be improved, the exposure proportion of catalytic active sites of the enzyme is low, and the like. At present, the development and immobilization method of micro-nano carrier materials needs to be improved. The development of a novel micro-nano material carrier with high porosity and large specific surface area reduces the preparation cost, simplifies the preparation method and is urgent to construct a high-efficiency immobilized enzyme catalytic reaction system.
The endoplasmic reticulum of the cell is an important organelle, and efficient enzyme catalytic reaction is carried out at any time, so that the guarantee is provided for the normal metabolism and life maintenance of the cell. The construction of the immobilized enzyme system bionic by the endoplasmic reticulum of the cells can provide favorable microenvironment for the enzyme, ensure the active conformation and the efficient substance transfer of the enzyme and facilitate the subsequent separation and the repeated use of the immobilized enzyme material. However, the construction of such biomimetic immobilized enzyme materials is still a great challenge at present. Therefore, the development of the bionic micro-nano structure carrier-biological enzyme hybrid material has high research value and has very important practical significance for promoting the industrial application of the immobilized enzyme.
Disclosure of Invention
Aiming at the problems and the requirements in the prior art, the invention provides a bionic cuprous oxide nanowire mesoscopic crystal-biological enzyme hybrid material and a preparation method thereof, which can greatly improve the specific activity of the immobilized enzyme, can be simply separated and recycled and can be repeatedly used, and can be used as an efficient wastewater treatment catalytic preparation.
The invention is realized as follows:
a cuprous oxide mesoscopic crystal-biological enzyme hybrid material is characterized in that biological enzyme is fixed on the surface of a cuprous oxide mesoscopic crystal nanowire through a covalent bond; the cuprous oxide mesoscopic crystal is obtained by taking cuprous oxide nanowires as construction units and performing mutual penetration and self-assembly growth in an orthogonal mode.
The biological enzyme is selected from laccase, horseradish peroxidase, alcohol dehydrogenase and galactose glycolase.
The size of the cuprous oxide mesoscopic crystal-biological enzyme hybrid material is 10-100 microns.
The preparation method of the cuprous oxide mesoscopic crystal-biological enzyme hybrid material is slightly improved by a reference method (J Mater Sci, 2016, 51: 3979-:
(1) preparing cuprous oxide mesoscopic crystal dispersion liquid, wherein cuprous oxide nanowires are used as construction units of the cuprous oxide mesoscopic crystals, and the cuprous oxide mesoscopic crystals are mutually penetrated through and grow in a self-assembly manner in an orthogonal mode;
(2) activating biological enzyme selected from laccase, horseradish peroxidase, alcohol dehydrogenase and galactose glargine;
(3) and (3) fixing the biological enzyme on the cuprous oxide mesoscopic crystal nanowire to obtain the cuprous oxide mesoscopic crystal-biological enzyme hybrid material.
In the step (1), the preparation method of the cuprous oxide mesoscopic crystal dispersion liquid comprises the following steps:
(1) dissolving copper acetate, perylene-3, 4,9, 10-tetracarboxylic anhydride and o-aminoanisole in deionized water, and reacting the obtained mixed solution in a closed reaction kettle at 150-200 ℃ to obtain cuprous oxide mesoscopic crystal particles, wherein the concentration of the copper acetate is 0.1-0.3 g/ml, the concentration of the perylene-3, 4,9, 10-tetracarboxylic anhydride is 1-2 mg/ml, and the concentration of the o-aminoanisole is 0.1-1 mmol/ml;
(2) adding the prepared cuprous oxide mesoscopic crystal particles and N-hydroxysuccinimide (NHS) into a buffer solution, stirring and activating to obtain a cuprous oxide mesoscopic crystal dispersion liquid, wherein the concentration of the cuprous oxide mesoscopic crystal nanoparticles is 0.5-3 mg/ml, the concentration of NHS is 0.1-0.5 mg/ml, and the pH value of the buffer solution is 6-9.
In the step (2), the activating treatment method of the biological enzyme comprises the following steps: adding a biological enzyme molecule and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) into a buffer solution, and stirring and activating at 20-40 ℃; wherein, the concentration of the biological enzyme molecules is 0.1-3 mg/ml, the concentration of the EDC is 0.1-0.5 mg/ml, and the pH value of the buffer solution is 4-6.
And (3) adding the activated biological enzyme particles into the cuprous oxide mesoscopic crystal dispersion liquid at the temperature of 20-40 ℃, stirring and washing to obtain cuprous oxide mesoscopic crystal-biological enzyme hybrid particles.
The catalytic activity of the immobilized enzyme is improved by more than 10 times compared with that of the biological enzyme, the specific activity is greatly improved, the immobilized enzyme can be repeatedly used, the storage stability is high, the preparation method is simple, the cost is low, the large-scale production is easy, the immobilized enzyme can be used as an efficient wastewater treatment catalytic preparation, and the application prospect in biological environment restoration is wide.
Drawings
FIG. 1 is a scanning electron microscope image of mesoscopic crystals of a sub-oxide nano-wire prepared in example 1 of the present invention;
FIG. 2 is an XRD pattern of mesocrystals of the sub-oxide nanowires prepared in example 1 of the present invention;
FIG. 3 is a transmission electron microscope image of mesoscopic crystals of the sub-oxide nano-wires prepared in example 1 of the present invention;
FIG. 4 is a scanning electron microscope image of the cuprous oxide nanowire mesoscopic crystal-laccase prepared in example 1 of the present invention;
FIG. 5 is a transmission electron microscope image of a single nanowire on a cuprous oxide nanowire mesoscopic crystal-laccase prepared in example 1 of the present invention;
FIG. 6 shows the results of enzyme activity determination of cuprous oxide nanowire mesoscopic crystal-laccase prepared in example 1 of the present invention;
FIG. 7 is the reusability results of cuprous oxide nanowire mesoscopic crystal-laccase prepared in example 1 of the present invention;
FIG. 8 is a scanning electron microscope image of cuprous oxide nanowire mesoscopic crystal-horseradish peroxidase hybrid material prepared in example 2 of the present invention;
FIG. 9 shows the results of the enzyme activity assay of the cuprous oxide nanowire mesoscopic crystal horseradish peroxidase prepared in example 2 of the present invention;
FIG. 10 is the result of the storage stability of the cuprous oxide nanowire mesoscopic crystal-horseradish peroxidase prepared in example 2 of the present invention;
FIG. 11 is a scanning electron microscope image of cuprous oxide nanoparticle-laccase prepared in example 3 of the present invention;
FIG. 12 shows the results of enzyme activity assay of cuprous oxide nanoparticle-laccase prepared in example 3 of the present invention;
FIG. 13 is a scanning electron micrograph of copper oxide nanoflower-laccase prepared according to example 4 of the present invention;
FIG. 14 is a result of enzyme activity assay of copper oxide nanoflower-laccase prepared in example 4 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described below by referring to the accompanying drawings and examples.
Example 1: cuprous oxide nanowire mesoscopic crystal
0.2 mg of copper acetate powder and 0.08 mg of perylene-3, 4,9, 10-tetracarboxylic anhydride powder were weighed out, dissolved in 40 ml of deionized water, and 160. mu.l of anthranilic ether (10 mg/ml) was added and mixed well. And transferring the mixed solution into a hydrothermal reaction kettle with the volume of 50 ml, keeping the temperature at 180 ℃, and reacting for 15 hours. And after the reaction is finished, naturally cooling to room temperature, and centrifugally washing with deionized water for three times to obtain precipitates, namely the cuprous oxide nanowire mesoscopic crystals. And (3) representing the appearance of the cuprous oxide mesoscopic crystal by using an SEM (figure 1), wherein the cuprous oxide mesoscopic crystal is integrally in an octahedron shape and is formed by orderly self-assembling nanowires. FIG. 2 is an XRD pattern of cuprous oxide mesoscopic crystals, all diffraction peaks corresponding one-to-one to the standard cubic cuprous oxide crystal planes, indicating that the sample is cubic cuprous oxide. Fig. 3 is a TEM negative staining pattern of any one of the nanowires on the octahedron, the diameter of the nanowire is about 80 nm, and the nanowire is black and no obvious bright spot is seen in the pattern.
Preparing a cuprous oxide nanowire mesoscopic crystal-laccase hybrid material: 2 ml of phosphate buffer solution (pH 7.3) was measured, and 0.3 mg of N-hydroxysuccinimide and 1 mg of cuprous oxide nanowire mesoscopic crystal were weighed and added to the above buffer solution to obtain a cuprous oxide mesoscopic crystal dispersion. 1 ml of laccase solution (1 mg/ml) was measured, 0.2 mg of carbodiimide powder was added thereto, and the reaction was stirred for 15 minutes to obtain a laccase solution. The dispersion and the enzyme solution were mixed and reacted for 2 hours with stirring at a rate of 200 rpm. And after the reaction is finished, fully centrifuging and washing a reaction product to obtain the cuprous oxide nanowire mesoscopic crystal-laccase hybrid material, and measuring the specific activity of the cuprous oxide nanowire mesoscopic crystal-laccase hybrid material. And (3) representing that the mesoscopic crystal of the laccase-cuprous oxide nanowire is in an octahedral shape by using SEM (figure 4), wherein the mesoscopic crystal is formed by orderly self-assembling by taking a nanometer as a unit. The octahedral structure has a size of about 15 microns. FIG. 5 is a TEM negative staining pattern of a single nanowire dropped on an octahedron, the nanowire is black, the surface of the nanowire has clearly visible white bright spots, and the bright spots are single laccase or laccase aggregates, which prove that the laccase is successfully bound on the surface of the nanowire. FIG. 6 is the result of enzyme activity determination of cuprous oxide nanowire mesoscopic crystal immobilized laccase, and it can be known from the figure that the relative enzyme activity of the immobilized laccase is about 1085% of that of laccase. FIG. 7 shows the reusability of the enzyme, and the results show that the cuprous oxide nanowire mesoscopic crystal immobilized laccase can still retain 60% of the initial activity after repeated catalytic reaction for 10 times, which indicates that the immobilized enzyme material has good stability.
Example 2: preparation of cuprous oxide nanowire mesoscopic crystal-horseradish peroxidase hybrid material
1 ml of phosphate buffer solution (pH 7.3) was measured, and 0.3 mg of N-hydroxysuccinimide and 1 mg of cuprous oxide nanowire mesoscopic crystal were weighed and added to the above buffer solution to obtain a cuprous oxide mesoscopic crystal dispersion. 1 ml of horseradish peroxidase solution (0.5 mg/ml) was measured, and 0.2 mg of carbodiimide powder was added thereto, and the reaction was stirred for 15 minutes to obtain a bio-enzyme solution. The dispersion and the enzyme solution were mixed and reacted for 2 hours with stirring at a rate of 200 rpm. And after the reaction is finished, fully centrifuging and washing a reaction product to obtain the cuprous oxide nanowire mesoscopic crystal-horseradish peroxidase hybrid material, and measuring the specific activity of the cuprous oxide nanowire mesoscopic crystal-horseradish peroxidase hybrid material. FIG. 8 is an SEM image of cuprous oxide nanowire mesoscopic crystal-horseradish peroxidase hybrid material, which still retains intact octahedral morphology. FIG. 9 is relative enzyme activity determination data, and the result shows that the enzyme activity of the hybrid material is 400% of that of horseradish peroxidase, and the cuprous oxide nanowire mesoscopic crystal is proved to be a universal immobilized enzyme carrier, so that the activity of the immobilized enzyme can be greatly improved. FIG. 10 is the storage stability data of the hybrid material, and after 60 days of storage, the immobilized horseradish peroxidase can retain 85% of the relative enzyme activity per month, while the activity of the natural horseradish peroxidase almost loses all the activity.
Example 3: preparation of cuprous oxide nanoparticle-laccase hybrid material
1 ml of phosphate buffer solution (pH 7.3) was measured, and 0.3 mg of N-hydroxysuccinimide and 1 mg of cuprous oxide nanoparticles were weighed and added to the above buffer solution to obtain a cuprous oxide nanoparticle dispersion liquid. 1 ml of laccase solution (0.6 mg/ml) was measured, and 0.2 mg of carbodiimide powder was added thereto, and the reaction was stirred for 15 minutes to obtain a bio-enzyme solution. The dispersion and the enzyme solution were mixed and reacted for 2 hours with stirring at a rate of 200 rpm. And after the reaction is finished, fully centrifuging and washing a reaction product to obtain the cuprous oxide nanoparticle-laccase hybrid material, and measuring the specific activity of the cuprous oxide nanoparticle-laccase hybrid material. FIG. 11 is an SEM image of a cuprous oxide nanoparticle-laccase hybrid material, with a particle size of about 300 nm. FIG. 12 shows that the relative enzyme activity of the hybrid material is about 135% of that of natural laccase, and the relative enzyme activity of cuprous oxide nano-particle-laccase is far lower than that of cuprous oxide nano-wire mesoscopic crystal-laccase (1085%).
Example 4: preparation of copper oxide nanoflower-laccase hybrid material
2 ml of phosphate buffer solution (pH 7.3) was measured, and 0.15 mg of N-hydroxysuccinimide and 1 mg of copper oxide nanoflower were weighed and added to the above buffer solution to obtain a copper oxide nanoflower dispersion. 1 ml of laccase solution (0.6 mg/ml) was measured, and 0.1 mg of carbodiimide powder was added thereto, and the reaction was stirred for 15 minutes to obtain a bio-enzyme solution. The dispersion and the enzyme solution were mixed and reacted for 2 hours with stirring at a rate of 200 rpm. And after the reaction is finished, fully centrifuging and washing a reaction product to obtain the cuprous oxide nanoflower-laccase hybrid material, and measuring the specific activity of the cuprous oxide nanoflower-laccase hybrid material. FIG. 13 is an SEM image of copper oxide nanoflower-laccase hybrid material, with the nanoflower-like structure size of 4 microns. FIG. 14 shows that the relative enzyme activity of the copper oxide nanoflower-laccase is 178% of that of the natural laccase, which is also far lower than that of the cuprous oxide nanowire mesoscopic crystal-laccase (1085%).
Claims (8)
1. A cuprous oxide mesoscopic crystal-biological enzyme hybrid material is characterized in that: immobilizing biological enzyme on the surface of the cuprous oxide mesoscopic crystal nanowire through a covalent bond; the cuprous oxide mesoscopic crystal is obtained by taking cuprous oxide nanowires as construction units and performing mutual penetration and self-assembly growth in an orthogonal mode.
2. The cuprous oxide mesoscopic crystal-bio-enzyme hybrid material of claim 1, wherein: the biological enzyme is selected from laccase, horseradish peroxidase, alcohol dehydrogenase and galactose glycolase.
3. The cuprous oxide mesoscopic crystal-bio-enzyme hybrid material of claim 1, wherein: the size of the cuprous oxide mesoscopic crystal-biological enzyme hybrid material is 10-100 microns.
4. The preparation method of the cuprous oxide mesoscopic crystal-bio-enzyme hybrid material as claimed in claim 1, which is characterized by comprising the following steps:
(1) preparing cuprous oxide mesoscopic crystal dispersion liquid, wherein cuprous oxide nanowires are used as construction units of the cuprous oxide mesoscopic crystals, and the cuprous oxide mesoscopic crystals are mutually penetrated through and grow in a self-assembly manner in an orthogonal mode;
(2) activating the biological enzyme;
(3) and (3) fixing the biological enzyme on the cuprous oxide mesoscopic crystal nanowire to obtain the cuprous oxide mesoscopic crystal-biological enzyme hybrid material.
5. The method according to claim 4, wherein: in the step (1), the preparation method of the cuprous oxide mesoscopic crystal dispersion liquid comprises the following steps:
(1) dissolving copper acetate, perylene-3, 4,9, 10-tetracarboxylic anhydride and o-aminoanisole in deionized water, and reacting the obtained mixed solution in a closed reaction kettle at 150-200 ℃ to obtain cuprous oxide mesoscopic crystal particles, wherein the concentration of the copper acetate is 0.1-0.3 g/ml, the concentration of the perylene-3, 4,9, 10-tetracarboxylic anhydride is 1-2 mg/ml, and the concentration of the o-aminoanisole is 0.1-1 mmol/ml;
(2) adding the prepared cuprous oxide mesoscopic crystal particles and N-hydroxysuccinimide (NHS) into a buffer solution, stirring and activating to obtain a cuprous oxide mesoscopic crystal dispersion liquid, wherein the concentration of the cuprous oxide mesoscopic crystal nanoparticles is 0.5-3 mg/ml, the concentration of NHS is 0.1-0.5 mg/ml, and the pH value of the buffer solution is 6-9.
6. The preparation method according to claim 4, wherein in the step (2), the biological enzyme activation treatment method comprises: adding a biological enzyme molecule and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) into a buffer solution, and stirring and activating at 20-40 ℃; wherein, the concentration of the biological enzyme molecules is 0.1-3 mg/ml, the concentration of the EDC is 0.1-0.5 mg/ml, and the pH value of the buffer solution is 4-6.
7. The preparation method according to claim 4, wherein in the step (3), the activated biological enzyme particles are added into the cuprous oxide mesoscopic crystal dispersion liquid at 20-40 ℃, stirred and washed to obtain cuprous oxide mesoscopic crystal-biological enzyme hybrid particles.
8. The method according to claim 4, wherein: the biological enzyme is selected from laccase, horseradish peroxidase, alcohol dehydrogenase and galactose glycolase.
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| CN105498558A (en) * | 2015-12-17 | 2016-04-20 | 浙江工业大学 | Hybrid membrane loaded with silver nanoparticles and preparation method and application thereof |
| CN106893576A (en) * | 2017-02-15 | 2017-06-27 | 中国科学院福建物质结构研究所 | A kind of organic inorganic hybridization thermal discoloration compound |
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| CN105498558A (en) * | 2015-12-17 | 2016-04-20 | 浙江工业大学 | Hybrid membrane loaded with silver nanoparticles and preparation method and application thereof |
| CN106893576A (en) * | 2017-02-15 | 2017-06-27 | 中国科学院福建物质结构研究所 | A kind of organic inorganic hybridization thermal discoloration compound |
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| Synthesis of Cu2O nanowire mesocrystals using PTCDA as a modifier and their superior peroxidase-like activity;Ga Long Li等;《J Mater Sci》;20160113;第51卷;3979-3988 * |
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