CN108059184B - Method for preparing ZnO nanoparticles by taking recombinant collagen as biomineralization template - Google Patents

Method for preparing ZnO nanoparticles by taking recombinant collagen as biomineralization template Download PDF

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CN108059184B
CN108059184B CN201711459404.7A CN201711459404A CN108059184B CN 108059184 B CN108059184 B CN 108059184B CN 201711459404 A CN201711459404 A CN 201711459404A CN 108059184 B CN108059184 B CN 108059184B
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肖建喜
何会霞
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Lanzhou Biological Technology Development Co.,Ltd.
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Abstract

The invention discloses a method for preparing ZnO nanoparticles by taking recombinant collagen as a biomineralization template, which comprises the following steps: (1) preparing recombinant collagen by utilizing a biological genetic engineering technology: determining the sequence of recombinant collagen; synthesizing nucleic acid encoding recombinant collagen; preparing and purifying the recombinant collagen; (2) preparing a ZnO nano material: preparing a uniformly mixed solution of recombinant collagen, zinc nitrate hexahydrate and sodium hydroxide; secondly, placing the mixed solution in a thermostat at 25 ℃; purifying and drying the prepared nano material. The invention adopts recombinant collagen as a biological template and zinc nitrate hexahydrate as a raw material to prepare the ZnO nano material with controllable size and shape at room temperature. The method is simple and convenient, is easy to operate, and the prepared ZnO nano material has excellent degradation capability on various organic dyes and has great application prospect in treatment of dye wastewater.

Description

Method for preparing ZnO nanoparticles by taking recombinant collagen as biomineralization template
Technical Field
The invention belongs to the technical field of preparation of biological inorganic materials, and particularly relates to a method for preparing ZnO nanoparticles by taking recombinant collagen as a biomineralization template.
Background
With the rapid development of global economy, people pay attention to the protection of the environment and pay particular attention to the problem of water pollution. The printing and dyeing wastewater in China has the characteristics of large discharge amount, more substances difficult to degrade, complex organic matter components, deep chroma and the like, and an efficient treatment method is urgently needed to be developed. The organic dye is a main component of printing and dyeing wastewater, such as a representative cationic dye rhodamine B, which has strong fluorescence property in a solution, and even if the concentration is low, the light transmittance of a water body is reduced, so that the ecological environment is seriously damaged. At present, the treatment method of dye wastewater comprises coagulation method, biochemical method, flocculation method, electrochemical precipitation, photochemical catalytic oxidation, membrane separation method, adsorption method and the like. The photocatalytic oxidation method is a method for effectively oxidatively decomposing organic pollutants under light irradiation by utilizing the property that a semiconductor oxide material can be excited under light irradiation. Researches show that zinc oxide with different micro-morphologies prepared by different methods has different effects on oxidative degradation of organic dyes.
Biomineralization refers to the process of preparing inorganic mineral substances with specific morphology and functions by the regulation and control of biomacromolecules under mild physiological conditions. Different from industrial production conditions, the biomineralization general conditions are very mild, the energy consumption is low, and the pollution is less. Biomineralization thus provides a new strategy for the preparation of inorganic materials with unique structures and properties.
Disclosure of Invention
The invention aims to provide a method for preparing ZnO nanoparticles by taking recombinant collagen as a biomineralization template, aiming at the defects in the prior art.
In order to achieve the purpose, the invention discloses the following technical scheme:
a method for preparing ZnO nanoparticles by taking recombinant collagen as a biomineralization template comprises the following steps:
(1) preparation of recombinant collagen by biological gene engineering technology
Determining the sequence of the recombinant collagen:
the sequence of the recombinant collagen is as follows:
GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGRPGKRGKQGQKGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGFPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKYGPPGPPGPPGPPGPPGPPGPPGPPGPPGPP, the recombinant collagen has good triple helix structure, and the temperature of thermal change is near 37 ℃;
② synthesizing nucleic acid encoding recombinant collagen:
synthesizing nucleic acid for encoding recombinant collagen in the step I, constructing a plasmid introduced into the nucleic acid, and transforming the plasmid into a escherichia coli BL21-DE3 strain;
preparing and purifying the recombinant collagen:
adding 50 μ L of bacterial liquid into 100mL LB liquid medium containing antibiotics, shaking overnight at constant temperature, culturing, transferring to 1Continuously carrying out amplification culture in an L LB culture medium containing antibiotics in a constant-temperature shaking table at 37 ℃; to be OD600Adjusting the temperature of a shaking table to 25 ℃ when the value reaches the range of 0.8-1, adding 1mM IPTG (isopropyl thiogalactoside) for induction expression, and culturing at constant temperature overnight; centrifuging the bacterial liquid in a low-temperature centrifuge, and collecting thalli; dissolving the bacteria with buffer A, wherein the buffer A is 20mM imidazole, 20mM sodium phosphate and 0.5M sodium chloride, and the pH value is 7.4; carrying out cell disruption by using an ultrasonic cell disruption instrument, and putting a bacterial suspension in an ice bath during ultrasonic treatment to prevent protein denaturation caused by overhigh temperature; and centrifuging the crushed suspension again to separate cell debris from the protein solution, wherein the centrifugation conditions are as follows: 14000rpm,4 ℃ and 30-50 min; collecting the supernatant, i.e. the crude protein solution, for further purification by liquid chromatography; then freeze-drying to obtain white flocculent solid; storing the solid in a refrigerator at-20 deg.C, and calibrating the concentration by weighing method;
(2) preparation of ZnO nano material
Preparing a uniformly mixed solution of recombinant collagen, zinc nitrate hexahydrate and sodium hydroxide:
adding 15-179mg of zinc nitrate hexahydrate and 0-20mg of collagen powder into 1mL of water, uniformly mixing, and slowly stirring for 5-120min to obtain colorless transparent uniform liquid; slowly dropwise adding a sodium hydroxide solution into the solution to obtain white floccules;
② preparing nano-ZnO under mild biomineralization condition:
placing the flocculent mixture in a thermostat at 20-37 deg.C for 1-40 days to obtain white precipitate;
purifying and drying the prepared nano material:
centrifuging the product under 12000rpm, and removing the supernatant to obtain solid; dispersing solid with deionized water, centrifuging for 3-5 times, and drying in a drying oven at constant temperature of 20-37 deg.C.
As a preferred technical scheme of the invention, the collagen powder in the step (2) is added in an amount of 0.1-5mg, and the mass fraction of the collagen is 0.01-0.5 wt%.
As a preferred technical scheme of the invention, the method comprises the following stepsThe solid zinc nitrate hexahydrate in the step (2) is added with the amount of 15-119mg, [ Zn ]2+]From 0.05 to 0.4 mol/L.
As a preferred technical scheme of the invention, the flocculent mixture in the second step of the step (2) is placed in a thermostat with the temperature of 25-37 ℃ for 1-10 days.
The invention has the beneficial effects that:
firstly, the research adopts recombinant collagen as a biological template and zinc nitrate hexahydrate as a raw material to prepare the ZnO nano material with controllable size and shape under mild conditions.
Secondly, the method does not need post-treatment in the whole synthesis process, is simple and convenient, is easy to operate, has a great application prospect, and can provide a foundation for large-scale production of the zinc oxide nano material.
Thirdly, the recombinant collagen is used as a biological template to regulate and control the zinc oxide nano crystal form to obtain the zinc oxide nano particles with specific shapes, and the zinc oxide nano particles have excellent photodegradation capability on organic dyes such as rhodamine B and the like.
Drawings
FIG. 1 is a powder X-ray polycrystalline diffraction (XRD) pattern of the prepared ZnO nanomaterial;
FIG. 2 is an X-ray photoelectron spectroscopy (XPS) chart of the prepared ZnO nanomaterial;
FIG. 3 is a thermogravimetric analysis (TGA) graph of the prepared ZnO nanomaterial;
FIG. 4 is a scanning electron microscope, transmission electron microscope, electron diffraction and energy scattering X-ray analysis chart of the prepared ZnO nano-material;
FIG. 5 is a graph showing the effect of different concentrations of recombinant collagen on the morphology of ZnO nanoparticles;
FIG. 6 is a graph showing the effect of nano ZnO on the catalytic degradation of rhodamine B at different ultraviolet irradiation times;
FIG. 7 shows the difference in the degradation capability of ZnO nanoparticles prepared using recombinant collagen of different concentrations as templates for rhodamine B;
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the drawings in the specification, and it is obvious that the described embodiments are only a part of the present invention, and not all of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1A method for preparing ZnO nanoparticles with recombinant collagen as biomineralization template
A method for preparing ZnO nanoparticles by using collagen as a biomineralization template comprises the following steps:
(1) preparation of recombinant collagen by biological gene engineering technology
Determining the sequence of the recombinant collagen:
the sequence of the recombinant collagen is as follows:
GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGRPGKRGKQGQKGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGFPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKYGPPGPPGPPGPPGPPGPPGPPGPPGPPGPP, the recombinant collagen has good triple helix structure, and the temperature of thermal change is near 37 ℃;
② synthesizing nucleic acid encoding recombinant collagen:
synthesizing nucleic acid for encoding recombinant collagen in the step I, constructing a plasmid introduced into the nucleic acid, and transforming the plasmid into a escherichia coli BL21-DE3 strain;
preparing and purifying the recombinant collagen:
adding 50 mu L of bacterial liquid into 100mL of LB liquid culture medium containing antibiotics, carrying out enrichment culture by a constant temperature shaking table overnight, transferring to 1L of LB culture medium containing antibiotics, and continuing the enrichment culture in the constant temperature shaking table at 37 ℃; to be OD600Adjusting the temperature of a shaking table to 25 ℃ when the value reaches the range of 0.8-1, adding 1mM IPTG (isopropyl thiogalactoside) for induction expression, and culturing at constant temperature overnight; centrifuging the bacterial liquid in a low-temperature centrifuge, and collecting thalli; dissolving the bacteria with buffer A, wherein the buffer A is 20mM imidazole, 20mM sodium phosphate and 0.5M sodium chloride, and the pH value is 7.4;
carrying out cell disruption by using an ultrasonic cell disruption instrument, and putting a bacterial suspension in an ice bath during ultrasonic treatment to prevent protein denaturation caused by overhigh temperature; and centrifuging the crushed suspension again to separate cell debris from the protein solution, wherein the centrifugation conditions are as follows: 14000rpm,4 ℃ and 30-50 min; collecting the supernatant, i.e. the crude protein solution, for further purification by liquid chromatography; then freeze-drying to obtain white flocculent solid; storing the solid in a refrigerator at-20 deg.C, and calibrating the concentration by weighing method;
(2) preparation of ZnO nano material
Preparing a uniformly mixed solution of recombinant collagen, zinc nitrate hexahydrate and sodium hydroxide:
adding 15-179mg of zinc nitrate hexahydrate and 0-20mg of collagen powder into 1mL of water, uniformly mixing, and slowly stirring for 5-120min to obtain colorless transparent uniform liquid; slowly dropwise adding a sodium hydroxide solution into the solution to obtain white floccules;
② preparing nano-ZnO under mild biomineralization condition:
placing the flocculent mixture in a thermostat at 20-37 deg.C for 1-40 days to obtain white precipitate;
purifying and drying the prepared nano material:
centrifuging the product under 12000rpm, and removing the supernatant to obtain solid; dispersing solid with deionized water, centrifuging for 3-5 times, and drying in a drying oven at constant temperature of 20-37 deg.C.
Wherein the addition amount of the collagen powder in the step (2) is 1mg, and the mass fraction of the collagen is 0.1 wt%.
Wherein the addition amount of the zinc nitrate hexahydrate in the step (2) is 29.7mg, [ Zn ]2+]The concentration of (2) is 0.1 mol/L.
Wherein the flocculent mixture in the second step of the step (2) is placed in a thermostat with the temperature of 25 ℃ for 8 days.
Fig. 1 is a powder X-ray polycrystalline diffraction (XRD) pattern of the prepared ZnO nanomaterial, in which diffraction peaks correspond to diffraction data of ZnO. Fig. 2 is an X-ray photoelectron spectroscopy (XPS) graph of the prepared ZnO nanomaterial, in which diffraction peaks correspond to diffraction data of ZnO. FIG. 3 is a thermogravimetric analysis (TGA) chart of the prepared ZnO nanomaterial, and when the concentration of the added collagen is 0.01 wt%, the thermogravimetric loss is 2.2%; when the collagen concentration increased to 0.2 wt%, the thermogravimetric loss increased to 8.3%. These data indicate that we successfully prepared ZnO nanomaterials using collagen as a template. FIG. 4 is the scanning electron microscope and transmission of the prepared ZnO nano material
Analysis graphs of a radio-electron microscope, electron diffraction and energy scattering X rays show that the zinc oxide nanoparticles are uniform in shape and are apple-shaped, and collagen and ZnO are uniformly distributed in the apples; FIG. 5 is the effect of different concentrations of recombinant collagen on the morphology of ZnO nanoparticles. The concentration of the a-d collagen is 0.01 wt%, 0.05 wt%, 0.1 wt% and 0.2 wt% in sequence. The collagen can well regulate and control the morphology of the ZnO nanoparticles.
Example 2 experiment for degrading organic dye by using recombinant collagen as biomineralization template to prepare ZnO nanoparticles
1) Preparing a uniformly mixed solution of zinc oxide nanoparticles and an organic dye;
preparing 4-100mg/L organic dye solution, adding 2-10mg of the zinc oxide nanoparticles prepared in the example 1, and reacting for 10-120min in a dark place to ensure that the solution is balanced in absorption and analysis;
2) the zinc oxide nano particles degrade organic dye under the illumination condition;
irradiating the mixed solution with ultraviolet lamp for 0.5-48 hrs;
3) tracking the degradation level of the organic dye in different time periods by using an ultraviolet spectrophotometer;
analyzing the ultraviolet absorption conditions of the organic dye at different irradiation times by using an UV-1750 ultraviolet spectrophotometer;
as shown in fig. 6 (effect diagram of different time of catalytic degradation of rhodamine B by nano ZnO): after 3 hours of illumination, rhodamine B in the solution is completely decomposed.
As shown in fig. 7 (a graph comparing the degradation effect of nano ZnO with different morphologies on rhodamine B): the zinc oxide with different shapes has obvious difference on the photodegradation capability of rhodamine B, and the catalytic effect of the zinc oxide is obviously related to the shapes of the zinc oxide.
Sequence listing
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Claims (4)

1. A method for preparing ZnO nanoparticles by taking recombinant collagen as a biomineralization template is characterized by comprising the following steps: the method comprises the following steps:
(1) preparation of recombinant collagen by biological gene engineering technology
(2) Preparation of ZnO nano material
Preparing a uniformly mixed solution of recombinant collagen, zinc nitrate hexahydrate and sodium hydroxide:
adding 15-179mg of zinc nitrate hexahydrate and collagen powder into 1ml of water, wherein the addition amount of the collagen powder is more than 0 and less than or equal to 20mg, uniformly mixing, and slowly stirring for 5-120min to obtain colorless, transparent and uniform liquid: slowly dropwise adding a sodium hydroxide solution into the solution to obtain white floccules;
② preparing nano-ZnO under mild biomineralization condition:
placing the flocculent mixture in a thermostat at 20-37 deg.C for 1-40 days to obtain white precipitate;
purifying and drying the prepared nano material:
the product was separated by centrifugation at 12000rpm, and the supernatant was discarded, leaving a solid: dispersing solid with deionized water, centrifuging and purifying for 3-5 times, and drying in a constant temperature drying oven at 20-37 deg.C;
the recombinant collagen in the step (1) is prepared by the following steps:
determining the sequence of the recombinant collagen:
the sequence of the recombinant collagen is as follows:
GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAGAQGRPGKRGKQGQKGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGFPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKYGPPGPPGPPGPPGPPGPPGPPGPPGPPGPP, the recombinant collagen has good triple helix structure, and the temperature of thermal change is near 37 ℃;
② synthesizing nucleic acid encoding recombinant collagen:
synthesizing nucleic acid for coding the recombinant collagen in the first step in the step (1), constructing a plasmid introduced with the nucleic acid, and transforming the plasmid into escherichia coli BL21-DE 3;
preparing and purifying the recombinant collagen:
adding 50 mu L of bacterial liquid into 100mL of LB liquid culture medium containing antibiotics, carrying out enrichment culture by a constant temperature shaking table overnight, transferring to 1L of LB culture medium containing antibiotics, and continuing the enrichment culture in the constant temperature shaking table at 37 ℃; adjusting the temperature of the shaking table to 25 ℃ when the OD 600 value reaches the range of 0.8-1, adding 1mM IPTG (isopropyl thiogalactoside) for induction expression, and culturing at constant temperature overnight; centrifuging the bacterial liquid in a low-temperature centrifuge, and collecting thalli; dissolving the bacteria with buffer A, wherein the buffer A is 20mM imidazole, 20mM sodium phosphate and 0.5M sodium chloride, and the pH value is 7.4; carrying out cell disruption by using an ultrasonic cell disruption instrument, and putting a bacterial suspension in an ice bath during ultrasonic treatment to prevent protein denaturation caused by overhigh temperature; and centrifuging the crushed suspension again to separate cell debris from the protein solution, wherein the centrifugation conditions are as follows: 14000rpm,4 ℃ and 30-50 min; collecting the supernatant, i.e. the crude protein solution, for further purification by liquid chromatography; then freeze-drying to obtain white flocculent solid; the solid is stored in a refrigerator at the temperature of minus 20 ℃, and the concentration is calibrated by a weighing method when the solid is used.
2. The method for preparing ZnO nanoparticles by using the recombinant collagen as the biomineralization template as the claimed claim 1, wherein the method comprises the following steps: the addition amount of the collagen powder in the step (2) is 0.1-5mg, and the mass fraction of the collagen is 0.01-0.5 wt%.
3. The method for preparing ZnO nanoparticles by using the recombinant collagen as the biomineralization template as the claimed claim 1, wherein the method comprises the following steps: the solid zinc nitrate hexahydrate in the step (2) is added with the amount of 15-119mg, [ Zn ]2+]From 0.05 to 0.4 mol/L.
4. The method for preparing ZnO nanoparticles by using the recombinant collagen as the biomineralization template as the claimed claim 1, wherein the method comprises the following steps: placing the flocculent mixture in the second step in the step (2) in a thermostat with the temperature of 25-37 ℃ for 1-10 days.
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