CN108059184A - A kind of method that ZnO nanoparticle is prepared using recombined collagen as biomineralization template - Google Patents
A kind of method that ZnO nanoparticle is prepared using recombined collagen as biomineralization template Download PDFInfo
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- CN108059184A CN108059184A CN201711459404.7A CN201711459404A CN108059184A CN 108059184 A CN108059184 A CN 108059184A CN 201711459404 A CN201711459404 A CN 201711459404A CN 108059184 A CN108059184 A CN 108059184A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G9/00—Compounds of zinc
- C01G9/02—Oxides; Hydroxides
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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Abstract
The invention discloses a kind of methods that ZnO nanoparticle is prepared using recombined collagen as biomineralization template, specifically comprise the following steps：(1) biology gene engineering technology Prepare restructuring collagen is utilized：1. determine the sequence of recombined collagen；2. the nucleic acid of composite coding recombined collagen；3. the preparation and purification of recombined collagen；(2) preparation of ZnO nano material：1. the preparation of recombined collagen and zinc nitrate hexahydrate and sodium hydroxide homogeneous mixture solotion；2., mixed solution is placed in 25 DEG C of insulating boxs；3. purify the nano material that simultaneously prepared by kept dry.For the present invention using recombined collagen as biological template, zinc nitrate hexahydrate is prepared for the ZnO nano material of size and morphology controllable as raw material at room temperature.The present invention is simple and convenient, and easily operated, the ZnO nano material of preparation shows a variety of organic dyestuff excellent degradation capability, has great application prospect in the processing of waste water from dyestuff.
Description
Technical field
The invention belongs to Inorganic biomatetials preparing technical fields, and in particular to a kind of using recombined collagen as biological ore deposit
Change the method that template prepares ZnO nanoparticle.
Background technology
With the fast development of global economy, people start to pay attention to the protection of environment, and water pollution problems is especially paid attention to.I
State's dyeing waste water has the characteristics that discharge capacity is big, hard-degraded substance is more, organic components are complicated, colourity is deep, and it is efficient to be badly in need of exploitation
Processing method.Organic dyestuff is the main component of dyeing waste water, such as representative cationic dyes rhodamine B, it
In solution there is strong fluorescence property, even if concentration is very low to will also result in the reduction of water body light transmittance, seriously destroy ecological environment.
At present, the processing method of waste water from dyestuff includes Coagulation Method, biochemical process, flocculence, electrochemical deposition, photochemical catalysis oxidation, film
Partition method and absorption method etc..Photocatalytic oxidation is the characteristic that can be excited under light illumination using semiconductor oxide materials,
Organic pollution is effectively subjected to oxygenolysis under light illumination.Research shows, diverse microcosmic appearance prepared by distinct methods
Zinc oxide there is different degrees of effect to the oxidative degradation of organic dyestuff.
Biomineralization refers to organism under mild physiological condition, by the regulating and controlling effect of large biological molecule, prepares special
The process of the inorganic mineral of the looks that shape and function.It is different from industrial process conditions, biomineralization general condition as mild as a dove, consumption
Energy is low, pollution is few.Therefore, biomineralization provides new strategy to prepare the inorganic material with unique texture and performance.
The content of the invention
The purpose of the present invention is being directed to deficiency of the prior art, one kind is provided using recombined collagen as biomineralization mould
The method that plate prepares ZnO nanoparticle, using recombined collagen as biological template, zinc nitrate hexahydrate, sodium hydroxide are made
For raw material, the ZnO nano material of size and morphology controllable is prepared in a mild condition.
To achieve the above object, the invention discloses following technical solutions：
A kind of method that ZnO nanoparticle is prepared using recombined collagen as biomineralization template, includes the following steps：
(1) biology gene engineering technology Prepare restructuring collagen is utilized
1. determine the sequence of recombined collagen：
The sequence of recombined collagen is：
GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGP
AGPQGPRGEQGPQGLPGKDGEAGAQGRPGKRGKQGQKGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERG
FPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKD
GQPGKPGKYGPPGPPGPPGPPGPPGPPGPPGPPGPPGPP, the recombined collagen possess good triple helices structure,
Thermal change temperature is close to 37 DEG C；
2. the nucleic acid of composite coding recombined collagen：
The nucleic acid of composite coding step 1. recombined collagen, structure import the plasmid of above-mentioned nucleic acid, and plasmid is converted
E. coli bl21-DE3 bacterial strains；
3. the preparation and purification of recombined collagen：
50 μ L bacterium solutions is taken to be added in LB fluid nutrient mediums of the 100mL containing antibiotic, constant-temperature table carries out Zengjing Granule overnight
Afterwards, it is transferred in LB culture mediums of the 1L containing antibiotic, continues amplification cultivation in 37 DEG C of constant-temperature tables；Treat OD600Value reaches 0.8-
Shaking table temperature is adjusted to 25 DEG C, adds in 1mM IPTG induced expressions by 1 scope, and constant temperature is incubated overnight；By bacterium solution in refrigerated centrifuge
Thalline is collected in middle centrifugation；By thalline A buffer solutions, A buffer solutions are 20mM imidazoles, 20mM sodium phosphates, 0.5M sodium chloride,
PH is 7.4；Clasmatosis is carried out using ultrasonic cell disruption instrument, bacterial suspension is put in ice bath when ultrasonic, to prevent temperature
It is excessively high to cause albuminous degeneration；The suspension crushed is centrifuged again, cell fragment is made to be separated with protein solution, centrifugal condition：
14000rpm, 4 DEG C, 30-50min；It collects supernatant, i.e. crude protein solution, is further purified by liquid chromatogram；By
It is lyophilized, obtain white fluffy solid；This solid is put in -20 DEG C of refrigerators and preserves, and demarcates concentration by weight method during use；
(2) preparation of ZnO nano material
1. the preparation of recombined collagen and zinc nitrate hexahydrate and sodium hydroxide homogeneous mixture solotion：
15-179mg zinc nitrate hexahydrates and 0-20mg collagen powder are added in 1mL water, is uniformly mixed, slowly stirs
After mixing 5-120min, water white transparency and uniform liquid are obtained；Sodium hydroxide solution is slowly added dropwise into the solution again, obtains white
The floccule of color；
2. nanoscale ZnO is prepared under the conditions of mild biomineralization：
Gained cloud mixture is placed in 20-37 DEG C of insulating box, places 1-40 days, obtains white precipitate；
3. purify the nano material that simultaneously prepared by kept dry：
By product by centrifuging, centrifugal condition 12000rpm, abandoning supernatant leaves solid；And use deionization
Water dispersible solid, then centrifugal purification 3-5 times, it is dry in 20-37 DEG C of thermostatic drying chamber.
As a preferred technical solution of the present invention, the collagen powder addition described in step (2) is 0.1-
5mg, collagen quality fraction are 0.01-0.5wt%.
As a preferred technical solution of the present invention, the zinc nitrate hexahydrate solids loading content described in step (2) is
15-119mg, [Zn2+] concentration distribution from 0.05 to 0.4mol/L.
As a preferred technical solution of the present invention, in step (2) the 2. the cloud mixture described in step in 25-37
DEG C insulating box in, place 1-10 days.
The beneficial effects of the present invention are：
1. this research is using recombined collagen as biological template, zinc nitrate hexahydrate is as raw material, in temperate condition
The lower ZnO nano material for preparing size and morphology controllable.
2. the present invention need not be post-processed in entire building-up process, simple and convenient, easily operated, there is very big answer
With prospect, basis can be provided for large-scale production nano zinc oxide material.
3. regulating and controlling zinc-oxide nano crystal form as biological template by recombined collagen, the zinc oxide nano of specific morphology is obtained
Rice corpuscles shows the organic dyestuff such as rhodamine B remarkable light degradation ability.
Description of the drawings
Fig. 1 is X-ray powder polycrystalline diffraction (XRD) figure of the ZnO nano material prepared；
Fig. 2 is x-ray photoelectron spectroscopy (XPS) figure of the ZnO nano material prepared；
Fig. 3 is thermogravimetric analysis (TGA) figure of the ZnO nano material prepared；
Fig. 4 is the scanning electron microscope of the ZnO nano material prepared, transmission electron microscope, electronic diffraction and Energy Dispersive X point
Analysis figure；
Fig. 5 is influence of the recombined collagen of various concentration to ZnO nano granule-morphology；
Fig. 6 be nano-ZnO to the catalytic degradation of rhodamine B different time of ultraviolet irradiation design sketch；
Fig. 7 is the degradation energy of the ZnO nano particle that is prepared by template of the recombined collagen of various concentration to rhodamine B
The difference of power；
Specific embodiment
The technical solution in the embodiment of the present invention is clearly and completely described below in conjunction with Figure of description, is shown
So, described embodiment is only the part of the present invention rather than the whole of invention.Based on the embodiments of the present invention,
Those of ordinary skill in the art's all other embodiments obtained without making creative work, belong to this hair
The scope of bright protection.
A kind of method that ZnO nanoparticle is prepared using recombined collagen as biomineralization template of embodiment 1
A kind of method that ZnO nanoparticle is prepared using collagen as biomineralization template, includes the following steps：
(1) biology gene engineering technology Prepare restructuring collagen is utilized
1. determine the sequence of recombined collagen：
The sequence of recombined collagen is：
GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKGETGP
AGPQGPRGEQGPQGLPGKDGEAGAQGRPGKRGKQGQKGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERG
FPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKD
GQPGKPGKYGPPGPPGPPGPPGPPGPPGPPGPPGPPGPP, the recombined collagen possess good triple helices structure,
Thermal change temperature is close to 37 DEG C；
2. the nucleic acid of composite coding recombined collagen：
The nucleic acid of composite coding step 1. recombined collagen, structure import the plasmid of above-mentioned nucleic acid, and plasmid is converted
E. coli bl21-DE3 bacterial strains；
3. the preparation and purification of recombined collagen：
50 μ L bacterium solutions is taken to be added in LB fluid nutrient mediums of the 100mL containing antibiotic, constant-temperature table carries out Zengjing Granule overnight
Afterwards, it is transferred in LB culture mediums of the 1L containing antibiotic, continues amplification cultivation in 37 DEG C of constant-temperature tables；Treat OD600Value reaches 0.8-
Shaking table temperature is adjusted to 25 DEG C, adds in 1mM IPTG induced expressions by 1 scope, and constant temperature is incubated overnight；By bacterium solution in refrigerated centrifuge
Thalline is collected in middle centrifugation；By thalline A buffer solutions, A buffer solutions are 20mM imidazoles, 20mM sodium phosphates, 0.5M sodium chloride,
PH is 7.4；
Clasmatosis is carried out using ultrasonic cell disruption instrument, bacterial suspension is put in ice bath when ultrasonic, to prevent temperature
It is excessively high to cause albuminous degeneration；The suspension crushed is centrifuged again, cell fragment is made to be separated with protein solution, centrifugal condition：
14000rpm, 4 DEG C, 30-50min；It collects supernatant, i.e. crude protein solution, is further purified by liquid chromatogram；By
It is lyophilized, obtain white fluffy solid；This solid is put in -20 DEG C of refrigerators and preserves, and demarcates concentration by weight method during use；
(2) preparation of ZnO nano material
1. the preparation of recombined collagen and zinc nitrate hexahydrate and sodium hydroxide homogeneous mixture solotion：
15-179mg zinc nitrate hexahydrates and 0-20mg collagen powder are added in 1mL water, is uniformly mixed, slowly stirs
After mixing 5-120min, water white transparency and uniform liquid are obtained；Sodium hydroxide solution is slowly added dropwise into the solution again, obtains white
The floccule of color；
2. nanoscale ZnO is prepared under the conditions of mild biomineralization：
Gained cloud mixture is placed in 20-37 DEG C of insulating box, places 1-40 days, obtains white precipitate；
3. purify the nano material that simultaneously prepared by kept dry：
By product by centrifuging, centrifugal condition 12000rpm, abandoning supernatant leaves solid；And use deionization
Water dispersible solid, then centrifugal purification 3-5 times, it is dry in 20-37 DEG C of thermostatic drying chamber.
The addition of collagen powder wherein described in step (2) is 1mg, and the mass fraction of collagen is
0.1wt%.
The addition of zinc nitrate hexahydrate wherein described in step (2) is 29.7mg, [Zn2+] concentration be 0.1mol/
L。
Wherein in step (2) the 2. cloud mixture described in step in 25 DEG C of insulating boxs, place 8 days.
Fig. 1 is X-ray powder polycrystalline diffraction (XRD) figure of the ZnO nano material prepared, diffraction maximum and ZnO in the collection of illustrative plates
Diffraction data correspond to.Fig. 2 is x-ray photoelectron spectroscopy (XPS) figure of the ZnO nano material prepared, diffraction maximum in the collection of illustrative plates
It is corresponding with the diffraction data of ZnO.Fig. 3 is thermogravimetric analysis (TGA) figure of the ZnO nano material prepared, when addition collagen
Concentration is 0.01wt%, thermogravimetric weight loss 2.2%；It is 0.2wt% when collagen concentration increases, thermogravimetric weight loss increase is
8.3%.These statistics indicate that, we successfully prepare ZnO nano material using collagen as template.Fig. 4 is that the ZnO prepared receives
Rice material scanning electron microscope, thoroughly
The analysis chart of radio mirror, electronic diffraction and Energy Dispersive X, it will be seen that zinc oxide nano from figure
Rice corpuscles pattern is homogeneous, and in apple shape, collagen and ZnO are uniformly distributed in apple；Fig. 5 is the recombinant collagen of various concentration
Influence of the albumen to ZnO nano granule-morphology.The concentration of a-d collagens is followed successively by 0.01wt%, 0.05wt%, 0.1wt%
And 0.2wt%.Show that collagen can be very good the pattern of regulation and control ZnO nanoparticle.
Embodiment 2 is a kind of to degrade by biomineralization template preparation ZnO nanoparticle of recombined collagen under photocatalysis
Organic dyestuff is tested
1) preparation of zinc oxide nano-particle and organic dyestuff homogeneous mixture solotion；
The organic dye solution of 4-100mg/L is prepared, zinc oxide nano-particle 2-10mg prepared by embodiment 1 is added in, keeps away
Light reaction 10-120min makes solution reach absorption and parsing balance；
2) zinc oxide nano-particle degradating organic dye under illumination condition；
With ultra violet lamp mixed solution 0.5-48hrs；
3) Degradation Level of ultraviolet specrophotometer tracking different time sections organic dyestuff；
The UV absorption situation of different irradiation time organic dyestuff is analyzed with UV-1750 ultraviolet specrophotometers；
As shown in Fig. 6 (nano-ZnO is to the design sketch of the catalytic degradation different time of rhodamine B)：After when illumination 3 is small,
Rhodamine B in solution has been decomposed completely.
As shown in Fig. 7 (nano-ZnO of different-shape compares figure to the degradation effect of rhodamine B)：The oxidation of different-shape
For zinc to the light degradation ability of rhodamine B there are notable difference, the catalytic effect of zinc oxide and its pattern are significantly correlated.
Sequence table
<110>Lanzhou University
<120>A kind of method that ZnO nanoparticle is prepared using recombined collagen as biomineralization template
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 270
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Gly Ser Pro Gly Leu Pro Gly Pro Arg Gly Glu Gln Gly Pro Thr Gly
1 5 10 15
Pro Thr Gly Pro Ala Gly Pro Arg Gly Leu Gln Gly Leu Gln Gly Leu
20 25 30
Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Pro Ala Gly Pro Arg
35 40 45
Gly Leu Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Leu Ala Gly
50 55 60
Lys Ala Gly Glu Ala Gly Ala Lys Gly Glu Thr Gly Pro Ala Gly Pro
65 70 75 80
Gln Gly Pro Arg Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp
85 90 95
Gly Glu Ala Gly Ala Gln Gly Arg Pro Gly Lys Arg Gly Lys Gln Gly
100 105 110
Gln Lys Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Gly Asp
115 120 125
Arg Gly Glu Thr Gly Pro Val Gly Pro Arg Gly Glu Arg Gly Glu Ala
130 135 140
Gly Pro Ala Gly Lys Asp Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly
145 150 155 160
Val Glu Gly Gln Asn Gly Gln Asp Gly Leu Pro Gly Lys Asp Gly Lys
165 170 175
Asp Gly Gln Asn Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp
180 185 190
Gly Gln Asn Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly
195 200 205
Gln Asp Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly Leu
210 215 220
Pro Gly Lys Asp Gly Lys Asp Gly Gln Pro Gly Lys Pro Gly Lys Tyr
225 230 235 240
Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly
245 250 255
Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro
260 265 270
Claims (5)
- A kind of 1. method that ZnO nanoparticle is prepared using recombined collagen as biomineralization template, it is characterised in that：This method Include the following steps：(1) biology gene engineering technology Prepare restructuring collagen is utilized(2) preparation of ZnO nano material1. the preparation of recombined collagen and zinc nitrate hexahydrate and sodium hydroxide homogeneous mixture solotion：15--179mg zinc nitrate hexahydrates and 0-20mg collagen powder are added in 1ml water, is uniformly mixed, is slowly stirred After 5-120min, water white transparency and uniform liquid are obtained；Sodium hydroxide solution is slowly added dropwise into the solution again, obtains white Floccule；2. nanoscale ZnO is prepared under the conditions of mild biomineralization：Gained cloud mixture is placed in 20-37 DEG C of insulating box, places 1-40 days, obtains white precipitate；3. purify the nano material that simultaneously prepared by kept dry：By product by centrifuging, centrifugal condition 12000rpm, abandoning supernatant leaves solid；And divided with deionized water Solid, then centrifugal purification 3-5 times are dissipated, is drying to obtain in 20-37 DEG C of thermostatic drying chamber.
- 2. a kind of side that ZnO nanoparticle is prepared using recombined collagen as biomineralization template according to claim 1 Method, it is characterised in that:Recombined collagen is prepared by following steps in the step (1)：1. determine the sequence of recombined collagen：The sequence of recombined collagen is：GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKG ETGPAGPQGPRGEQGPQGLPGKDGEAGAQGRPGKRGKQGQKGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKD GERGFPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGK DGKDGQPGKPGKYGPPGPPGPPGPPGPPGPPGPPGPPGPPGPP, the recombined collagen possess good triple helices Structure, thermal change temperature is close to 37 DEG C；2. the nucleic acid of composite coding recombined collagen：The nucleic acid of composite coding step 1. recombined collagen, structure import the plasmid of above-mentioned nucleic acid, and plasmid is converted large intestine Bacillus BL21-DE3 bacterial strains；3. the preparation and purification of recombined collagen：50 μ L bacterium solutions is taken to be added in LB fluid nutrient mediums of the 100mL containing antibiotic, after constant-temperature table carries out Zengjing Granule overnight, are turned It moves on in LB culture mediums of the 1L containing antibiotic, continues amplification cultivation in 37 DEG C of constant-temperature tables；Treat OD600Value reaches 0.8-1 models It encloses, shaking table temperature is adjusted to 25 DEG C, adds in 1mM IPTG induced expressions, constant temperature is incubated overnight；By bacterium solution in refrigerated centrifuge Thalline is collected in centrifugation；By thalline A buffer solutions, A buffer solutions are 20mM imidazoles, 20mM sodium phosphates, 0.5M sodium chloride, pH For 7.4；Clasmatosis is carried out using ultrasonic cell disruption instrument, bacterial suspension is put in ice bath when ultrasonic, to prevent temperature mistake Height causes albuminous degeneration；The suspension crushed is centrifuged again, cell fragment is made to be separated with protein solution, centrifugal condition： 14000rpm, 4 DEG C, 30-50min；It collects supernatant, i.e. crude protein solution, is further purified by liquid chromatogram；By It is lyophilized, obtain white fluffy solid；This solid is put in -20 DEG C of refrigerators and preserves, and demarcates concentration by weight method during use.
- 3. a kind of side that ZnO nanoparticle is prepared using recombined collagen as biomineralization template according to claim 1 Method, it is characterised in that：Collagen powder addition described in step (2) is 0.1-5mg, and collagen quality fraction is 0.01-0.5wt%.
- 4. a kind of side that ZnO nanoparticle is prepared using recombined collagen as biomineralization template according to claim 1 Method, it is characterised in that：Zinc nitrate hexahydrate solids loading content described in step (2) is 15-119mg, [Zn2+] concentration distribution From 0.05 to 0.4mol/L.
- 5. a kind of side that ZnO nanoparticle is prepared using recombined collagen as biomineralization template according to claim 1 Method, it is characterised in that：In step (2) the 2. the cloud mixture described in step in 25-37 DEG C of insulating box, place 1-10 My god.
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| CN115521373A (en) * | 2022-06-06 | 2022-12-27 | 胶原蛋白(武汉)生物科技有限公司 | Triple-helix recombinant humanized type I collagen, preparation method and application thereof |
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| CN115521373B (en) * | 2022-06-06 | 2024-04-19 | 胶原蛋白(武汉)生物科技有限公司 | Triple helix recombinant humanized type I collagen, preparation method and application thereof |
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