CN110283810A - Enzyme/calcium salt/graphene oxide functional form nanocomposite and preparation method thereof - Google Patents
Enzyme/calcium salt/graphene oxide functional form nanocomposite and preparation method thereof Download PDFInfo
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 93
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 93
- 239000002114 nanocomposite Substances 0.000 title claims abstract description 54
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 45
- 159000000007 calcium salts Chemical class 0.000 title claims abstract description 43
- 229910021389 graphene Inorganic materials 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 239000002131 composite material Substances 0.000 claims abstract description 14
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 8
- 239000013078 crystal Substances 0.000 claims abstract description 7
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 claims abstract description 7
- 238000001179 sorption measurement Methods 0.000 claims abstract description 4
- 108010029541 Laccase Proteins 0.000 claims description 21
- 102000003846 Carbonic anhydrases Human genes 0.000 claims description 15
- 108090000209 Carbonic anhydrases Proteins 0.000 claims description 15
- 239000000463 material Substances 0.000 claims description 11
- 102000003425 Tyrosinase Human genes 0.000 claims description 8
- 108060008724 Tyrosinase Proteins 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- VSGNNIFQASZAOI-UHFFFAOYSA-L calcium acetate Chemical compound [Ca+2].CC([O-])=O.CC([O-])=O VSGNNIFQASZAOI-UHFFFAOYSA-L 0.000 claims description 4
- 239000001639 calcium acetate Substances 0.000 claims description 4
- 229960005147 calcium acetate Drugs 0.000 claims description 4
- 235000011092 calcium acetate Nutrition 0.000 claims description 4
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 239000013049 sediment Substances 0.000 claims description 4
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 239000002023 wood Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 31
- 238000000034 method Methods 0.000 abstract description 17
- 239000002086 nanomaterial Substances 0.000 abstract description 8
- 238000002474 experimental method Methods 0.000 abstract description 5
- 108010093096 Immobilized Enzymes Proteins 0.000 abstract description 4
- 238000010521 absorption reaction Methods 0.000 abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 4
- 150000001875 compounds Chemical class 0.000 abstract description 4
- 230000004044 response Effects 0.000 abstract description 4
- 230000001066 destructive effect Effects 0.000 abstract description 3
- 230000001681 protective effect Effects 0.000 abstract description 3
- 230000000845 anti-microbial effect Effects 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 12
- 238000011160 research Methods 0.000 description 11
- 230000008859 change Effects 0.000 description 8
- 230000008901 benefit Effects 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 239000001506 calcium phosphate Substances 0.000 description 3
- 229910000389 calcium phosphate Inorganic materials 0.000 description 3
- 235000011010 calcium phosphates Nutrition 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 229910002804 graphite Inorganic materials 0.000 description 3
- 239000010439 graphite Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 241000446313 Lamella Species 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- -1 carbonic acid Acid anhydride Chemical class 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 239000011147 inorganic material Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002048 multi walled nanotube Substances 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/04—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium
- B01J20/048—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium containing phosphorus, e.g. phosphates, apatites, hydroxyapatites
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/20—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising free carbon; comprising carbon obtained by carbonising processes
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0059—Catechol oxidase (1.10.3.1), i.e. tyrosinase
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03001—Catechol oxidase (1.10.3.1), i.e. tyrosinase
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- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/01001—Carbonate dehydratase (4.2.1.1), i.e. carbonic anhydrase
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Abstract
The present invention provides a kind of enzyme/calcium salt/graphene oxide functional form nanocomposites and preparation method thereof, and wherein nanocomposite includes the component of following mass percentage: biological enzyme 20-40%, synthos crystal 32-70% and graphene oxide 5-30%.The present invention uses dynamic response method, without the fierce experimental method such as high temperature, and meet environmentally protective route without reference to any organic reagent, obtained enzyme/calcium salt/graphene oxide functional form nanocomposite compound with regular structure, in the case where non-destructive enzyme conformation and GO nanostructure, the raising of immobilized enzyme enzyme activity and the raising of thermal stability and storage-stable are realized, while also there is certain antibacterial and absorption property, can be widely applied to antimicrobial composite material and adsorption composite material.
Description
Technical field
The present invention relates to chemical material technical fields, in particular to a kind of enzyme/calcium salt/graphene oxide functional form
Nanocomposite and preparation method thereof.
Background technique
Enzyme is a kind of biocatalyst, has many advantages, such as that selectivity is strong, reaction condition is mild, simple process.Traditional enzyme is solid
Certain enzyme performance can be improved in the method for determining, and with the rapid development of modern society, various cleverly preparation processes are by variety classes
Material fixed applied to enzyme, the performance after enzyme is fixed is also higher and higher.Discovery multi-walled carbon nanotube is used as admittedly in research before
The carrier of laccase is determined for accelerating electron transmission, keeps laccase activity 85.3%；And utilize magnetic ferroferric oxide nanometer particle
Anchoring fiber element enzyme makes its enzyme activity conservation rate reach 99.1%, although being able to maintain by enzyme fixing means constantly improving
The higher activity of enzyme, but seldom will appear the phenomenon that fixed enzyme activity is higher than free enzyme activity.
With the rapid development of material science and constantly progressing greatly for preparation process, Material Field is developed more and more novel
Nano material, they often have certain special microstructure, a superior performance etc., these features and advantages are by novel nano
Material has pushed the study frontier of materials science field to.Wherein in graphite family graphene oxide (GO) due to unique nano junction
Structure and excellent performance can form unique three-D space structure with other materials, make its various performance enhancement, and associated materials are answered
With having a high potential, thus become the hot spot of every field research.
With the protrusion of inorganic material advantage, multiple material simultaneously immobilized enzyme relevant report whether with synergy compared with
It is few, it is worth further investigation.In conclusion for enzyme-field of nanometer material technology, also in the exploratory stage, and by enzyme-calcium salt-oxygen
Graphite alkene combines, and the research for forming composite nano materials has not been reported.
Therefore, prepare a kind of enzyme/calcium salt/graphene oxide functional form nanocomposite, in non-destructive enzyme conformation and
In the case where GO nanostructure, improves enzyme/calcium salt/graphene oxide functional form nanocomposite enzyme activity and improve its thermostabilization
Property and storage stability.
Summary of the invention
The present invention is based at least one above-mentioned technical problem, proposes a kind of enzyme/calcium salt/graphene oxide functional form
Nanocomposite and preparation method thereof generates a kind of enzyme/calcium salt/oxidation under the conditions of by enzyme, GO existing for the divalent calcium ions
Graphene functional form nanocomposite, wherein GO provides carrier, while the conjugate as enzyme for enzyme, can be by the Optimality of GO
Matter is widely applied.The composite material nanometer compound with regular structure, realizes the raising of enzyme activity, thermal stability and storage-stable, simultaneously
Also there is certain antibacterial and absorption property.
In view of this, the invention proposes a kind of enzyme/calcium salt/graphene oxide functional form nanocomposites, including with
The component of lower mass percentage: biological enzyme 20-40%, synthos crystal 32-70% and graphene oxide 5-30%.
In the technical scheme, while enzyme/calcium salt/graphene oxide functional form nanocomposite can improve enzyme activity,
The thermal stability and storage-stable of nanocomposite are improved, which is expected in catalysis, materialogy, electrification
, biology etc. are multi-field to play a significant role.
According to the second aspect of the invention, enzyme/calcium salt/graphene oxide functional form nanocomposite is proposed in antibacterial
It is applied in composite material and adsorption composite material.
In the technical scheme, GO is the lamella of C-C composition, and electronics can be received and be transmitted well, thus
Promote enzyme activity, while GO has suction-operated and antibacterial action, thallus is adsorbed in nanocomposite by it, so that nanometer
Composite material is easier to contact thallus, realizes that " cutting " to thallus acts on, so that the antibacterial effect of composite material is more preferable；
Enzyme/calcium salt/graphene oxide functional form nanocomposite simultaneously, destructive enzyme conformation and GO nanostructure, do not improve it
Thermal stability and storage stability.
According to the third aspect of the present invention, a kind of enzyme/calcium salt/graphene oxide functional form nanocomposite is proposed
Preparation method, comprising the following steps:
(1) it disperses graphene oxide in the phosphate buffer of 0.05-0.2mol/L, is uniformly mixed；
(2) biological enzyme is added in the mixed liquor of step (1) formation, is mixed；
(3) calcium salt soln is added to in the mixed liquor of step (2) formation 25-30 DEG C, 100-150 rpm, shaking table reacts 24-48
h；
(4) sediment in separating step (3) solution, the sediment washed and is freeze-dried 4-6 h at -20 DEG C.
In the technical scheme, enzyme/calcium salt/graphene oxide functional form nanocomposite is that sulfuric acid is added in PBS
The solution such as calcium, calcium chloride or calcium acetate generate synthos crystal, and then react and obtain with enzyme, GO.And in the present invention
The pH of PBS changes according to enzyme not of the same race, i.e., first GO is dispersed in PBS, and enzyme is added after being uniformly dispersed and mixes, adds
Calcium salt soln is put into shaking table and reacts, and stands a few hours later, and centrifugation will be freeze-dried after precipitation and separation.Therefore experimentation
It is mild simple, it using dynamic response method, can carry out at room temperature, without the fierce experimental method such as high temperature, and without reference to any
Organic reagent, equipment is simple, meets environmentally protective route.
In the above-mentioned technical solutions, it is preferable that biological enzyme: calcium salt: the mass percent of graphene oxide is 8-10:10-
25:1-5.
In the above-mentioned technical solutions, it is preferable that biological enzyme includes laccase, carbonic anhydrase or tyrosinase.
In the above-mentioned technical solutions, it is preferable that calcium salt includes the soluble divalent calcium such as calcium sulfate, calcium acetate or calcium chloride
Salt.
In the above-mentioned technical solutions, it is preferable that the pH value of phosphate buffer is 5.6-8.5.
In the above-mentioned technical solutions, it is preferable that step (3) shaking table reaction condition is at a temperature of 25 DEG C, and 140 rpm turn
Speed reacts 48 h.
By above technical scheme, the invention proposes a kind of enzyme/calcium salt/graphene oxide functional form nanocomposites
And preparation method thereof, it has the following technical effect that
(1) GO is not only that enzyme provides carrier, and GO can be used as the conjugate of enzyme in the present invention, and the composite material of formation is further
The advantageous property of GO is widely applied；
(2) enzyme/calcium salt/graphene oxide functional form nanocomposite compound with regular structure, realize the raising of immobilized enzyme enzyme activity with
And thermal stability and storage-stable improve, while also having certain antibacterial and absorption property；
(3) experimentation of the present invention is mildly simple, using dynamic response method, can carry out at room temperature, fierce real without high temperature etc.
Proved recipe method, and without reference to any organic reagent；Equipment is simple simultaneously, meets environmentally protective route.
Detailed description of the invention
Fig. 1 shows enzyme/calcium salt in embodiment 1/graphene oxide functional form nanocomposite scanning electron figure.
Fig. 2 shows tyrosinases in enzyme/calcium salt in root embodiment 1/graphene oxide functional form nanocomposite
Thermal stability change curve.
Fig. 3 shows the laccase thermal stability change curve of embodiment according to the present invention 2.
Fig. 4 shows the laccase storage-stable change curve of embodiment according to the present invention 2.
Fig. 5 shows the carbonic anhydrase thermal stability change curve of embodiment according to the present invention 3.
Fig. 6 shows the carbonic anhydrase storage-stable change curve of embodiment according to the present invention 3.
Specific embodiment
To better understand the objects, features and advantages of the present invention, with reference to the accompanying drawing and specific real
Applying mode, the present invention is further described in detail.It should be noted that in the absence of conflict, implementation of the invention
Feature in example and embodiment can be combined with each other.
In the following description, numerous specific details are set forth in order to facilitate a full understanding of the present invention, still, the present invention may be used also
To be implemented using other than the one described here other modes, therefore, protection scope of the present invention is not by described below
Specific embodiment limitation.
Embodiment 1
A kind of enzyme/calcium salt/graphene oxide functional form nanocomposite, the component including following mass percentage: junket ammonia
Sour enzyme 27.17%, calcium phosphate crystal 67.39%, GO 5.43%.
(1) preparation of material
The PBS(0.06 mol/L for being 8.0 in pH by 4 mg GO ultrasonic dissolutions) in, sequentially add 20 mg tyrosinases, 0.667
The calcium chloride solution of 240 mM of mL shakes up, and reacts 48 h at room temperature.Precipitation and separation, washing are placed in vacuum refrigeration later
4 h are freeze-dried in drying instrument, rear that the nanocomposite dried is observed using scanning electron microscope, nanostructure is such as
Fig. 1.The composite nanostructure is the even cluster being made of 80-120 nm club shaped structure as shown in Figure 1.
(2) laccase activity measuring method and outcome research in nanocomposite
Taking pH is 8.2 3.5 mL of citrate-phosphate disodium hydrogen buffer, and the standard substrate solution of 0.5 mL, 2 mM is added, then
The starting reaction of 1 mL testing sample solution is added, reaction solution is filtered with filter after reaction specific time and terminates reaction, uses purple
Outer spectrophotometer measures the variation of absorbance before and after reaction at 475nm, and calculates enzyme activity.Finally obtain tyrosinase enzyme activity
72% is improved than free enzyme activity.
(3) tyrosinase thermal stability determination method and outcome research in nanocomposite
Nanocomposite and free enzyme solutions (enzyme molecule containing same quality) 10 mL are in test tube in difference Example 1
In, be put into 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, keep the temperature 1 h respectively in 80 DEG C of environment.Solution after measurement heat treatment
Enzyme activity, record data, calculate relative to 30 DEG C when enzyme activity conservation rate and make Fig. 2.By attached drawing 2 it is found that in the present invention
Enzyme/calcium salt/graphene oxide functional form nanocomposite tyrosinase storage-stable is far superior to free tyrosinase.
Embodiment 2
A kind of enzyme/calcium salt/graphene oxide functional form nanocomposite, the component including following mass percentage: laccase
40%, calcium phosphate crystal 32%, GO 28%.
(1) preparation method of nanocomposite
The PBS(0.2 mol/L for being 5.7 in pH by 2 mg GO ultrasonic dissolutions) in, sequentially add 15 mg laccases, 0.667 mL
The calcium chloride solution of 120 mM shakes up, and 48 h are reacted in 25 DEG C, 140rpm shaking table, later by precipitation and separation, washing be placed in-
6 h are freeze-dried in 30 DEG C of vacuum freeze drier.
(2) laccase activity measuring method and outcome research in nanocomposite
Taking pH is 4.6 3.5 mL of citrate-phosphate disodium hydrogen buffer, and the ABTS solution of 0.5 mL, 2 mM is added, adds
The starting of 1 mL testing sample solution is reacted, and is filtered reaction solution with filter after reaction specific time and is terminated reaction, uses ultraviolet point
The variation of light photometric determination reaction front and back absorbance, and calculate enzyme activity.Laccase activity is finally obtained to improve than free enzyme activity
27%。
(3) laccase thermal stability determination method and outcome research in nanocomposite
Nanocomposite and free laccase solution (enzyme molecule containing same quality) 10 mL are in examination in difference Example 2
Guan Zhong, be put into 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, keep the temperature 1 h respectively in 80 DEG C of environment.It is molten after measurement heat treatment
The enzyme activity of liquid, record data, calculate relative to 30 DEG C when enzyme activity conservation rate and map.Laccase thermal stability in the present embodiment
Variation is such as attached drawing 3, by attached drawing 3 it is found that enzyme/calcium salt/graphene oxide functional form nanocomposite laccase in the present invention
Thermal stability is far superior to free laccase.
(4) laccase storage-stable measuring method and outcome research in nanocomposite
Nanocomposite in embodiment 2 and free laccase solution are placed on experimental bench, it is without any processing, enzyme is measured daily
Variation living.The numerical value of measurement analyzes change of numerical value on the basis of first day enzyme activity numerical value compared with the numerical value of measurement in first day
Change and calculate opposite enzyme activity, is calculated with percentage, calculated percentage is mapped, the variation of storage-stable can be clearly seen.
Laccase storage-stable variation such as attached drawing 4 in the present embodiment.By attached drawing 4 it is found that enzyme/calcium salt/graphene oxide in the present invention
The laccase storage-stable of functional form nanocomposite is far superior to free laccase.
Embodiment 3
A kind of enzyme/calcium salt/graphene oxide functional form nanocomposite, the component including following mass percentage: carbonic acid
Acid anhydride enzyme 40%, calcium phosphate crystal 32%, GO 28%.
(1) preparation method of nanocomposite
The PBS(0.2 mol/L for being 8.5 in pH by 5 mg GO ultrasonic dissolutions) in, sequentially add 15 mg carbonic anhydrases, 0.667
The calcium acetate solution of 120 mM of mL shakes up, and 48 h are reacted in 25 DEG C, 140rpm shaking table, later by precipitation and separation, washing juxtaposition
6 h are freeze-dried in -30 DEG C of vacuum freeze drier.
(2) carbonic anhydrase enzyme activity determination method and outcome research in nanocomposite
By PBS(pH=7 of the enzyme solution to be measured of 0.3 mL, 2.4 mL), the p-NPA(0.02 M of 0.3 mL configures in nitrile) three
Person mixes, and reaction after a certain period of time, measures the difference of absorbance, calculates enzyme activity.Finally obtain carbonic anhydrase enzyme activity ratio
Free enzyme activity improves 137%.
(3) carbonic anhydrase thermal stability determination method and outcome research experimental method such as embodiment 2 in nanocomposite.
Carbonic anhydrase thermal stability variation such as attached drawing 5 in the present embodiment.By attached drawing 5 it is found that enzyme/calcium salt/graphite oxide in the present invention
The carbonic anhydrase thermal stability of olefinic functionality type nanocomposite is far superior to free carbonic anhydrase.
(4) carbonic anhydrase storage-stable measuring method and outcome research experimental method such as embodiment in nanocomposite
2.Carbonic anhydrase storage-stable variation such as attached drawing 6 in the present embodiment.By attached drawing 6 it is found that enzyme/calcium salt/oxidation in the present invention
The carbonic anhydrase storage-stable of graphene functional form nanocomposite is far superior to free carbonic anhydrase.
In conclusion the invention proposes a kind of enzyme/calcium salt/graphene oxide functional form nanocomposite and its preparations
Method, the present invention in GO be only enzyme provide carrier, at the same time as the conjugate of enzyme, the nanocomposite of formation can be by GO
Advantageous property be widely applied；Furthermore experimentation of the present invention is mildly simple, using dynamic response method, can carry out at room temperature,
Without the fierce experimental method such as high temperature, and without reference to any organic reagent；Finally, present device is simple, meet green ring
Route is protected, obtained enzyme/calcium salt/graphene oxide functional form nanocomposite compound with regular structure realizes immobilized enzyme enzyme activity
Raising and thermal stability and storage-stable improve, while also having certain antibacterial and absorption property, can be widely applied to
Antimicrobial composite material and adsorption composite material.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (7)
1. a kind of enzyme/calcium salt/graphene oxide functional form nanocomposite, which is characterized in that contain including following quality percentage
The component of amount: biological enzyme 20-40%, synthos crystal 32-70% and graphene oxide 5-30%.
2. a kind of enzyme/calcium salt/graphene oxide functional form nanocomposite according to claim 1 is in antibacterial composite wood
It is applied in material and adsorption composite material.
3. a kind of preparation method of enzyme/calcium salt/graphene oxide functional form nanocomposite according to claim 1,
Characterized by comprising the following steps:
(1) it disperses graphene oxide in the phosphate buffer of 0.05-0.2mol/L, is uniformly mixed；
(2) biological enzyme is added in the mixed liquor of step (1) formation, is mixed；
(3) calcium salt soln is added to in the mixed liquor of step (2) formation 25-30 DEG C, 100-150 rpm, shaking table reacts 24-48
h；
(4) sediment in separating step (3) solution, the sediment washed and is freeze-dried 4-6 h at -20 DEG C.
4. a kind of preparation method of enzyme/calcium salt/graphene oxide functional form nanocomposite according to claim 3,
It is characterized in that, the biological enzyme: the calcium salt: the mass percent of the graphene oxide is 8-10:10-25:1-5.
5. a kind of preparation method of enzyme/calcium salt/graphene oxide functional form nanocomposite according to claim 3,
It is characterized in that, the biological enzyme includes laccase, carbonic anhydrase or tyrosinase.
6. a kind of preparation method of enzyme/calcium salt/graphene oxide functional form nanocomposite according to claim 3,
It is characterized in that, the calcium salt includes calcium sulfate, calcium acetate or calcium chloride.
7. a kind of preparation method of enzyme/calcium salt/graphene oxide functional form nanocomposite according to claim 3,
It is characterized in that, the pH value of the phosphate buffer is 5.6-8.5.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104164414A (en) * | 2014-08-21 | 2014-11-26 | 郑州大学 | Enzyme/graphene oxide functional hybrid nanocomposite and preparation method thereof |
| CN108727752A (en) * | 2018-06-11 | 2018-11-02 | 中原工学院 | A method of preparing high strength poly vinyl alcohol composite hydrogel using in-situ fibrillation |
-
2019
- 2019-04-02 CN CN201910262421.4A patent/CN110283810A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104164414A (en) * | 2014-08-21 | 2014-11-26 | 郑州大学 | Enzyme/graphene oxide functional hybrid nanocomposite and preparation method thereof |
| CN108727752A (en) * | 2018-06-11 | 2018-11-02 | 中原工学院 | A method of preparing high strength poly vinyl alcohol composite hydrogel using in-situ fibrillation |
Non-Patent Citations (2)
| Title |
|---|
| JEAN CLAUDE MUNYEMANA ET AL.: ""A trypsin–calcium carbonate hybrid nanosphere based enzyme reactor with good stability and reusability"", 《NEW J. CHEM.》 * |
| 李从海: ""脂肪酶-无机杂化纳米花的制备及其催化性能的研究"", 《中国优秀博硕士学位论文全文数据库电子期刊 工程科技I辑》 * |
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