CN116376890A - Preparation method of graphene oxide hydrogel immobilized enzyme - Google Patents
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 138
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- 238000002360 preparation method Methods 0.000 title claims abstract description 23
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- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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Abstract
The invention relates to the technical field of enzyme engineering, and discloses a preparation method of graphene oxide hydrogel immobilized enzyme. According to the preparation method provided by the invention, firstly, polyethyleneimine crosslinking is introduced into graphene oxide, then intermolecular acting force is further improved through glutaraldehyde modification, and finally, enzyme is covalently bound and loaded on the surface of hydrogel, so that the immobilized enzyme with good cyclic batch usability and catalytic activity is prepared.
Description
Technical Field
The invention relates to the technical field of enzyme engineering, in particular to a preparation method of graphene oxide hydrogel immobilized enzyme.
Background
The biological enzyme has the advantages of high catalytic efficiency, high selectivity, mild reaction condition and the like. However, free enzyme catalysis suffers from poor stability and difficult recovery, which makes it limited in industrial applications. The enzyme immobilization technology limits the free enzyme in a certain space or is attached to a solid structure and cannot move freely, so that the stability and the recoverability of the enzyme are improved. The carrier material is the basis and key for enzyme immobilization, and its performance affects the enzyme catalytic efficiency. In recent years, novel materials such as porous silicon materials, nano materials (such as graphene oxide, carbon nano tubes and the like), magnetic materials, metal Organic Frameworks (MOFs) and the like are used for enzyme immobilization, so that the stability and the recyclability of enzymes are improved.
Graphene oxide is a two-dimensional nanomaterial with a monoatomic layer, has the advantages of large specific surface area, abundant surface functional groups, good biocompatibility and the like, and is considered to be an ideal immobilized enzyme carrier material. For example, graphene oxide-supported lipases exhibit excellent catalytic performance in gas phase reactions. However, graphene oxide is used as a nanomaterial, immobilized enzyme prepared based on the graphene oxide is easy to cause carrier residue or loss in the catalytic process, and the usability of the circulating batch is poor.
The hydrogel is a kind of aqueous macroscopic three-dimensional porous structure gel material, and has wide application in the fields of electronics, materials, biomedicine and the like. The graphene oxide is assembled to construct a macroscopic porous hydrogel material, and the macroscopic porous hydrogel material is used for enzyme immobilization, so that the recoverability of graphene oxide immobilized enzyme is expected to be improved; meanwhile, the macroscopic three-dimensional porous structure has good fluid characteristics and can be used for constructing a bioreactor.
Currently, graphene oxide hydrogel enzyme immobilization mainly adopts a co-assembly immobilization method, namely an embedding method, namely, graphene oxide and biomolecules are self-assembled to construct hydrogel by utilizing acting forces (such as hydrogen bonds, van der Waals forces, electrostatic interactions and the like) of the graphene oxide and the biomolecules, and enzymes are wrapped in the hydrogel to realize enzyme immobilization. However, since the acting force between the biomolecules and the graphene oxide is weak, the hydrogel constructed by the co-assembly method has poor mechanical properties, resulting in poor cyclic batch usability of the immobilized enzyme; meanwhile, the immobilized enzyme has lower catalytic activity because the enzyme is embedded in the hydrogel.
Therefore, development of a new preparation method of graphene oxide hydrogel immobilized enzyme is needed, so that the immobilized enzyme has good recycling batch usability and catalytic activity.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of graphene oxide hydrogel immobilized enzyme. According to the preparation method provided by the invention, firstly, polyethyleneimine crosslinking is introduced into graphene oxide, then intermolecular acting force is further improved through glutaraldehyde modification, and finally, enzyme is covalently bound and loaded on the surface of hydrogel, so that the immobilized enzyme with good cyclic batch usability and catalytic activity is prepared.
The specific technical scheme of the invention is as follows:
the invention provides a preparation method of graphene oxide hydrogel immobilized enzyme, which comprises the following steps:
(1) Preparation of crosslinked gel: dissolving graphene oxide, regulating pH to be alkaline, adding a polyethyleneimine solution, uniformly mixing, and reacting to obtain polyethyleneimine crosslinked graphene oxide hydrogel;
(2) Glutaraldehyde modification: placing the hydrogel prepared in the step (1) into glutaraldehyde solution for reaction to obtain glutaraldehyde-modified graphene oxide hydrogel;
(3) Enzyme loading: and (3) adding the hydrogel prepared in the step (2) into an enzyme solution, and carrying out an immobilization reaction to obtain the graphene oxide hydrogel immobilized enzyme.
The immobilized enzyme in the prior art is prepared by embedding the enzyme in a skeleton matrix, such as hydrogel, and has the problems of low enzyme activity and poor mechanical properties of the skeleton; the enzyme is fixed on the surface of the skeleton matrix through surface adsorption, so that the problem that the surface adsorption force is weak and the enzyme is easy to fall off exists; in the prior art, enzymes are immobilized on the surface of a skeleton matrix through covalent bond connection, and the problems are that the method is complex and the loss of enzyme activity is easy to cause. In the preparation method of the graphene oxide hydrogel immobilized enzyme, firstly, polyethyleneimine is introduced into graphene oxide through the step (1) to crosslink, so that the polyethyleneimine crosslinked graphene oxide hydrogel is obtained. After the graphene oxide hydrogel is introduced into polyethyleneimine for crosslinking, the polyethyleneimine and the graphene oxide act together to form a skeleton matrix of the immobilized enzyme, and the acting force between the graphene oxide lamellar structures is increased due to crosslinking among polyethyleneimine molecular chains. Then, the intermolecular force is further improved by modifying the hydrogel matrix by glutaraldehyde in the step (2), and the enzyme is immobilized and loaded on the surface of the hydrogel by covalent bonding of the enzyme in the step (3) and the reactive groups of the aldehyde group and the amino group on the surface of the hydrogel, so that the immobilized enzyme with excellent recycling batch usability and catalytic activity is prepared, and the preparation process is simple.
Compared with the immobilized enzyme obtained by embedding the enzyme, the immobilized enzyme prepared by the preparation method provided by the invention has good mechanical properties and can maintain excellent enzyme activity; compared with immobilized enzyme obtained by surface adsorption, the immobilized enzyme has good immobilization performance and is not easy to fall off; in contrast to the immobilized enzymes with covalently linked surfaces, the method of the invention is simple and does not cause enzyme inactivation during covalent linkage.
Preferably, in the above embodiment of the present invention, in the step (3), the enzyme is amidase. The enzyme is easy to inactivate through covalent bond connection between the enzyme and the skeleton matrix, and is easy to fall off through surface adsorption of the ligase. The preparation method provided by the invention is characterized in that amidase and skeleton matrix graphene oxide hydrogel form covalent bonds to carry out fixed connection, and the immobilized enzyme can keep the activity equivalent to that of free enzyme. The research team finds that the excellent activity of the amidase immobilized enzyme is firstly that the amino groups on the graphene oxide can enhance the surface hydrophilicity, so that the contact and digestion of the amidase immobilized enzyme and a substrate are promoted through hydrogen bond formation.
As a preferable mode of the above technical scheme of the invention, in the step (1), the concentration of the polyethyleneimine solution is 1.25-20 mg/mL.
As a preferable mode of the technical scheme, in the step (1), the concentration of graphene oxide in the polyethyleneimine-crosslinked graphene oxide hydrogel is 1-10 mg/mL.
In the graphene oxide hydrogel immobilized enzyme prepared by the preparation method provided by the invention, the polyethylene imine crosslinked graphene oxide hydrogel is used as a skeleton matrix, the amount of graphene oxide in the skeleton has a great influence on the mechanical properties of the prepared hydrogel, and the concentration of graphene oxide in the polyethylene imine crosslinked graphene oxide hydrogel obtained in the step (1) is 1-10 mg/mL, so that the mechanical properties of the finally obtained immobilized enzyme hydrogel are in a good state. If the amount of graphene oxide is too large, the acting force between lamellar structures is insufficient, the mechanical properties of the prepared hydrogel are slightly poor, and if the amount of graphene oxide is too small, colloid cannot be formed, and the hydrogel immobilized enzyme cannot be prepared.
In the step (1), the pH is adjusted by using a sodium hydroxide solution as a regulator, and the end point of the adjustment is 7.8-9.0.
As a preferable mode of the above technical scheme of the present invention, in the step (1), the temperature of the reaction is 85-100 ℃.
As a preferable mode of the above technical scheme of the invention, in the step (1), the reaction time is 5-8 h.
As a preferable mode of the above technical scheme of the present invention, in the step (2), the concentration of the glutaraldehyde solution is 0.1 to 1wt%.
As a preferable mode of the above technical scheme of the invention, in the step (2), the reaction time is 1.5-2.5 h.
Compared with the prior art, the invention has the following technical effects:
according to the preparation method of the immobilized enzyme, firstly, polyethyleneimine is introduced into graphene oxide for crosslinking, then intermolecular acting force is further improved through glutaraldehyde modification, and finally, the enzyme is covalently bound and loaded on the surface of the hydrogel, so that the immobilized enzyme with good cyclic batch usability and catalytic activity is prepared. Compared with the immobilized enzyme obtained by embedding the enzyme, the immobilized enzyme prepared by the preparation method provided by the invention has good mechanical properties and can maintain excellent enzyme activity; compared with immobilized enzyme obtained by surface adsorption, the immobilized enzyme has good immobilization performance and is not easy to fall off; in contrast to the immobilized enzymes with covalently linked surfaces, the method of the invention is simple and does not cause enzyme inactivation during covalent linkage.
Drawings
FIG. 1 is a macroscopic view of the graphene oxide hydrogel immobilized enzyme prepared in example 2 (1) of the present invention;
FIG. 2 is a graph showing the result of a scanning electron microscope of the graphene oxide hydrogel prepared in example 2 (1) of the present invention;
FIG. 3 is a Fourier-infrared spectrum of graphene oxide hydrogel prepared in example 2 (1) and graphene oxide prepared in example 1 of the present invention;
FIG. 4 is a graph showing the progress of the conversion rate of the graphene oxide hydrogel immobilized enzyme catalyzed synthesis of L-4-fluorophenylglycine according to example 6 of the present invention;
FIG. 5 is a graph showing the results of enzyme activities of the graphene oxide hydrogel immobilized enzyme of example 6 according to the present invention for different times of use.
Detailed Description
The invention is further described below with reference to examples.
Example 1 preparation of graphene oxide
Graphene oxide references were prepared using the modified Hummers method (Journal of the American Chemical Society,1958,80 (6): 1339-1341).
The preparation process comprises the following steps: weighing 0.75g NaNO 3 34mL of concentrated H 2 SO 4 And placing 1g of graphite flake on the triangular flask, placing the triangular flask on an ice-water mixture for continuous stirring, slowly adding 5g of potassium permanganate in batches when the temperature of the system is reduced to 0 ℃, still placing the triangular flask in an ice bath condition and continuously stirring, keeping the temperature of the system at not higher than 35 ℃, and observing the formation of thick slurry after stirring for 2 hours. Then slowly adding 50mL of ice water for a plurality of times, controlling the water adding speed to ensure that the system temperature is not higher than 100 ℃, stirring for 2 hours, and slowly adding 4mL of H for four times 2 O 2 Stirring was carried out until no gas was generated, and the solution changed from dark brown to bright yellow. Repeatedly washing the mixed liquid with dilute hydrochloric acid and deionized water until the graphene oxide sheets fall off, centrifuging at 4000r/min, and removing the precipitate to finally obtain the graphene oxide nano sheet dispersion liquid. Subpackaging into dialysis bags, dialyzing with ultrapure water for 48 hr, and fixing volume for use.
Example 2 preparation of graphene oxide hydrogels and performance testing thereof
(1) Firstly, taking 5mL of graphene oxide (5 mg/mL) subjected to dialysis in example 1, adding 50 mu L of 1M NaOH solution to adjust the pH of the graphene oxide to 8.0, slowly adding 5mL of polyethyleneimine solution with the concentration of 2.5mg/mL, uniformly mixing, carrying out ultrasonic treatment for 20min, subpackaging into 3mL glass bottles, putting into a polytetrafluoroethylene reaction kettle, and placing into a 90 ℃ air-blast oven for reaction for 6h. And taking out the graphene oxide hydrogel after the reaction is finished, dialyzing for 24 hours by using ultrapure water, and finally removing unreacted graphene oxide and polyethyleneimine. The concentration of graphene oxide in the obtained polyethyleneimine crosslinked graphene oxide hydrogel is 5mg/mL.
And (3) placing the prepared graphene oxide hydrogel in glutaraldehyde solution with the concentration of 0.5wt% for reaction for 2 hours to obtain the graphene oxide hydrogel with the surface modified with aldehyde groups, and then washing with buffer solution/pure water to remove surface proteins and free aldehyde groups.
The 5g weight is placed above the graphene oxide hydrogel, and the property of the graphene oxide hydrogel is as shown in figure 1, and the graphene oxide hydrogel has no splitting phenomenon and good mechanical properties; and the morphology of the gel is observed by using a scanning electron microscope, as shown in fig. 2, the constructed graphene oxide hydrogel has a rich pore structure, and is beneficial to mass transfer of a substrate.
(2) Firstly, taking 5mL of graphene oxide (5 mg/mL) subjected to dialysis in example 1, adding 50 mu L of 1M NaOH solution to adjust the pH of the graphene oxide to 9.0, slowly adding 5mL of polyethyleneimine solution with the concentration of 1.25mg/mL, uniformly mixing, carrying out ultrasonic treatment for 20min, subpackaging into 3mL glass bottles, putting into a polytetrafluoroethylene reaction kettle, and placing into a 90 ℃ air-blast oven for reaction for 5h. And taking out the graphene oxide hydrogel after the reaction is finished, dialyzing for 24 hours by using ultrapure water, and finally removing unreacted graphene oxide and polyethyleneimine. The concentration of graphene oxide in the obtained polyethyleneimine crosslinked graphene oxide hydrogel is 1mg/mL.
And (3) placing the prepared graphene oxide hydrogel in 0.1wt% glutaraldehyde solution for reaction for 1.5 hours to obtain the graphene oxide hydrogel with aldehyde groups modified on the surface, and then washing with buffer solution/pure water to remove surface proteins and free aldehyde groups.
The 5g weight is placed above the graphene oxide hydrogel, so that the phenomenon of splitting does not occur, and the graphene oxide hydrogel has good mechanical properties; and observing the morphology of the gel by using a scanning electron microscope, and finding that the constructed graphene oxide hydrogel has a rich pore structure.
(3) Firstly, taking 5mL of graphene oxide (5 mg/mL) subjected to dialysis in the embodiment 1, adding 50 mu L of 1M NaOH solution to adjust the pH of the graphene oxide to 7.8, slowly adding 5mL of polyethyleneimine solution with the concentration of 20mg/mL, uniformly mixing, carrying out ultrasonic treatment for 20min, subpackaging into 3mL glass bottles, putting into a polytetrafluoroethylene reaction kettle, and placing into a blast oven at 90 ℃ for reaction for 8h. And taking out the graphene oxide hydrogel after the reaction is finished, dialyzing for 24 hours by using ultrapure water, and finally removing unreacted graphene oxide and polyethyleneimine. The concentration of graphene oxide in the obtained polyethyleneimine crosslinked graphene oxide hydrogel is 10mg/mL.
And (3) placing the prepared graphene oxide hydrogel in a glutaraldehyde solution with the weight percent of 1.0 for reaction for 2.5 hours to obtain the graphene oxide hydrogel with the surface modified with aldehyde groups, and then washing the graphene oxide hydrogel with buffer solution/pure water to remove surface proteins and free aldehyde groups.
The 5g weight is placed above the graphene oxide hydrogel, so that the phenomenon of splitting does not occur, and the graphene oxide hydrogel has good mechanical properties; and observing the morphology of the gel by using a scanning electron microscope, and finding that the constructed graphene oxide hydrogel has a rich pore structure.
(4) Firstly, 10mL of graphene oxide (5 mg/mL) subjected to dialysis in example 1 is taken, 50 mu L of 1M NaOH solution is added to adjust the pH of the graphene oxide to 8.0, 5mL of polyethyleneimine solution with the concentration of 2.5mg/mL is slowly added, uniformly mixed, ultrasonically treated for 20min, packaged into a 3mL glass bottle, placed into a polytetrafluoroethylene reaction kettle, and placed into a 90 ℃ blast oven for reaction for 6h. And taking out the graphene oxide hydrogel after the reaction is finished, dialyzing for 24 hours by using ultrapure water, and finally removing unreacted graphene oxide and polyethyleneimine. The concentration of graphene oxide in the obtained polyethyleneimine crosslinked graphene oxide hydrogel is 13mg/mL.
And (3) placing the prepared graphene oxide hydrogel in glutaraldehyde solution with the concentration of 0.5wt% for reaction for 2 hours to obtain the graphene oxide hydrogel with the surface modified with aldehyde groups, and then washing with buffer solution/pure water to remove surface proteins and free aldehyde groups.
Placing a 5g weight above the graphene oxide hydrogel, wherein slight cracks appear; and the morphology of the gel is observed by using a scanning electron microscope, and the obtained graphene oxide hydrogel has few holes and compact structure.
(5) Firstly, taking 5mL of graphene oxide (5 mg/mL) subjected to dialysis in the embodiment 1, adding 50 mu L of 1M NaOH solution to adjust the pH of the graphene oxide to 8.0, slowly adding 5mL of polyethyleneimine solution with the concentration of 6mg/mL, uniformly mixing, carrying out ultrasonic treatment for 20min, subpackaging into 3mL glass bottles, putting into a polytetrafluoroethylene reaction kettle, and placing into a blast oven at 90 ℃ for reaction for 6h. After completion of the reaction, it was found that hydrogel formation was impossible.
Example 3 Fourier infrared Spectrometry of graphene oxide hydrogels
Fourier infrared characterization was performed on the graphene oxide hydrogel prepared in example 2 (1) and the graphene oxide prepared in example 1. As a result, as shown in FIG. 3, there were three peaks of 3400, 1657 and 1575cm-1, which are more than graphene oxide hydrogel, for the ammonia-based surface in-bending (amide II), carbon-carbon double bond stretching, and carbon-carbon double bond stretching (amide I), respectively. The peak position of 1727cm < -1 > of the graphene oxide two-dimensional nano sheet is missing, and the peak is for carbon-oxygen double bonds in carboxyl, and the missing peak proves that amino groups on the polyethyleneimine react with the carboxyl. These evidence indicate that the graphene oxide two-dimensional nanoplatelets are successfully combined with polyethyleneimine to form graphene oxide hydrogels, thus indicating that the graphene oxide hydrogels are rich in amino groups.
EXAMPLE 4 expression and purification of amidase
Amidase is expressed in bacillus subtilis WB800, glycerol tubes for preserving strains are taken out from-80 ℃, sterilized inoculation operation is carried out in an ultra-clean bench after thawing at room temperature, bacterial liquid in a small amount of glycerol tubes is dipped in an inoculating loop to streak on LB solid culture medium containing ampicillin resistance, and then the culture medium is placed in a constant temperature incubator at 37 ℃ for 10-12h after sealing a flat plate by a sealing film. Complete single colonies were picked with an inoculating loop, added to a final concentration of 50. Mu.g/mL of kana-resistant LB liquid medium, and shake-cultured in a shaker at 37℃for 12h at 200 r/min. The cultured seed solution was then inoculated into sterilized LB liquid medium containing Canada resistance at an inoculum size of 1%, and cultured in a shaking table at 37℃for 150 r/min. When the OD value of the thallus concentration is about 0.4, IPTG is taken and added to the thallus concentration for induction, and the final concentration of the IPTG is 0.1mmol/L. Then the bacterial liquid is placed in a shaking table at 37 ℃ and 150r/min for culturing for 12 hours.
The overexpressed recombinant amidase was isolated by centrifugation and purified by cell-free extraction using 80% saturated ammonium sulfate, DEAE-IEX chromatography and butyl HIC chromatography. The method comprises the following steps: and (3) placing the crude enzyme solution obtained after the fermentation broth is centrifuged on ice, taking ammonium sulfate with saturation of 30%, 50%, 60%, 70%, 80% and 90%, and salting out and precipitating. Namely, every 100mL of crude enzyme solution, 16.4g, 11.7g, 6.0g, 6.2g, 6.5g and 6.7g of ammonium sulfate are slowly added in sequence, and each time ammonium sulfate is added, the solution is centrifuged (4 ℃,12000r/min and 10 min), and the supernatant and the precipitate are respectively collected. Collecting supernatant, and adding ammonium sulfate for further precipitation; while the precipitated proteins were solubilized with a small amount of phosphate buffer (pH 8.0,0.05 mol/L). Preparing Buffer A: naH2PO4-Na2HPO4 solution (0.05 mol/L) at pH 8.0; buffer B:1mol/L NaCl solution, prepared with Buffer A. After Buffer A and Buffer B were formulated, both were filtered with a 0.22 μm water membrane. The enzyme solution after ammonium persulfate precipitation was applied to a Buffer A equilibrated DEAE-Sepharose FF column (1.6X10 cm, GE), and eluted with a gradient of phosphate Buffer containing 0-1.0mol/L NaCl, and the eluate was collected by a separate tube. And then performing SDS-PAGE electrophoresis analysis to judge which eluent is the enzyme solution, and collecting the eluent for dialysis. Since a large amount of salt ions are introduced during gradient elution, the method is not suitable for preserving enzymes, so that the dialysis bag is utilized to remove salt, the dialysis solution is PBS solution which is the same as Buffer A, and the PBS solution is required to be placed on ice, then placed in an explosion-proof refrigerator and dialyzed for 12 hours.
Example 5 preparation of graphene oxide hydrogel immobilized enzyme of amidase
The graphene oxide hydrogel prepared in example 2 (1) was placed in the purified amidase solution obtained in example 4, and was subjected to a fixation reaction for 2 hours, followed by separation and washing, to obtain a graphene oxide hydrogel immobilized enzyme of amidase.
Example 6 Performance test of graphene oxide hydrogel immobilized enzyme of amidase
(1) The graphene oxide hydrogel of amidase prepared in example 5 is used for carrying enzyme, and the immobilized enzyme carries enzyme up to 2175mg/g.
(2) Enzyme activity determination: 20mL of the reaction system was added with 50mg of the amidase-oxidized graphene hydrogel immobilized enzyme or amidase-free enzyme prepared in example 5, N-phenylacetyl-DL-p-fluorophenylglycine (20 mmol/L), glycine-sodium hydroxide buffer (pH 9.0). Reacting at 40deg.C under 200r/min, taking out immobilized enzyme or free enzyme, and detecting supernatant by high performance liquid chromatography.
The enzyme activity measurement is carried out when the reaction time is 0, 30, 60, 90, 120, 150, 180 and 210min, the conversion rate progress of the immobilized enzyme of the oxidized graphene hydrogel of amidase and the catalytic synthesis of L-4-fluorophenyl glycine by the free enzyme is shown in figure 4, the free amidase reaches the reaction equilibrium in 180min, the immobilized amidase reaches the equilibrium in 210min, and the conversion rate is similar to that of the free enzyme.
(3) Determining the reuse rate of the oxidized graphene hydrogel immobilized enzyme of amidase: 20mL of the reaction system was added 50mg of the amidase oxidized graphene hydrogel immobilized enzyme prepared in example 5, N-phenylacetyl-DL-p-fluorophenylglycine (20 mmol/L), and glycine-sodium hydroxide buffer (pH 9.0). The reaction is carried out for 30min at 40 ℃ and 200r/min, the immobilized enzyme is taken out, and the supernatant is taken out and the enzyme activity is detected by HPLC. The above operation was repeated 6 times, and the samples were periodically taken by HPLC.
The results of the enzyme activity of the graphene oxide hydrogel immobilized enzyme for different times are shown in FIG. 5. As shown in fig. 5, the graphene oxide hydrogel immobilized enzyme of amidase still retains 96% of the initial enzyme activity after the immobilized enzyme is recycled 6 times.
The graphene oxide hydrogel prepared by the invention is not only limited to amidase load, but also widely applied to immobilization of other enzyme species, such as alpha-glucan phosphorylase, phosphoglucomutase, phosphoglucose isomerase and the like.
The raw materials and equipment used in the invention are common raw materials and equipment in the field unless specified otherwise; the methods used in the present invention are conventional in the art unless otherwise specified.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent transformation of the above embodiment according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.
Claims (10)
1. The preparation method of the graphene oxide hydrogel immobilized enzyme is characterized by comprising the following steps of: the method comprises the following steps:
(1) Preparation of crosslinked gel: dissolving graphene oxide, regulating pH to be alkaline, adding a polyethyleneimine solution, uniformly mixing, and reacting to obtain polyethyleneimine crosslinked graphene oxide hydrogel;
(2) Glutaraldehyde modification: placing the hydrogel prepared in the step (1) into glutaraldehyde solution for reaction to obtain glutaraldehyde-modified graphene oxide hydrogel;
(3) Enzyme loading: and (3) adding the hydrogel prepared in the step (2) into an enzyme solution, and carrying out an immobilization reaction to obtain the graphene oxide hydrogel immobilized enzyme.
2. The method for preparing the graphene oxide hydrogel immobilized enzyme according to claim 1, which is characterized in that: in step (3), the enzyme is amidase.
3. The method for preparing the graphene oxide hydrogel immobilized enzyme according to claim 1, which is characterized in that: in the step (1), the concentration of the polyethyleneimine solution is 1.25-20 mg/mL.
4. The method for preparing the graphene oxide hydrogel immobilized enzyme according to claim 1, which is characterized in that: in the step (1), the concentration of graphene oxide in the polyethyleneimine crosslinked graphene oxide hydrogel is 1-10 mg/mL.
5. The method for preparing the graphene oxide hydrogel immobilized enzyme according to claim 1, which is characterized in that: in the step (1), the end point of the pH adjustment is 7.8-9.0.
6. The method for preparing the graphene oxide hydrogel immobilized enzyme according to claim 1 or 5, wherein the method comprises the following steps: in the step (1), the regulator used for regulating the pH is sodium hydroxide solution.
7. The method for preparing the graphene oxide hydrogel immobilized enzyme according to claim 1, which is characterized in that: in the step (1), the reaction temperature is 85-100 ℃.
8. The method for preparing the graphene oxide hydrogel immobilized enzyme according to claim 1 or 7, wherein the method comprises the following steps: in the step (1), the reaction time is 5-8 hours.
9. The method for preparing the graphene oxide hydrogel immobilized enzyme according to claim 1, which is characterized in that: in the step (2), the concentration of the glutaraldehyde solution is 0.1-1wt%.
10. The method for preparing the graphene oxide hydrogel immobilized enzyme according to claim 1, which is characterized in that: in the step (2), the reaction time is 1.5-2.5 h.
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