CN112877321A - Method for preparing graphene immobilized enzyme and application thereof - Google Patents
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 46
- 229910021389 graphene Inorganic materials 0.000 title claims abstract description 45
- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims abstract description 27
- 108090000790 Enzymes Proteins 0.000 claims abstract description 27
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 102100038238 Aromatic-L-amino-acid decarboxylase Human genes 0.000 claims abstract description 12
- 108010035075 Tyrosine decarboxylase Proteins 0.000 claims abstract description 12
- 238000006114 decarboxylation reaction Methods 0.000 claims abstract description 7
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims description 18
- 238000006555 catalytic reaction Methods 0.000 claims description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 14
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 claims description 12
- 239000003054 catalyst Substances 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000003431 cross linking reagent Substances 0.000 claims description 11
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 claims description 10
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 claims description 10
- 229960003638 dopamine Drugs 0.000 claims description 9
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims description 9
- 239000007787 solid Substances 0.000 claims description 8
- 239000000758 substrate Substances 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 7
- 239000008367 deionised water Substances 0.000 claims description 7
- 229910021641 deionized water Inorganic materials 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 6
- 238000009210 therapy by ultrasound Methods 0.000 claims description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 5
- FDRCDNZGSXJAFP-UHFFFAOYSA-M sodium chloroacetate Chemical compound [Na+].[O-]C(=O)CCl FDRCDNZGSXJAFP-UHFFFAOYSA-M 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- 239000005515 coenzyme Substances 0.000 claims description 4
- 229960001327 pyridoxal phosphate Drugs 0.000 claims description 4
- -1 carboxyl activated graphene oxide powder Chemical class 0.000 claims description 3
- 239000006185 dispersion Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 2
- 239000013522 chelant Substances 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
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- 239000000843 powder Substances 0.000 claims description 2
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- 238000002360 preparation method Methods 0.000 abstract description 6
- 102000004031 Carboxy-Lyases Human genes 0.000 abstract description 5
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- 230000002195 synergetic effect Effects 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 9
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
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- 150000001299 aldehydes Chemical class 0.000 description 1
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- 238000005804 alkylation reaction Methods 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000002210 biocatalytic effect Effects 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000009920 chelation Effects 0.000 description 1
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- 230000001186 cumulative effect Effects 0.000 description 1
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- 150000004985 diamines Chemical class 0.000 description 1
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- 125000003700 epoxy group Chemical group 0.000 description 1
- 238000006266 etherification reaction Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 208000030603 inherited susceptibility to asthma Diseases 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
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- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
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- 239000002086 nanomaterial Substances 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229920001690 polydopamine Polymers 0.000 description 1
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- 238000003786 synthesis reaction Methods 0.000 description 1
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/88—Lyases (4.)
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/001—Amines; Imines
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- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01028—Aromatic-L-amino-acid decarboxylase (4.1.1.28), i.e. tryptophane-decarboxylase
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Abstract
The invention discloses a method for preparing a graphene immobilized enzyme and application thereof.A dopa decarboxylase technology is fixed by a covalent bonding method, graphene oxide with high specific surface area is used as a carrier to modify the surface of the graphene oxide, a functional group capable of performing specific covalent reaction with enzyme is introduced to the surface of the carrier, and the functional group is bonded and fixed with the enzyme under mild conditions; and the stability and the repeated utilization rate of the enzyme are improved through a synergistic catalytic effect. The preparation technology can be applied to the field of enzyme decarboxylation, is environment-friendly and pollution-free, simple in post-treatment, high in conversion rate, capable of being repeatedly used and particularly suitable for fixing decarboxylase.
Description
Technical Field
The invention belongs to the field of biocatalytic organic synthesis, and particularly relates to a method for preparing a graphene immobilized enzyme and application thereof.
Background
Decarboxylase is a type of enzyme that reacts extremely rapidly and is subject to product inhibition. Dopa decarboxylase is able to catalyze the decarboxylation of Dopa (DP) to produce Dopamine (DA). In medicine, dopamine can be used for treating shock, heart failure, bronchial asthma and other diseases. In addition, dopamine can be self-polymerized into polydopamine under alkaline conditions, has good stability and water compatibility, and can be used for modifying nano materials. However, dopamine can severely inhibit the activity of enzyme, so that the high-efficiency production cannot be realized.
Graphene has a large specific surface area, a unique two-dimensional structure and chemical stability, and excellent electrical, mechanical, thermal and mechanical properties, and has become a new field for currently developing novel catalyst carriers. Graphene Oxide (GO), a graphene derivative, contains a plurality of groups, such as hydroxyl, carboxyl, epoxy group and carbonyl, is easily modified by chemical modification, and can be well dispersed in a solvent. The decarboxylase has many free functional groups such as hydroxyl, phenolic hydroxyl, carboxyl, amino, sulfydryl and the like on the surface, and can react with carboxyl, aldehyde, ketone and the like, such as etherification, alkylation, acylation and the like, so as to be covalently modified on the graphene surface.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for preparing a graphene immobilized enzyme and application thereof, the method selects high specific surface area graphene oxide as a carrier, applies a covalent bonding method to fix dopa decarboxylase technology, modifies the surface of the graphene oxide, introduces a functional group capable of carrying out specific covalent reaction with enzyme on the surface of the carrier, and carries out bonding fixation with the enzyme under mild conditions; the obtained immobilized enzyme has high reaction conversion rate and high stability through a synergistic catalytic effect. In addition, the preparation technology is environment-friendly and pollution-free, has simple post-treatment and high conversion rate, can be repeatedly utilized, and has important significance for expanding the application and development of the industrial technology of the enzyme-catalyzed decarboxylation.
In order to solve the problems of the prior art, the invention adopts the technical scheme that:
a method for preparing graphene immobilized enzyme comprises the following steps:
step 1, taking 1mg/mL graphene oxide GO aqueous dispersion, adding NaOH and sodium chloroacetate solids to keep the concentration of NaOH and sodium chloroacetate at 3-4mg/mL, and carrying out water bath ultrasonic reaction for 1-2 h; neutralizing with dilute HCl, washing with deionized water to neutrality, freeze-drying to obtain surface carboxyl activated graphene oxide powder, and hermetically storing for later use;
and 3, adding 5mg of surface active group modified functionalized graphene oxide powder into 5mL of PBS (50mmol/L, pH7.0), carrying out ultrasonic treatment for 5-10min, adding a PBS (50mmol/L, pH7.0) containing Dopa Decarboxylase (DDC) to maintain the initial concentration of the dopa decarboxylase and the functionalized graphene oxide in the mixed solution at 0.2-0.6:1, reacting at 4 ℃ for 3-5h, carrying out centrifugal separation, washing to obtain graphene immobilized enzyme catalyst particles, and freeze-drying and storing at-20 ℃ to obtain the graphene immobilized enzyme.
The improvement is that the cross-linking agent in the step 2 is polyethanol diamine (PEG-NH)2) 6-arm Polyethanoldiamine (6-br-PEG-NH)2) Methoxy polyethanolamine (mPEG-NH)2) Or N-hydroxysuccinimide (NHS).
The graphene immobilized enzyme is applied to catalyzing the decarboxylation of Dopa (DP) to generate dopamine.
As an improvement, the specific application steps are as follows: dissolving graphene immobilized enzyme in PBS (50mmol/L, pH7.0) containing 2mmol/L coenzyme pyridoxal phosphate PLP, incubating at 40 ℃ for 5min, adding substrate dopa solid for catalysis, controlling the concentration of substrate dopa not to exceed 10mg/mL (the substrate concentration is high, the higher the concentration of the generated product is, the enzyme activity can be seriously inhibited, the repeated use times of the enzyme are influenced), centrifuging and taking out the immobilized enzyme after reacting for 10-15min, re-dissolving the immobilized enzyme in the PBS (50mmol/L, pH7.0) containing coenzyme pyridoxal phosphate PLP, then carrying out catalysis, taking out the enzyme after each reaction for 10-15min, carrying out a new round of catalysis, and calculating the repeated use rate after a period of catalysis times.
Has the advantages that:
compared with the prior art, the method for preparing the graphene immobilized enzyme and the application thereof have the following specific advantages:
the functionalized active graphene immobilized enzyme catalyst prepared by the method can be used for the decarboxylation catalysis of dopa, and has the characteristics of high conversion rate, mild reaction conditions, easy product separation, environmental protection and the like; the method is suitable for immobilization of various decarboxylases, and the prepared immobilized decarboxylase catalyst is not easy to inactivate and fall off, has high reaction conversion rate and high reuse rate, and has wide application prospect particularly in the field of catalysis with product inhibition.
Drawings
FIG. 1 shows the effect of different kinds of cross-linking agents on the catalysis of immobilized enzymes;
fig. 2 is an electron microscope picture of a functionalized active graphene immobilized enzyme, (a) graphene before enzyme immobilization, (b) graphene after enzyme immobilization;
fig. 3 shows the catalytic results of the functionalized active graphene immobilized enzyme, (a) the continuous catalytic results of the immobilized enzyme, wherein the dashed line of the decrease indicates the consumption of the substrate per reaction of the immobilized enzyme, and the solid line of the increase indicates the production of the reaction product per reaction, (b) the catalytic results of the free enzyme, wherein the line of the decrease indicates the consumption of the substrate in the continuous catalytic reaction of the free enzyme, and the broken line of the increase indicates the production of the product.
Detailed Description
The present invention will be described in detail below with reference to the drawings and embodiments, and the embodiments of the present invention are not to be considered limited to the description. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
The materials and the technical means which are not mentioned in the following examples are all conventional in the field and do not need to be specifically explained.
Example 1 preparation of functionalized graphene oxide
Weighing 100mg of graphene oxide GO, dissolving in 100mL of deionized water, and carrying out ultrasonic treatment for 1h to prepare 1mg/mL of water dispersion; adding 4g of NaOH and 3g of sodium chloroacetate, and carrying out water bath ultrasound for 30 min; adding 100mL of 0.1 mol/L HCl for neutralization, washing with deionized water to neutrality, and freeze-drying to obtain the graphene oxide powder with surface carboxyl activated.
4 cross-linking agents needed by immobilization are selected, and are respectively PEG-NH2，6-br-PEG-NH2, mPEG-NH2NHS. Adding 10mg of the obtained acid activated graphene oxide powder into 10mL of aqueous solution containing different cross-linking agents (the concentration of the cross-linking agent is 20 mg/mL), and carrying out ultrasonic treatment for 30 min; adding the aqueous solution into 10mL of 10mg/mL EDC aqueous solution, and stirring at 800rpm for 16h at 25 ℃ to fully perform a chelation reaction; centrifugally separating the solution, and repeatedly washing the obtained solid with deionized water until the pH value of the washing liquid is neutral; and then vacuum drying is carried out for 24h, so as to obtain the functionalized graphene oxide powder modified by the surface active groups.
Example 2 preparation of graphene immobilized enzyme catalyst
With PEG-NH2-GO-DDC as an example to illustrate the preparation process of graphene immobilized enzyme catalyst, 10mg PEG-NH was taken2Adding the modified functionalized graphene oxide powder into 10mL of PBS buffer solution (50mmol/L, pH7.0), ultrasonically dispersing for 20min, and adding 1mL of PBS buffer solution of dopa decarboxylase with the concentration of 4mg/mLStirring the solution (50mmol/L, pH7.0) at 4 deg.C and 200rpm for 4h, centrifuging at 4000rpm, washing to obtain graphene immobilized enzyme catalyst particles, freeze drying for 24h, and storing at-20 deg.C for use. The electron micrographs of the functionalized graphene oxide powder before and after enzyme immobilization are shown in fig. 2.
Example 3 preparation of functionalized active graphene immobilized enzyme catalyst for catalytic reaction
1mg of different cross-linking agents are taken to prepare a functionalized active graphene immobilized enzyme catalyst and equivalent amount of free enzyme powder for catalytic reaction. The reaction system and the detection method were the same as in example 4 except for the continuous catalysis step. The relative activities of the graphene immobilized enzyme catalyst prepared by different cross-linking agents and free enzyme are shown in fig. 2.
Example 4 continuous high-efficiency catalysis of immobilized enzymes
1mg of PEG-NH was taken2Dissolving the modified functionalized graphene oxide immobilized enzyme catalyst particles in 1mL of PBS (50mmol/L, pH7.0) containing 5mmol/L PLP, incubating at 35 ℃ for 5min, adding 10mg of dopa solid for catalysis, reacting for 10min, centrifuging at 4 ℃ and 6000rpm for 2min, pouring out the supernatant, taking out the immobilized enzyme, dissolving in 1mL of PBS (50mmol/L, pH7.0) containing 5mmol/L PLP again, repeating the above catalysis steps, and finishing the reaction after continuously catalyzing for 10 times. The reaction was stopped by pipetting 400. mu.l of the supernatant from each reaction into 800. mu.l of a 1% (v/v) solution of trifluoroacetic acid. By high performance liquid chromatography (using Agilent TC-C18 chromatographic column (150 mm × 4.6 mm,5 μm), acetonitrile (96: 4) as mobile phase, 0.1% trifluoroacetic acid solution, and flow rate of 1.0 ml/min-1The detection wavelength is 280nm, and the column temperature is room temperature). After repeated use for 10 times, the cumulative yield of the dopamine reaches 64.02 g/L, and the enzyme activity is kept to be 68.25 percent of the original enzyme activity (shown in figure 3 (a)). The catalytic results of the same amount of free enzyme are shown in FIG. 3 (b), and when the concentration of dopamine reaches 21.8g/L, the activity of dopa decarboxylase is severely inhibited and the reaction is terminated.
According to the invention, the enzyme is fixed on the insoluble material in a covalent bonding mode of functional graphene oxide and dopa decarboxylase, so that the enzyme is conveniently taken out of a reaction system, and the product inhibition effect is effectively slowed down. In addition, a proper cross-linking agent is screened, so that the enzyme can be immobilized in a covalent cross-linking mode, and the enzyme can be close to dopa decarboxylase through charge action with hydroxyl of substrate dopa, the decarboxylation reaction of the enzyme is promoted to be rapidly carried out, and the macro expression shows that the activity reduction after conventional immobilization is not shown.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Claims (4)
1. A method for preparing graphene immobilized enzyme is characterized by comprising the following steps:
step 1, taking 1mg/mL graphene oxide GO aqueous dispersion, adding NaOH and sodium chloroacetate solids to keep the concentration of NaOH and sodium chloroacetate at 3-4mg/mL, and carrying out water bath ultrasonic reaction for 1-2 h; neutralizing with dilute HCl, washing with deionized water to neutrality, freeze drying to obtain surface carboxyl activated graphene oxide powder, and hermetically storing for later use;
step 2, adding the graphene oxide powder with the surface carboxyl activated obtained in the step 1 into an aqueous solution containing a cross-linking agent, performing ultrasonic treatment for 20-30min, and then adding into an aqueous solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to maintain the final concentration of EDC at 1.2-3 mg/mL; violently stirring for 12-16h at normal temperature to fully chelate the reaction, centrifugally separating, and repeatedly washing the obtained solid with deionized water until the pH value of the washing liquid is neutral; drying for 15-24h in a vacuum environment to obtain functionalized graphene oxide modified by surface active groups;
and 3, adding 5mg of functionalized graphene oxide powder modified by surface active groups into 5mL of PBS buffer, performing ultrasonic treatment for 5-10min, adding the powder into the PBS buffer containing dopa decarboxylase to maintain the initial concentration of the dopa decarboxylase and the functionalized graphene oxide in the mixed solution at 0.2-0.6:1, reacting for 3-5h at 4 ℃, performing centrifugal separation, washing to obtain graphene immobilized enzyme catalyst particles, and storing the particles at-20 ℃ after freeze drying to obtain the graphene immobilized enzyme.
2. The method for preparing graphene immobilized enzyme according to claim 1, wherein the cross-linking agent in step 2 is polyethanediamine, 6-arm polyethanediamine, methoxypolyethanolamine or N-hydroxysuccinimide.
3. The application of the graphene immobilized enzyme obtained based on the claim 1 in catalyzing the decarboxylation of dopa to generate dopamine.
4. Use according to claim 3, characterized in that the steps are as follows: dissolving graphene immobilized enzyme in PBS buffer solution containing 2mmol/L coenzyme pyridoxal phosphate PLP, incubating for 5min at 40 ℃, adding substrate dopa solid for catalysis, controlling the concentration of the substrate dopa not to exceed 10mg/mL, reacting for 10-15min, centrifuging the immobilized enzyme, taking out, dissolving in PBS buffer solution containing coenzyme pyridoxal phosphate PLP again, then catalyzing, taking out the enzyme for a new round of catalysis after 10-15min of each reaction, and calculating the reuse rate after a period of catalysis times.
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| CN114591941A (en) * | 2022-03-09 | 2022-06-07 | 北京理工大学 | Preparation of epoxy functionalized covalent organic framework-based covalent immobilized Cyt c-based biocatalytic material |
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