CN116622693A - Immobilized enzyme encapsulated in situ by covalent organic frameworks, preparation method and application thereof - Google Patents

Immobilized enzyme encapsulated in situ by covalent organic frameworks, preparation method and application thereof Download PDF

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CN116622693A
CN116622693A CN202310534064.9A CN202310534064A CN116622693A CN 116622693 A CN116622693 A CN 116622693A CN 202310534064 A CN202310534064 A CN 202310534064A CN 116622693 A CN116622693 A CN 116622693A
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situ
covalent organic
immobilized enzyme
encapsulated
organic framework
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董育斌
冯杰
任文秀
于浩
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Shandong Normal University
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Shandong Normal University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
    • C12Q2326/10—Benzidines
    • C12Q2326/12—3,3',5,5'-Tetramethylbenzidine, i.e. TMB
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)

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  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

The invention belongs to the technical field of immobilized enzymes, and relates to an immobilized enzyme in-situ packaged by a covalent organic framework, and a preparation method and application thereof. Adding 2, 5-dimethoxy-1, 4-dicarboxaldehyde benzene and horseradish peroxidase into water, mixing uniformly, then adding 1,3, 5-tri (4-aminophenyl) benzene aqueous solution containing acetic acid, stirring and reacting for 18-36 h at room temperature. The invention not only can improve the stability of the loaded enzyme, but also can keep the extremely high activity of the enzyme.

Description

Immobilized enzyme encapsulated in situ by covalent organic frameworks, preparation method and application thereof
Technical Field
The invention belongs to the technical field of immobilized enzymes, and relates to an immobilized enzyme in-situ packaged by a covalent organic framework, and a preparation method and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Most of the natural enzymes are composed of proteins, and have complex and unstable structures, severe requirements on catalytic conditions, high storage requirements, complex preparation process and high cost. By loading enzymes on MOFs or on COFs, the interference of external conditions on enzymes can be reduced, and the stability of the enzymes can be improved. However, MOFs carriers are not highly water/chemically stable and have the potential for toxic metal ion leaching, so they are inferior to enzyme-containing frameworks with COFs as carriers in terms of stability and biocompatibility. The inventor researches and knows that at present, common immobilization methods of enzymes on COFs are a surface adsorption method, a covalent bonding method and a pore channel diffusion method. However, according to the research of the inventor, the methods have the problems that the stability of the loaded enzyme is poor, the enzyme activity is influenced by adopting an organic solvent, and the like.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide the immobilized enzyme encapsulated in situ by the covalent organic framework, and the preparation method and the application thereof, which not only can improve the stability of the loaded enzyme, but also can keep the extremely high activity of the enzyme.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
on the one hand, the preparation method of the immobilized enzyme with the covalent organic framework in-situ encapsulation comprises the steps of adding 2, 5-dimethoxy-1, 4-dicarboxaldehyde benzene (DMTP) and horseradish peroxidase (Horseradish Peroxidase, HRP) into water, uniformly mixing, then adding a 1,3, 5-tris (4-aminophenyl) benzene (TPB) aqueous solution containing acetic acid, and stirring and reacting for 18-36 h at room temperature.
The preparation process can regulate the crystal size, form and crystallinity of the reacted DMTP and TPB in water, and the HRP is wrapped in porous crystal to produce new cavities around enzyme to form interaction with the HRP, so that the covalent organic frame in-situ encapsulated immobilized enzyme is nanometer level. This interaction and size not only protects the HRP from harsh environments, but also makes it more difficult for HRP to detach from COFs. Thereby ensuring that the immobilized enzyme encapsulated in situ by the covalent organic framework has higher activity and reusability.
Experiments have unexpectedly found that the immobilized enzyme in-situ encapsulated by the covalent organic framework provided by the invention can have the characteristic of high-temperature (100-153 ℃) enzyme activity, and HRP basically loses activity under the high-temperature condition.
In another aspect, an immobilized enzyme encapsulated in situ by a covalent organic framework is obtained by the above preparation method.
In a third aspect, the use of an immobilized enzyme encapsulated in situ by a covalent organic framework as described above for detecting peroxides.
In a fourth aspect, a detection kit comprises an immobilized enzyme and a chromogenic substrate encapsulated in situ by a covalent organic framework as described above.
The beneficial effects of the invention are as follows:
1. in the preparation process, the crystal size, the morphology and the crystallinity of DMTP and TPB are regulated in water by HRP, and simultaneously HRP is wrapped in a porous crystal to generate new cavities which tightly surround enzymes and interact with the HRP, and the prepared immobilized enzyme in-situ packaged by the covalent organic frameworks has the nano-scale size, so that the enzyme can be protected from severe environment, and the enzyme is more difficult to separate from COFs, so that the obtained immobilized enzyme in-situ packaged by the covalent organic frameworks has higher activity and reusability.
2. The preparation process is carried out at room temperature and in water, so that the reaction condition is mild, and the reduction of the enzyme activity can be avoided.
3. Experiments show that the covalent organic framework in-situ encapsulated immobilized enzyme prepared by the invention can maintain extremely high activity in a protein decomposition agent, boiling water (100 ℃) and boiling DMF (153 ℃).
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic diagram of an immobilized enzyme encapsulated in situ by a covalent organic framework prepared in accordance with an embodiment of the present invention;
FIG. 2 is an XRD pattern of an immobilized enzyme encapsulated in situ by a covalent organic framework prepared in accordance with an embodiment of the present invention;
FIG. 3 is an SEM image of an in situ encapsulated immobilized enzyme of a covalent organic framework prepared according to an embodiment of the present invention, the scale bar representing 500nm;
FIG. 4 is an EDX map of an immobilized enzyme encapsulated in situ by a covalent organic framework prepared in accordance with an embodiment of the present invention;
FIG. 5 is a graph showing the enzyme activity of an immobilized enzyme encapsulated in situ by a covalent organic framework prepared in the embodiment of the invention, wherein A is a schematic diagram of a peroxidase catalysis mechanism, B is a fluorescence curve of different samples over time, C is an ultraviolet-visible absorption intensity curve of different systems at different time intervals, D is a graph showing product conversion conditions after the free HRP, HRP-COF, HRP@ZIF-8 and HRP@COF are respectively treated for 1 hour in a proteolytic agent, 100 ℃ boiling water and 153 ℃ boiling Dimethylformamide (DMF), and error bars represent standard deviations (n=3).
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In view of the problems of poor stability, serious enzyme activity reduction and the like of the existing immobilized enzyme adopting the COFs, the invention provides an immobilized enzyme in-situ packaged by a covalent organic framework, and a preparation method and application thereof.
In an exemplary embodiment of the invention, a preparation method of immobilized enzyme encapsulated in situ by a covalent organic framework is provided, 2, 5-dimethoxy-1, 4-dicarboxaldehyde benzene and horseradish peroxidase are added into water to be uniformly mixed, then 1,3, 5-tris (4-aminophenyl) benzene aqueous solution containing acetic acid is added, and stirring reaction is carried out for 18-36 h at room temperature.
The room temperature refers to the temperature of the indoor environment, and is generally 15-30 ℃.
In some embodiments, the molar ratio of 2, 5-dimethoxy-1, 4-dicarboxybenzene to 1,3, 5-tris (4-aminophenyl) benzene is 2.50 to 3.50:2. Preferably 2.50 to 3.00:2, more preferably 2.80 to 2.90:2.
In some embodiments, the mass ratio of 2, 5-dimethoxy-1, 4-dicarboxaldehyde to horseradish peroxidase is 1.5-2.0:1. Preferably 1.5 to 1.8:1.
In some embodiments, the 1,3, 5-tris (4-aminophenyl) benzene is added to the acetic acid in an amount of 15 to 25:1, g: l. Preferably 18 to 22:1, g: l.
In another embodiment of the invention, an immobilized enzyme encapsulated in situ by a covalent organic framework is provided, which is obtained by the preparation method.
In a third aspect, the use of an immobilized enzyme encapsulated in situ by a covalent organic framework as described above for detecting peroxides.
Specifically, the peroxide is hydrogen peroxide.
In a fourth aspect, a detection kit comprises an immobilized enzyme and a chromogenic substrate encapsulated in situ by a covalent organic framework as described above.
Specifically, the chromogenic substrate is 3,3', 5' -Tetramethylbenzidine (TMB).
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail below with reference to specific examples and comparative examples.
Examples
The preparation method of the immobilized enzyme encapsulated in situ by the covalent organic framework comprises the following steps:
(1) DMTP (48 mg) was dispersed in 60mL of water and labeled as solution (1).
(2) HRP (30 mg) was dispersed in 30mL of water, added to the solution (1), mixed and stirred for one hour, and the mixed and stirred solution was labeled as solution (2).
(3) TPB (60 mg) was dispersed in a solution containing 60mL of water and 3mL of Hac (acetic acid) and labeled as solution (3), and solution (3) was added to solution (2), followed by stirring the mixed solution at room temperature for 24 hours.
The mechanism is shown in figure 1, and the enzyme exists in the whole framework of the COFs and becomes a part of the COFs, so that the enzyme load stability is ensured, the protection of the enzyme is better realized, and the whole load step suddenly changes more simply and conveniently by one-pot synthesis; the synthesis process of the enzyme has mild condition and uses water as a solvent, so that the activity of the enzyme is well maintained, and the pollution of the organic reagent to the environment can be reduced.
The HRP@COF prepared in this example is shown in FIG. 2. FIG. 3 shows that the HRP@COF prepared in this example is nano-sized. Since HRP contains iron element, the position distribution of iron element can be regarded as the position distribution of HRP on COF, and as can be seen from fig. 4, HRP is uniformly loaded on COF.
The crystals obtained were first left to stand in a solution of sodium dodecyl sulfate SDS (0.2 g/2ml deionized water) at 70℃for 10 minutes, and the free enzymes on the crystal surfaces were washed off. Three centrifugation/washing cycles were then performed in water. The activity of horseradish peroxidase is determined by measuring the decomposition rate of hydrogen peroxide using pyrogallol as hydrogen donor, which can be converted into yellow product red gallic acid. In a typical assay, solution A contains 76 μLKH 2 PO 3 (100mm,pH6.0)、38μLH 2 O 2 (5% w/w in deionized water), 76. Mu.L of pyrogallol (5% w/w in deionized water), and 1.8ml of PBS buffer (pH 7.4). The crystals were added to solution A and the absorbance of the solution was monitored at 420nm in 30s increments using an ultraviolet-visible spectrophotometer. In the case of enzyme activity measurement using free HRP, the amount of free enzyme added to solution A was adjusted to be equal to the amount of enzyme loaded into ZIF-8/HRP, and the loading efficiency was determined. As shown in FIG. 5 (D), free HRP enzyme and simple covalent-bond loading were achieved in a proteolytic agent, boiling water (100 ℃) and boiling DMF (153 ℃)The enzyme on COFs had been severely inactivated, but the enzyme-loaded COF obtained in this example still remained extremely reactive, indicating that COFs had excellent protection for the enzyme.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. The preparation process of covalent organic frame in-situ packed immobilized enzyme features that 2, 5-dimethoxy-1, 4-dicarboxaldehyde and horseradish peroxidase are mixed in water, and then 1,3, 5-tris (4-amino phenyl) benzene aqua containing acetic acid is added and reacted via stirring at room temperature for 18-36 hr.
2. The method for preparing the immobilized enzyme encapsulated in situ by the covalent organic framework according to claim 1, wherein the molar ratio of 2, 5-dimethoxy-1, 4-dicarboxybenzene to 1,3, 5-tris (4-aminophenyl) benzene is 2.50-3.50:2.
3. The method for preparing the immobilized enzyme encapsulated in situ by the covalent organic framework according to claim 2, wherein the molar ratio of 2, 5-dimethoxy-1, 4-dicarboxybenzene to 1,3, 5-tris (4-aminophenyl) benzene is 2.50-3.00:2, preferably 2.80-2.90:2.
4. The method for preparing the immobilized enzyme in-situ encapsulated by the covalent organic framework according to claim 1, wherein the mass ratio of the 2, 5-dimethoxy-1, 4-dicarboxyl benzene to the horseradish peroxidase is 1.5-2.0:1; preferably 1.5 to 1.8:1.
5. The method for preparing the immobilized enzyme in situ encapsulated by the covalent organic framework according to claim 1, wherein the ratio of the addition amount of 1,3, 5-tris (4-aminophenyl) benzene to acetic acid in the aqueous solution of 1,3, 5-tris (4-aminophenyl) benzene is 15-25:1, g: l is; preferably 18 to 22:1, g: l.
6. An immobilized enzyme encapsulated in situ by a covalent organic framework, characterized in that it is obtained by the preparation method according to any one of claims 1 to 5.
7. Use of the immobilized enzyme of claim 6 encapsulated in situ by a covalent organic framework for detecting peroxides.
8. The use according to claim 7, wherein the peroxide is hydrogen peroxide.
9. A test kit comprising the immobilized enzyme and the chromogenic substrate encapsulated in situ by a covalent organic framework of claim 6.
10. The test kit of claim 9, wherein the chromogenic substrate is 3,3', 5' -tetramethylbenzidine.
CN202310534064.9A 2023-05-10 2023-05-10 Immobilized enzyme encapsulated in situ by covalent organic frameworks, preparation method and application thereof Pending CN116622693A (en)

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