CN114505070B - Porous nano-enzyme, porous nano-enzyme crystal, preparation method and application thereof - Google Patents
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- CN114505070B CN114505070B CN202210344440.3A CN202210344440A CN114505070B CN 114505070 B CN114505070 B CN 114505070B CN 202210344440 A CN202210344440 A CN 202210344440A CN 114505070 B CN114505070 B CN 114505070B
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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Abstract
The invention relates to a porous nano enzyme, a porous nano enzyme crystal, a preparation method and application thereof, and relates to the field of catalysts. The porous nano-enzyme is self-supporting porous nano-enzyme (PNEs), has the advantages of light weight, flexibility, controllable shape, good stability and mechanical property and high catalytic efficiency, and the PNEs can be easily converted into porous nano-enzyme Crystals (CPNEs) through high-temperature (combustion) treatment and can be an excellent bionic system and an advanced functional material-coating after simple one-step impregnation.
Description
Technical Field
The invention relates to the field of catalysts, in particular to a porous nano-enzyme, a preparation method of the porous nano-enzyme, a porous nano-enzyme crystal, a preparation method of the porous nano-enzyme crystal, a porous nano-enzyme compound and application of the porous nano-enzyme and the porous nano-enzyme compound as a catalyst.
Background
The novel biological material (nano enzyme) with enzyme activity is considered as an excellent substitute for natural enzyme because of the advantages of adjustable catalytic activity, high stability, low cost, easy mass production and the like. The unique characteristic enables the nano-enzyme to have wide application prospect in various fields, in particular to nano-enzyme, biological sensor, disease diagnosis and treatment. Rare earth-based nanomaterials are becoming economically viable nanoenzymes due to their unique catalytic activity for a range of important biocatalytic reactions.
Cerium oxide (CeO) 2 ) As a rare earth nanoenzyme that mimics the structure and function of natural enzymes, a series of applications in the biomedical field have received a great deal of attention in recent years. However, cerium oxide-based nanoezymes generally cannot exhibit high catalytic performance due to poor stability, low recyclability, and loss of a specific microenvironment around the active site under complex conditions. Furthermore, the enzyme-like activity decreases sharply or even disappears when approaching neutral physiological conditions. Thus, it remains a great challenge to design multifunctional nanoenzymes with unique features, customizable functionality, excellent catalytic and therapeutic properties under physiological conditions.
Disclosure of Invention
Object of the Invention
The invention aims at providing a porous nano-enzyme, a preparation method of the porous nano-enzyme, a porous nano-enzyme crystal, a preparation method of the porous nano-enzyme crystal, a porous nano-enzyme compound and application of the porous nano-enzyme and the porous nano-enzyme compound as a catalyst.
The invention designs a huge 3D ultra-light porous serial nano enzyme library with an amorphous to high-crystalline quality structure by combining protein chains with metal ions, the preparation of the porous nano enzyme does not need any harsh condition, the porous nano enzyme can be carried out under physiological conditions (room temperature and neutral aqueous solution), the porous nano enzyme is self-supporting Porous Nano Enzyme (PNEs), and the porous nano enzyme has the advantages of light weight, flexibility, controllable shape, good stability and mechanical property and high catalytic efficiency, and the PNEs can be easily converted into porous nano enzyme Crystals (CPNEs) through high-temperature (combustion) treatment and can be easily converted into an excellent bionic system and advanced functional material-coating after simple one-step impregnation.
Solution scheme
In order to achieve the above purpose, the present invention provides the following technical solutions:
in a first aspect, the present invention provides a porous nano-enzyme, which is a protein-metal foam material formed by self-assembling a metal ion and protein conjugate under the action of sodium borohydride, and optionally, the metal ion and protein conjugate is formed by combining a metal ion and a protein.
Further, the metal ion includes Ce 3+ 、Ag + 、Ca 2+ 、Sr 2+ 、Cr 3+ 、Zn 2+ 、In 3+ 、Cu 2+ 、Fe 3+ 、Sn 4+ 、Cd 2+ 、Pb 2+ 、Co 2+ 、Mn 2+ 、Ni 2+ 、Ru 3+ 、Rh 3+ 、Pd 2+ 、Pt 3+ 、Au 3+ 、Hg 2+ 、Bi 3+ 、La 3+ Any one or more of the following; the metal ion is preferably Ce 3+ 、Ag + 、Ca 2+ Any one of them.
Further, the protein comprises any one or more of bovine serum albumin, lysozyme, lactoferrin, insulin, alpha-lactalbumin, human serum albumin, fibrinogen, betA-Amyloid, abeta peptide, prion protein, alpha-synuclein, cystatin C, huntingtin, immunoglobulin light chain, whey albumin, beta-lactoglobulin, ribonuclease A, cytochrome C, alphA-Amylase, horseradish peroxidase, pepsin, myoglobin, collagen, keratin, soy protein, lactoferrin, hemoglobin, DNA polymerase, casein, trypsin, chymotrypsin, thyroglobulin, transferrin, fibrinogen, goat serum, fetal calf serum, mouse serum, immunoglobulin, milk protein, ovalbumin, canavanin, fish skin collagen, superoxide dismutase, pancreatic lipase, laccase, histone, collagenase, cellulase, gluten, mucin, transglutaminase, beta-galactosidase; the protein is preferably Bovine Serum Albumin (BSA) or lysozyme.
Further, 0.01 to 0.5mol of metal ion is bound per 2 to 60g of protein. Alternatively, 0.05 to 0.5mol of metal ion is bound per 10 to 60g of protein; alternatively, 0.1 to 0.5mol of metal ion is bound per 30 to 60g of protein.
Further, 0.05mol of sodium borohydride is used for every 2 to 60g of protein. Alternatively, 0.05mol of sodium borohydride per 10-60 g of protein; alternatively, 0.05mol of sodium borohydride per 30-60 g of protein.
Further, the protein is a commercially available protein that unfolds upon interaction with a metal ion.
Further, the volume weight of the porous nano-enzyme is 78.4-196N/m 3 。
Further, the porous nano-enzyme has catalytic activity at pH 3-11, optionally high catalytic activity at pH 5-9.
Further, the porous nano-enzyme has high catalytic activity at 0-80 ℃, optionally 10-45 ℃.
High catalytic activity means that the catalytic efficiency reaches more than 80%.
In a second aspect, a method for preparing a porous nanoenzyme is provided, comprising: combining metal ions and protein in the solution, adding sodium borohydride to form porous protein-metal foam material, and freeze drying to obtain the porous nanometer enzyme.
Further, the method comprises the steps of: mixing and incubating protein buffer solution and metal ion buffer solution, and rapidly adding precooled NaBH after incubation 4 The aqueous solution is reacted at room temperature, and then the foam generated in a few seconds is freeze-dried to obtain the porous nano-enzyme.
Alternatively, the mixed incubation conditions are: 2-60 mg/mL protein buffer solution is mixed with 0.01-0.5M metal ion buffer solution, and incubated for 1-10 minutes; optionally, 20-60 mg/mL protein buffer is mixed with an equal volume of 0.05-0.5M metal ion buffer; optionally, the protein buffer is mixed with the metal ion buffer in equal volumes;
alternatively, naBH added 4 The concentration of the aqueous solution was 0.05M.
Alternatively, naBH 4 The aqueous solution is freshly preparedIs a solution of (a) and (b).
Optionally freeze drying for 12-28 h; the freeze drying time is optionally 24h.
Alternatively, the specific method may be: 2-60 mg/mL (optionally 20-60 mg/mL) of protein buffer solution (pH 7.2) is mixed with 0.01-0.5M (optionally 0.05-0.5M) of metal ion buffer solution, after incubation for 1-10 min, freshly prepared ice-cold 0.05M NaBH is added rapidly 4 The aqueous solution is reacted at room temperature, and then the foam generated in a few seconds is frozen and dried for 24 hours, so that the porous nano-enzyme can be obtained. Alternatively, the protein buffer is mixed with the metal ion buffer in equal volumes.
A preferred embodiment may be: the preparation method comprises the following steps: 60mg/mL protein buffer (pH 7.2) was mixed with an equal volume of 0.37M metal ion buffer, incubated for 1-10 min, and freshly prepared ice-cold 0.05M NaBH was added rapidly 4 The aqueous solution reacts at room temperature, and then foam generated in a plurality of seconds is freeze-dried for 24 hours to obtain the porous nano-enzyme; optionally, the metal ion buffer is Ce 3+ And (3) a buffer solution.
Further, the buffer solution in the protein buffer solution or the metal ion buffer solution adopts one or more of HEPES, tris (Tris (hydroxymethyl aminomethane)), citric acid-disodium hydrogen phosphate, ammoniA-Ammonium chloride buffer solution, acetic acid-sodium acetate buffer solution and phosphate buffer solution.
In a third aspect, a porous nano-enzyme crystal is provided, which is formed by burning a porous nano-enzyme in air, wherein the porous nano-enzyme is the porous nano-enzyme according to the first aspect or the porous nano-enzyme prepared by the preparation method according to the second aspect.
This is because the porous nano-enzyme has a structure that the content of organic protein is greatly reduced and the crystal structure is greatly increased after combustion. The porous nano-enzyme has the characteristic of nano-enzyme before combustion, but the porous nano-enzyme crystal obtained after combustion shows better chemical and thermal stability, and the specific surface area of the nano-enzyme after combustion is larger, so that the catalytic active center is more.
In a fourth aspect, a method for preparing a porous nano-enzyme crystal is provided, comprising: the porous nano enzyme prepared by the porous nano enzyme of the first aspect or the porous nano enzyme prepared by the preparation method of the second aspect is burnt in air to obtain porous nano enzyme crystals, and optionally, the burning time is 10-30 min.
Alternatively, the porous nano-enzyme is directly combusted in air for 10-30 minutes to obtain the porous nano-enzyme crystal.
Alternatively, the combustion temperature may be 400-700 ℃.
In a fifth aspect, there is provided a porous nano-enzyme complex comprising the porous nano-enzyme of the first aspect, or the porous nano-enzyme prepared by the preparation method of the second aspect, or the porous nano-enzyme crystal of the third aspect and the porous nano-enzyme crystal prepared by the preparation method of the fourth aspect, and a functional molecule supported on the porous nano-enzyme crystal carrier.
Further, the functional molecules comprise one or more of catalytic molecules, drug molecules, photosensitizers, small-molecule fluorescent dyes), toluidine blue O, amyloid binding molecules and hydrophobic fluorescent probes, optionally, the functional molecules comprise one or more of Prussian blue, tetracycline, methylene blue, rhodamine 6G, acridine orange, toluidine blue O, congo red, thioflavine T and 8-anilino-1-naphthalene sulfonate, and optionally, the functional molecules are loaded on the porous nano enzyme by a one-step soaking method.
In a sixth aspect, there is provided a porous nano-enzyme according to the first aspect, or a porous nano-enzyme prepared by the preparation method according to the second aspect, or a porous nano-enzyme crystal according to the third aspect, or a porous nano-enzyme crystal prepared by the preparation method according to the fourth aspect, or a porous nano-enzyme complex according to the fifth aspect, for use as a catalyst, optionally the catalyst comprising a bio-enzyme catalyst or a photocatalyst.
The technical scheme adopted by the invention is as follows: binding to proteins using metal ions and being bound by sodium borohydride (NaBH 4 ) In situ reductive assembly to form porous protein-metal foam (porous nanoenzyme). The porous nano enzyme can convert metal into metal oxide after being directly combusted in air,thereby forming a 3D protein-metal oxide foam (porous nano-enzyme crystals).
Advantageous effects
(1) The invention designs a huge 3D ultra-light and porous serial nano enzyme library with an amorphous to high-crystallization quality structure by combining protein chains with metal ions; the preparation process does not require any harsh conditions and can be carried out under physiological conditions (room temperature and neutral aqueous solution). The porous nano-enzyme is self-supporting porous nano-enzyme (PNEs) which is light, flexible, controllable in shape, good in stability and mechanical property, high in catalytic efficiency and possibly suitable for large-scale application. PMFs can be easily converted into porous nano-enzyme Crystals (CPNEs) by high temperature treatment, and can also become an excellent biomimetic system and advanced functional material-coating after simple one-step impregnation.
(2) The inventors of the present invention have unexpectedly found that NaBH is employed 4 The method can promote the protein to generate foam materials, the material is easier to recycle compared with the nano enzyme materials in micro-nano particle shape, and the generated foam materials have larger specific surface area and more contactable active sites, so that the catalytic efficiency is improved.
(3) The porous nano-enzyme, the porous nano-enzyme crystal or the porous nano-enzyme compound can be applied to the fields of biological enzyme, photocatalysis and the like.
Drawings
One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
FIG. 1 is a scanning electron microscope picture of porous nanoenzymes of examples 1 to 8 of the present invention, wherein (a) is lysozyme-Ce porous nanoenzyme of example 1; (b) an alphA-Amylase-Ce porous nanoenzyme of example 2; (c) BSA-Ce porous nano enzyme of example 3; (d) A catalase-Ce porous nanoenzyme of example 4; (e) ovalbumin-Ce porous nano-enzyme of example 5; (f) hemoglobin-Ce porous nanoenzyme of example 6; (g) whey protein-Ce porous nanoenzyme of example 7; (h) an egg white protein-Ce porous nano enzyme of example 8.
FIG. 2 is a transmission electron microscope picture of the porous nano-enzyme of example 3 of the present invention; wherein c and d are pictures under different multiples, which indicates that the porous nano-enzyme consists of protein-coated cerium oxide nano-particles;
FIG. 3 is a transmission electron microscope image of the porous nano-enzyme crystal and elemental analysis of example 16 of the present invention; the upper graph (larger graph) is an electron microscope image of corresponding element analysis represented by C, O, ce, N, S, and the overlay is a superposition graph of five elements;
FIG. 4 is an X-ray diffraction pattern of a porous nano-enzyme crystal of example 16 of the present invention;
FIG. 5 is a graph showing density of porous nano-enzyme as a function of BSA concentration prepared according to the method of example 3 of the present invention.
FIG. 6 is a thermogravimetric analysis and specific surface area characterization of porous nanoenzymes and porous nanoenzyme crystals; wherein a, b are the thermogravimetric analyses of the porous nano-enzyme of example 3 and the porous nano-enzyme crystal of example 16, respectively; c, d are nitrogen adsorption and desorption isotherms of the porous nano-enzyme of example 3 and the porous nano-enzyme crystal of example 16, respectively, and the inset shows pore volume distributions of the porous nano-enzyme and the porous nano-enzyme crystal, respectively.
FIG. 7 is a scanning electron microscope image of the porous nano-enzyme crystal of example 16 of the present invention after loading different functional molecules; wherein a-i are rhodamine 6G, congo red, prussian blue, thioflavine T, tetracycline, toluidine blue O, methylene blue, acridine orange and 8-anilino-1-naphthalene sulfonate respectively.
FIG. 8 is a method of soaking porous nanoenzyme in H according to example 18 of the present invention 2 O 2 Catalytic decomposition of solutions to O at different times 2 Is a photograph of the condition of (2).
FIG. 9 is a method of soaking porous nano-enzyme into H according to example 18 of the present invention 2 O 2 Solution catalyzed decomposition to O 2 The amount varies with time.
FIG. 10 shows the reusability of the porous nanoenzyme of example 3 of the present invention.
FIG. 11 shows the catalytic performance of the porous nanoenzyme of example 3 of the present invention at different pH conditions.
FIG. 12 shows the catalytic performance of the porous nanoenzyme of example 3 of the present invention at different temperatures.
FIG. 13 is a compressive stress-strain curve for a porous nanoenzyme of example 3 of the present invention at 30% compression; the embedded image is a field emission scanning electron microscope image of the foam after 10 cycles of compression.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.
In addition, numerous specific details are set forth in the following description in order to provide a better illustration of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.
The present invention will be described in detail below.
Reagents used in the examples of the present invention, e.g., BSA, lysozyme, alphA-Amylase, HEPES buffer, ceCl 3 ·7H 2 O and the like are all commercial products.
Example 1
60mg/mL lysozyme HEPES (10 mM) buffer solution (pH 7.2) with an equal volume of 0.37M CeCl 3 ·7H 2 After incubation at room temperature for 2 min with O HEPES (10 mM) buffer solution, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 The aqueous solution is reacted at room temperature, and then the foam generated in a few seconds is freeze-dried for 24 hours, so that the lysozyme-Ce porous nano enzyme can be obtained, as shown in figure 1 a.
Example 2
HEPES (10 mM) buffer (pH 7.2) of 60mg/mL alphA-Amylase with an equal volume of 0.37M CeCl 3 ·7H 2 After incubation at room temperature for 2 min with O HEPES (10 mM) buffer solution, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 The aqueous solution is reacted at room temperature, and then the foam generated within a few seconds is freeze-dried for 24 hours, so that the alphA-Amylase-Ce porous nano enzyme can be obtained, as shown in figure 1 b.
Example 3
HEPES (10 mM) buffer (pH 7.2) at 60mg/mL BSA with an equal volume of 0.37M CeCl 3 ·7H 2 After incubation at room temperature for 2 min with O HEPES (10 mM) buffer solution, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 The aqueous solution is reacted at room temperature, and then the foam generated in a few seconds is freeze-dried for 24 hours, so that the BSA-Ce porous nano enzyme can be obtained, as shown in figure 1c and figure 2.
Example 4
HEPES (10 mM) buffer (pH 7.2) at 60mg/mL catalase with an equal volume of 0.37M CeCl 3 ·7H 2 After incubation at room temperature for 2 min with O HEPES (10 mM) buffer solution, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 The aqueous solution is reacted at room temperature, and then the foam generated in a few seconds is freeze-dried for 24 hours to obtain the catalase-Ce porous nano enzyme, as shown in figure 1 d.
Example 5
HEPES (10 mM) buffer (pH 7.2) at 60mg/mL ovalbumin with an equal volume of 0.37M CeCl 3 ·7H 2 After incubation at room temperature for 2 min with O HEPES (10 mM) buffer solution, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 The aqueous solution is reacted at room temperature, and then the foam generated in a few seconds is freeze-dried for 24 hours, so that the ovalbumin-Ce porous nano enzyme can be obtained, as shown in figure 1 e.
Example 6
HEPES (10 mM) buffer (pH 7.2) at 60mg/mL hemoglobin with an equal volume of 0.37M CeCl 3 ·7H 2 After incubation at room temperature for 2 min with O HEPES (10 mM) buffer solution, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 The aqueous solution is reacted at room temperature, and then the foam generated within a few seconds is freeze-dried for 24 hours, so that the hemoglobin-Ce porous nano enzyme can be obtained, as shown in figure 1 f.
Example 7
HEPES (10 mM) buffer (pH 7.2) at 60mg/mL whey protein with an equal volume of 0.37M CeCl 3 ·7H 2 After incubation at room temperature for 2 min with O HEPES (10 mM) buffer solution, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 The aqueous solution is reacted at room temperature, and then the foam generated in a few seconds is frozen and dried for 24 hours, so that the whey protein-Ce porous nano enzyme can be obtained, and the whey protein-Ce porous nano enzyme is shown in figure 1 g.
Example 8
HEPES (10 mM) buffer (pH 7.2) with 60mg/mL egg white protein and an equal volume of 0.37M CeCl 3 ·7H 2 After incubation at room temperature for 2 min with O HEPES (10 mM) buffer solution, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 And (3) reacting at room temperature after the aqueous solution is prepared, and freeze-drying foam generated within a few seconds for 24 hours to obtain the egg white protein-Ce porous nano enzyme, as shown in figure 1 h.
According to FIG. 1, the results of the electron microscope scans of the porous proteases of examples 1 to 8 show that the protein-Ce nanoenzymes prepared from different proteins have different nanostructures.
Example 9
HEPES (10 mM) buffer (pH 7.2) at 30mg/mL BSA with an equal volume of 0.37M CeCl 3 ·7H 2 After incubation at room temperature for 2 min with O HEPES (10 mM) buffer solution, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 And (3) reacting the aqueous solution at room temperature, and freeze-drying foam generated within a few seconds for 24 hours to obtain the BSA-Ce porous nano enzyme.
Example 10
HEPES (10 mM) buffer solution (pH 7) of 10mg/mL BSA2) and equal volume of 0.37M CeCl 3 ·7H 2 After incubation at room temperature for 2 min with O HEPES (10 mM) buffer solution, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 And (3) reacting the aqueous solution at room temperature, and freeze-drying foam generated within a few seconds for 24 hours to obtain the BSA-Ce porous nano enzyme.
Example 11
HEPES (10 mM) buffer (pH 7.2) at 60mg/mL BSA with an equal volume of 0.1M CeCl 3 ·7H 2 After incubation at room temperature for 2 min with O HEPES (10 mM) buffer solution, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 And (3) reacting the aqueous solution at room temperature, and freeze-drying foam generated within a few seconds for 24 hours to obtain the BSA-Ce porous nano enzyme.
Example 12
HEPES (10 mM) buffer (pH 7.2) at 60mg/mL BSA with an equal volume of 0.5M CeCl 3 ·7H 2 After incubation at room temperature for 2 min with O HEPES (10 mM) buffer solution, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 And (3) reacting the aqueous solution at room temperature, and freeze-drying foam generated within a few seconds for 24 hours to obtain the BSA-Ce porous nano enzyme.
Example 13
60mg/mL BSAHEPES (10 mM) buffer (pH 7.2) with an equal volume of 0.37M AgNO 3 HEPES (10 mM) buffer solution was mixed and after incubation at room temperature for 2 minutes, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 And (3) reacting the aqueous solution at room temperature, and freeze-drying foam generated within a few seconds for 24 hours to obtain the BSA-Ag porous nano-enzyme.
Example 14
60mg/mL BSA HEPES (10 mM) buffer solution (pH 7.2) with an equal volume of 0.37M CaCl 2 ·5H 2 After incubation at room temperature for 2 min with O HEPES (10 mM) buffer solution, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 And (3) reacting the aqueous solution at room temperature, and freeze-drying foam generated within a few seconds for 24 hours to obtain the BSA-Ca porous nano-enzyme.
Example 15
60mg/mL BSA HEPES (10 mM) buffer solution (pH 7.2) and an equal volume0.37M CoCl of (C) 2 ·6H 2 After incubation at room temperature for 2 min with O HEPES (10 mM) buffer solution, freshly prepared ice-cold 0.05M NaBH was added rapidly 4 And (3) reacting the aqueous solution at room temperature, and freeze-drying foam generated within a few seconds for 24 hours to obtain the BSA-Co porous nano-enzyme.
Example 16
The porous nano enzyme of example 3 was directly burned in air for 20 minutes to obtain a porous nano enzyme crystal.
The inventors carried out transmission electron microscope analysis and X-ray diffraction patterns on the porous nano-enzyme crystal of the example, and the results are shown in FIG. 3 and FIG. 4, and the results show that the porous nano-enzyme crystal contains a large amount of CeO 2 A crystal structure.
The inventors also performed thermogravimetric analysis and specific surface area characterization on the porous nano-enzyme of example 3 and the porous nano-enzyme crystal of example 16, the heating rate of the thermogravimetric analysis was 10 ℃/min, and as a result, as shown in fig. 6, fig. 6a shows that the weight loss of the porous nano-enzyme of example 3 is about 70% in the thermogravimetric analysis experiment, mainly the weight lost after the thermal carbonization of protein; while the results in FIG. 6b show that the weight loss of the porous nano-enzyme crystals of example 16 is about 5%, indicating that the protein content in the porous nano-enzyme crystals is extremely low and the metal content is relatively increased. The results in FIGS. 6 c-6 d show that the specific surface area of the porous nano-enzyme crystals of example 16 is increased.
Example 17
The porous nano enzyme crystal of example 16 is respectively soaked into rhodamine 6G, congo red, prussian blue, thioflavin T, tetracycline, toluidine blue O, methylene blue, acridine orange and 8-anilino-1-naphthalene sulfonate solution for 0.5 hours, so that the porous nano enzyme crystal loaded with various functional molecules can be obtained, and a scanning electron microscope is respectively carried out, and the result is shown as figure 7, and the result shows that the porous nano enzyme crystal of the application can well load various functional molecules.
Example 18
Soaking the porous nanoenzyme BSA-Ce foam of example 3 into H 2 O 2 H is caused to be in solution 2 O 2 Decomposition to produce O 2 As a result, as shown in fig. 8,fig. 9 shows the same.
FIG. 8 illustrates that the porous nanoenzyme BSA-Ce foam can exhibit good catalytic performance over time.
FIG. 9 illustrates that porous nano-enzyme BSA-Ce foam material exhibits good catalytic performance relative to BSA, buffers, etc.
The inventors have also studied the effect of the change in BSA concentration on the volume weight of the produced porous nano-enzyme, as shown in FIG. 5, and the results show that the density of the porous nano-enzyme produced by using HEPES buffer solution of 2-60 mg/mL BSA is 8-16mg/cm as the concentration of BSA is increased gradually 3 The porous nano enzyme is porous, has small density and light weight.
The inventors studied the reproducible practicality of the porous nano-enzyme, the catalytic performance at different pH, and the catalytic performance at different temperatures using the porous nano-enzyme BSA-Ce foam material of example 3, and the results are shown in FIGS. 10 to 12.
The method for testing the catalytic performance comprises the following steps: the testing instrument is a portable dissolved oxygen monitor, H 2 O 2 At a concentration of 20mM, from 10min after the porous nano-enzyme was put in to decompose to produce O 2 The highest amount of (2) is defined as 100% relative activity.
FIG. 10 illustrates that the porous nanoenzyme has good catalytic performance after 20 cycles, and has good repeatability and practicability and is easy to recycle. The cyclic test method comprises the following steps: after each reaction, the reaction solution is centrifuged, the centrifugally collected porous nano-enzyme is washed three times by water and ethanol, and then dried for 12 hours at 80 ℃, the porous nano-enzyme is recovered, the recovered porous nano-enzyme is mixed with fresh centrifugal PBS buffer solution (pH 7.1), and the mixture is added into H 2 O 2 Carrying out the next catalytic reaction in the solution; and (3) carrying out centrifugation, washing, recovery and catalytic reaction for 20 times, and verifying the catalytic activity of the porous nano enzyme after each reaction recovery.
FIG. 11 shows that the porous nano-enzyme has catalytic activity in the pH range of 3-11, and the catalytic performance at pH 5-9 can be maintained above 80%, especially the catalytic performance is maintained almost 100% under the physiological condition of pH7, illustrating the porous nano-enzyme of the present inventionThe nano-enzyme has good catalytic performance under physiological conditions. Whereas conventional CeO 2 The catalyst (available from Shanghai Michelia Biochemical technologies Co., ltd.) had its enzyme-like activity disappeared at near physiological pH.
FIG. 12 illustrates that the porous nano-enzyme shows catalytic activity at 20-80 ℃, the catalytic efficiency at 20-55 ℃ can be maintained at more than 70%, and the catalytic efficiency at 20-45 ℃ can be maintained at more than 90%, indicating that the porous nano-enzyme shows catalytic performance in a wider temperature range, especially shows high catalytic efficiency at 20-45 ℃.
The inventors have also studied the mechanical stability of the porous nano-enzyme, taking the porous nano-enzyme of example 3 as an example, and carrying out compression pressure-strain test by an instrument, and the result shows that the porous nano-enzyme of example 3 still has no rupture under the condition of 30% of compression, and the field emission scanning electron microscope picture of the foam material after 10 cycles of compression is shown as an embedded graph of fig. 13, and the porous nano-enzyme has good form, which indicates that the porous nano-enzyme of the invention has good mechanical property.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (34)
1. A porous nano enzyme is a protein-metal foam material formed by self-assembling a metal ion and protein conjugate under the action of sodium borohydride, wherein the metal ion and protein conjugate is formed by combining a metal ion and protein;
the metal ion includes Ce 3+ 、 Ag + 、 Ca 2+ 、 Sr 2+ 、 Cr 3+ 、 Zn 2+ 、 In 3+ 、Cu 2+ 、 Fe 3+ 、 Sn 4+ 、 Cd 2+ 、 Pb 2+ 、 Co 2+ 、 Mn 2+ 、 Ni 2+ 、 Ru 3+ 、 Rh 3+ 、 Pd 2+ 、 Pt 3+ 、 Au 3+ 、 Hg 2+ 、 Bi 3+ 、 La 3+ Any one or more of the following;
the protein comprises any one or more of bovine serum albumin, lysozyme, lactoferrin, insulin, alpha-lactalbumin, human serum albumin, fibrinogen, betA-Amyloid protein, A beta peptide, alpha-synuclein, cystatin C, huntingtin, immunoglobulin light chain, beta-lactoglobulin, ribonuclease A, cytochrome C, alphA-Amylase, horseradish peroxidase, pepsin, myoglobin, collagen, keratin, soy protein, hemoglobin, DNA polymerase, casein, trypsin, chymotrypsin, thyroglobulin, transferrin, fibrinogen, goat serum, mouse serum, immunoglobulin, lactoprotein, ovalbumin, canavalin, fish skin collagen, superoxide dismutase, pancreatic lipase, laccase, histone, collagenase, cellulase, glutelin, mucin, transglutaminase and beta-galactosidase;
binding 0.01-0.5 mol of metal ions to every 2-60 g of protein;
the volume weight of the porous nano enzyme is 78.4-196N/m 3 ；
Mixing and incubating protein buffer solution and metal ion buffer solution, and rapidly adding precooled NaBH after incubation 4 The aqueous solution is reacted at room temperature, and then the foam generated in a few seconds is freeze-dried to obtain the porous nano-enzyme.
2. The porous nanoenzyme of claim 1 wherein the metal ion is Ce 3+ 、Ag + 、Ca 2+ Any one of them;
and/or the protein is any one or more of bovine serum albumin and lysozyme.
3. The porous nanoenzyme of claim 1 wherein 0.05 to 0.5mol of metal ion is bound per 10 to 60g of protein.
4. The porous nanoenzyme of claim 1, wherein 0.05 to 0.5mol of metal ion is bound per 30 to 60g of protein.
5. The porous nanoenzyme of claim 1, wherein 0.05mol of sodium borohydride is added per 2-60 g of protein.
6. The porous nano-enzyme according to claim 1, wherein 0.05mol of sodium borohydride is added per 10-60 g of protein.
7. The porous nano-enzyme according to claim 1, wherein 0.05mol of sodium borohydride is added per 30-60 g of protein.
8. The porous nanoenzyme of any one of claims 1-7, wherein said porous nanoenzyme has catalytic activity at pH 3-11.
9. The porous nanoenzyme of any one of claims 1-7, wherein the porous nanoenzyme has high catalytic activity at pH 5-9.
10. The porous nanoenzyme of any one of claims 1 to 7, wherein the porous nanoenzyme has high catalytic activity at 0-80 ℃.
11. The porous nanoenzyme of any one of claims 1 to 7, wherein the porous nanoenzyme has high catalytic activity at 10-45 ℃.
12. A method of preparing a porous nanoenzyme according to any one of claims 1 to 11, comprising: mixing metal ions and protein in the solution, adding sodium borohydride to form porous protein-metal foam material, and freeze drying to obtain the porous nanometer enzyme.
13. The preparation method according to claim 12, characterized by comprising: mixing and incubating protein buffer solution and metal ion buffer solution, and rapidly adding precooled NaBH after incubation 4 The aqueous solution is reacted at room temperature, and then the foam generated in a few seconds is freeze-dried to obtain the porous nano-enzyme.
14. The method according to claim 12, wherein the mixed incubation conditions are: 2-60 mg/mL protein buffer solution is mixed with 0.01-0.5M metal ion buffer solution, and incubated for 1-10 minutes.
15. The method of claim 12, wherein the protein buffer is mixed with the metal ion buffer in equal volumes.
16. The process according to claim 12, wherein NaBH is added 4 The concentration of the aqueous solution was 0.05M.
17. The process according to claim 16, wherein NaBH 4 The aqueous solution was freshly prepared.
18. The method according to claim 12, wherein the freeze-drying time is 12 to 28 hours.
19. The process according to claim 12, wherein the lyophilization time is 24 hours.
20. The preparation method according to claim 12, wherein the buffer in the protein buffer or the metal ion buffer is one or more of HEPES buffer, tris, citric acid-disodium hydrogen phosphate, ammoniA-Ammonium chloride buffer, acetic acid-sodium acetate buffer, and phosphate buffer.
21. According toThe method of claim 12, wherein the preparing step comprises: 2-60 mg/mL protein buffer solution and 0.01-0.5M metal ion buffer solution are mixed, after incubation for 1-10 minutes, freshly prepared ice-cold 0.05M NaBH is added rapidly 4 The aqueous solution is reacted at room temperature, and then the foam generated in a few seconds is freeze-dried for 24 to h, thus obtaining the porous nano-enzyme.
22. The method of claim 21, wherein the protein buffer has a pH of 7.2.
23. The method of preparing according to claim 22, wherein the preparing step comprises: 60mg/mL protein buffer solution is mixed with 0.37M metal ion buffer solution with equal volume, and after incubation for 1-10 minutes, freshly prepared ice-cooled 0.05M NaBH is added rapidly 4 The aqueous solution is reacted at room temperature, and then the foam generated in a few seconds is freeze-dried for 24 to h, thus obtaining the porous nano-enzyme.
24. The process according to claim 23, wherein the metal ion buffer is Ce 3+ And (3) a buffer solution.
25. A porous nano enzyme crystal, which is characterized in that the porous nano enzyme crystal is formed by burning porous nano enzyme in air, and the porous nano enzyme is prepared by the porous nano enzyme of any one of claims 1 to 11 or the preparation method of any one of claims 12 to 24.
26. A method for preparing a porous nano-enzyme crystal, comprising: burning the porous nano-enzyme according to any one of claims 1 to 11 or the porous nano-enzyme prepared by the preparation method according to any one of claims 12 to 24 in air to obtain the porous nano-enzyme crystal.
27. The method of claim 26, wherein the burning time is 10 to 30 minutes.
28. The method of claim 26, wherein the combustion temperature is 400-700 ℃.
29. A porous nano-enzyme complex comprising the porous nano-enzyme according to any one of claims 1 to 3, or the porous nano-enzyme produced by the production method according to any one of claims 12 to 24, or the porous nano-enzyme crystal according to claim 25, or the porous nano-enzyme crystal produced by the production method according to any one of claims 26 to 28, and a functional molecule supported on the porous nano-enzyme or porous nano-enzyme crystal.
30. The porous nanoenzyme complex of claim 29, wherein said functional molecule comprises one or more of a catalytic molecule, a drug molecule, a photosensitizer, a small fluorescent dye, toluidine blue O, an amyloid-binding molecule, and a hydrophobic fluorescent probe.
31. The porous nanoenzyme complex of claim 29, wherein said functional molecule comprises one or more of prussian blue, tetracycline, methylene blue, rhodamine 6G, acridine orange, toluidine blue O, congo red, thioflavin T, and 8-anilino-1-naphthalene sulfonate.
32. The porous nano-enzyme complex according to claim 29, wherein the functional molecule is supported on the porous nano-enzyme or porous nano-enzyme crystal by a one-step soaking method.
33. Use of a porous nanoenzyme according to any one of claims 1 to 11, or a porous nanoenzyme produced according to any one of claims 12 to 24, or a porous nanoenzyme crystal according to claim 25, or a porous nanoenzyme crystal produced according to any one of claims 26 to 28, or a porous nanoenzyme complex according to any one of claims 29 to 32, as a catalyst.
34. The use of claim 33, the catalyst comprising a photocatalyst.
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