CN110423742B

CN110423742B - Method for realizing enzyme immobilization through magnetic ferritin surface display

Info

Publication number: CN110423742B
Application number: CN201910698944.3A
Authority: CN
Inventors: 张瑜; 董亦馨; 周金花; 戚岚岚; 李迅; 王飞
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2019-07-31
Filing date: 2019-07-31
Publication date: 2022-10-18
Anticipated expiration: 2039-07-31
Also published as: CN110423742A

Abstract

A method for realizing enzyme immobilization by magnetic ferritin surface display is disclosed, which comprises the steps of constructing ferritin recombinant plasmids containing Ecoil short peptides and enzyme recombinant plasmids containing Kcoil short peptides; transforming the constructed recombinant plasmid into an escherichia coli expression strain TOP10 competent cell to obtain a corresponding genetic engineering strain; inoculating the genetically engineered bacteria into an LB culture medium for induced culture, and obtaining purified ferritin and enzyme by utilizing affinity chromatography and size exclusion chromatography; synthesis of Fe in the internal cavity of purified ferritin ₃ O ₄ (ii) a The obtained magnetic ferritin is mixed with enzyme, and magnetic ferritin immobilized enzyme can be formed through the interaction of the Ecoil helical peptide and the Kcoil helical peptide. Compared with the traditional covalent bond mode for realizing enzyme immobilization, the gene fusion mode avoids the damage of the enzyme activity center, obviously improves the effect of surface display enzyme and the enzyme activity, and greatly reduces the cost.

Description

Method for realizing enzyme immobilization through surface display of magnetic ferritin

Technical Field

The invention relates to the technical field of biotechnology and analysis, in particular to a method for realizing enzyme immobilization by surface display of magnetic ferritin.

Background

In industrial production, free enzyme is widely used, however, the free enzyme is not easy to separate after catalytic reaction, so that the free enzyme cannot be recycled and is high in cost, and the bottle neck is effectively solved by an enzyme immobilization technology. The immobilized enzyme has the advantages of low cost, easy control, easy separation from the reaction and reuse, possibly improved thermodynamics and pH stability, and the like. With the development of biotechnology and nanotechnology, immobilization of enzymes on carrier materials with excellent performance has become a new research hotspot, such as silicon nanotubes, carbon nanotube graphene, mesoporous materials, magnetic nanomaterials, and the like. The magnetic nano-immobilized enzyme has the characteristics of high specific surface area, excellent chemical stability, biocompatibility and the like, and the special magnetic property enables the magnetic nano-immobilized enzyme to be endowed with a new function, namely, the magnetic nano-immobilized enzyme can realize directional motion under the action of an external magnetic field, and is easy to separate from a substrate and reuse. Meanwhile, the movement mode and direction of the magnetic material immobilized enzyme are controlled by the change of the external magnetic field, the traditional mechanical stirring mode can be replaced, and the catalytic efficiency of the immobilized enzyme is improved, so that the magnetic nanoparticle immobilized enzyme has wide application prospect. Magnetic nanoparticle magnetic cores are primarily some species containing iron, manganese, cobalt and oxides thereof. Wherein, the preparation of the iron oxide is relatively simple, the toxicity is low, and the application is wider. When the grain size of the prepared magnetic core is less than 20nm, the prepared magnetic core has superparamagnetism, can be instantaneously magnetized under the action of an external magnetic field, and has no remanence after the magnetic field is removed, so the magnetic core is easy to recover and can be dispersed in a reaction system again.

From the relevant research at home and abroad, the immobilized enzyme prepared by different methods by directly using the iron oxide magnetic nanoparticles or various functionalized iron oxide magnetic nanoparticles as carriers has improved operation stability, but the activity of the immobilized enzyme is reduced to different degrees compared with that of free enzyme, and particularly, the activity is reduced to a greater degree in covalent bonding immobilization. However, the method of gene fusion is used to fix the target enzyme on the surface of the protein structure, so that the disadvantage of chemical bonding immobilization can be solved, and the advantage is more obvious if the target enzyme is made to have paramagnetism at the same time.

Ferritin is an iron storage protein whose shell is composed of 24 subunits, typically an H subunit (molecular weight about 21 kD) and an L subunit (molecular weight about 19.5 kD). Ferritin is a cage-like spherical structure with an outer diameter of about 12nm, a cavity with an inner diameter of about 8nm, and an inner iron core composed mainly of hydrated iron oxide (5 Fe) ₂ O ₃ ·9H ₂ O) is present. Ferritin is undoubtedly the first choice for the synthesis of magnetic nanoparticles containing iron oxides due to its own iron storage function. The ferritin has good stability, and can endure the high temperature of 70-80 ℃ and the action of various denaturants under the condition of keeping the structure unchanged. Compared with other nanoparticles, the magnetic ferritin has the following advantages: (1) The particle size is uniform, and the nano particles with good dispersity and uniform particle size can be prepared without additional condition control; (2) The core of the magnetoferritin is Fe ₃ O ₄ The nano particles have superparamagnetism due to the fact that the particle size of the inner shell is about 8nm, and separation of reaction is facilitated; (3) The ferritin has good biocompatibility, and can be fused with other functional genes for co-expression to form a functional nano-carrier. As a carrier of immobilized enzyme, ferritin is not only beneficial to synthesizing magnetic nanoparticles, but also has unique structural advantages that the N end of the subunit points to the outside of the shell-shaped structure, and the self-assembly of ferritin into a 24-polymer shell-shaped structure is not influenced finally through the gene sequence of fusion enzyme at the N end.

Coiled-coil is a generic term for a class of parallel or antiparallel left-handed supercoiled structures formed by two or more right-handed alpha-helices intertwined with each other that are present in a variety of native proteins. Typical coiled coils are based on repeating units of seven amino acid residues in series to form amphipathic right-handed alpha-helical molecules, which are then assembled to form a left-handed supercoiled bundle. With the continuous and deep knowledge of the coiled coil structure, the development and application of the function of the coiled coil are gradually increased, and the coiled coil structure is implemented in the aspects of basic research, industry, medicine and health and the like.

Based on the described advantages of ferritin and coiled coils, coiled coils can be used to display enzyme molecules on the surface of magnetic ferritin molecules for catalysis. Similarly, the mode of the magnetic ferritin nanoparticle immobilized enzyme can also be used for immobilizing other enzyme molecules by only changing the enzyme molecule connected with one helical peptide. The construction of the enzyme immobilization mode has potential application value, namely, the movement mode and direction of the magnetic material immobilized enzyme can be controlled by using the change of an external magnetic field during catalytic reaction, the traditional mechanical stirring mode is replaced, and directional movement can be realized after the reaction, so that the recovery and the reutilization of the enzyme are facilitated.

Disclosure of Invention

The technical problem to be solved is as follows: the invention provides a method for realizing enzyme immobilization by magnetic ferritin surface display, and provides a brand-new and effective immobilization strategy. The invention skillfully utilizes the characteristic that ferritin subunits can form a shell-shaped structure by self assembly in a solution, the beta-glucosidase and the ferritin subunits are immobilized through a pair of coiled coil peptides, and the recombined enzyme interacts with ferritin by utilizing the property that the coiled coil peptides Ecoil and Kcoil can be specifically combined in the solution, so that the enzyme is immobilized on the surface of the ferritin. The magnetic property of ferroferric oxide synthesized in the ferritin inner cavity is utilized to achieve the aim of enzyme recovery.

The technical scheme is as follows: a method for realizing enzyme immobilization by magnetic ferritin surface display comprises the following steps: (1) Constructing ferritin recombinant plasmid containing Ecoil short peptide and enzyme recombinant plasmid containing Kcoil short peptide: designing PCR primers to carry gene sequences coding the Ecoi short peptide and the Kcoil short peptide respectively, and fusing the gene sequences to the C end of a ferritin subunit and the N end of a beta-glucosidase subunit respectively through PCR to construct a ferritin recombinant plasmid containing the Ecoi short peptide and an enzyme recombinant plasmid containing the Kcoil short peptide; (2) Transforming the constructed recombinant plasmid into an escherichia coli expression strain TOP10 competent cell, and culturing at a constant temperature of 37 ℃ overnight to obtain a corresponding genetic engineering strain; (3) Inoculating the genetically engineered bacteria into an LB culture medium for induction culture, and obtaining purified ferritin and enzyme by utilizing affinity chromatography and size exclusion chromatography; synthesis of Fe in the internal cavity of purified ferritin ₃ O ₄ Obtaining magnetic ferritin; (4) Mixing the obtained magnetic ferritin with enzyme, and forming magnetic ferritin immobilized enzyme through the interaction of Ecoil helical peptide and Kcoil helical peptide; (5) The magnetic ferritin immobilized enzyme is adsorbed by a magnetic field, and the enzyme activity and the recovery efficiency are measured.

The amino acid sequence of the Ecoil short peptide is shown in SEQ ID NO. 1.

The amino acid sequence of the Kcoil short peptide is shown in SEQ ID NO. 2.

The PCR primers carrying the encoded Ecoil short peptide are shown in SEQ ID NO.3 and 4.

The PCR primers carrying the encoded Kcoil short peptide are shown in SEQ ID NO.5 and 6.

The above synthesis of Fe in the internal cavity of purified ferritin ₃ O ₄ Comprises the following steps: 2.0mg ferritin dissolved in 50mM Tris-HCl with pH 8.5 is degassed, 12.5mM ammonium ferrous sulfate solution, 4.17mM hydrogen peroxide solution, 100mM sodium hydroxide solution and 300mM sodium citrate solution which are degassed are prepared, the reaction is carried out in a water bath with the temperature of 65 ℃, 1.57mL ammonium ferrous sulfate solution and 1.57mL hydrogen peroxide solution are dropwise added into a reaction device, the pH value of the reaction is controlled to be 8.5 by dropwise adding sodium hydroxide solution, 5min is waited after 50min reaction is finished, 200 mu L sodium citrate solution is added to chelate redundant iron, then the sample is centrifuged at 10,000rpm and 10min, and supernatant is collected.

The magnetic ferritin surface immobilized enzyme is beta-glucosidase.

Has the advantages that: (1) The invention utilizes the property that Ecoil and Kcoil can be specifically combined in a solution, takes magnetic ferritin as a carrier, and fixes beta-glucosidase in a cellulase system on the surface of a synthesized magnetic ferritin shell structure, thereby realizing the magnetic ferritin immobilized enzyme. (2) The particle size is below 20nm, so the magnetic material has superparamagnetism and is easy to utilize the repeated use of the enzyme under the magnetic field condition; (3) Compared with the traditional covalent bond mode for realizing enzyme immobilization, the gene fusion mode avoids the damage of an enzyme activity center, obviously improves the effect of surface display enzyme and enzyme activity, and greatly reduces the cost.

Drawings

FIG. 1 is a schematic diagram showing the design of the magnetic ferritin surface display enzyme of the present invention.

FIG. 2 is a design drawing of the experiment of the present invention. Wherein HE is the English abbreviation of recombinant ferritin Ecoil-ferritin; KG is the English abbreviation for recombinase, kcoil-beta-glucosidase; the design scheme is as follows, ecoil spiral short peptide is fused at the C end of human ferritin, and Kcoil spiral short peptide is fused at the N end of beta-glucosidase;

FIG. 3 is a graph showing the results of the purification of recombinant ferritin Ecoil-ferritin. As shown in the figure, A is the result of polyacrylamide gel electrophoresis (SDS-PAGE) (wherein, lane 1 is after induction, 2 is soluble protein fraction, 3 is insoluble protein fraction, 4 is lysate supernatant, 5 is 250mM imidazole elution, 6 is protein after gel column chromatography purification); b is a graph of the results of size exclusion chromatography;

FIG. 4 is a TEM result of the recombinant ferritin Ecoil-ferritin;

FIG. 5 is a graph showing the purification results of recombinant enzyme Kcoil- β -glucosidase. As shown in the figure, A is a polyacrylamide gel electrophoresis (SDS-PAGE) result graph (wherein 1 is before induction, 2 is after induction, 3 is a soluble protein component, 4 is an insoluble protein component, 5 is lysate supernatant, 6 is after lysis buffer elution, 7 is after wash buffer elution, and 8 is a protein eluted by 250mM imidazole); b is a graph of size exclusion chromatography results;

FIG. 6 is a graph showing the results of the verification of the synthesized magnetic Ecoil-ferritin. As shown, a is the size exclusion chromatogram prior to iron loading; b is the size exclusion chromatogram after loading iron;

FIG. 7 is a graph of the results of polyacrylamide gel electrophoresis (SDS-PAGE) of the interaction of magnetic Ecoil-ferritin and Kcoil- β -glucosidase (binding of Ecoil-ferritin and Kcoil- β -glucosidase is demonstrated after elution in lane 1, wash buffer in lanes 2-4, HE (noHis) protein in lane 5, wash buffer in lanes 6-8, and 250mM imidazole in lane 9);

FIG. 8 shows the results of Native-PAGE (lane 1 is HE (noHis) protein, lane 2 is Kcoil- β glucosidase, and lane 3 is HE (noHis) protein and Kcoil- β glucosidase) for the interaction between magnetic Ecoil-ferritin and Kcoil- β glucosidase;

FIG. 9 is a graph showing the results of enzyme activity measurements of free enzyme and immobilized enzyme. As shown in the figure, A is a result graph of the influence of pH on the enzyme activity of the free enzyme; b is a result graph of the influence of temperature on the activity of the free enzyme; c is a result graph of the influence of time on the enzyme activity of the free enzyme; d is a result graph of the influence of pH on the enzyme activity of the immobilized enzyme; e is a result graph of the influence of temperature on the enzyme activity of the immobilized enzyme; f is a result graph of the influence of time on the enzyme activity of the immobilized enzyme;

fig. 10 is a graph showing the adsorption efficiency of magnetic field adsorption of magnetic immobilized enzyme (demonstrating that about 60% of the enzyme remains immobilized on the surface of magnetic ferritin after 3 uses).

Detailed Description

(1) Firstly, designing PCR primers to carry gene sequences coding the Ecoil short peptide and the Kcoil short peptide respectively (the amino acid sequences of the Ecoil short peptide and the Kcoil short peptide are shown in a table 1 and an attached figure 2), and fusing the gene sequences to the C end of ferritin and the N end of beta-glucosidase respectively through PCR to construct a human ferritin recombinant plasmid containing the Ecoil short peptide and a beta-glucosidase recombinant plasmid containing the Kcoil short peptide;

the primer names and primer sequences are shown in Table 2

TABLE 1 amino acid sequences of Ecoil short peptides and Kcoil short peptides

TABLE 2 primer names and primer sequences

The direction of the primer is 5'-3'

(2) Transforming the constructed recombinant plasmid into an escherichia coli expression strain TOP10 competent cell, and culturing at a constant temperature of 37 ℃ overnight to obtain corresponding genetic engineering bacteria;

(3) Inoculating the genetically engineered bacteria into 200mL LB culture medium for induction culture, wherein the final concentration of ampicillin is 50 mug/mL, performing shake culture at 37 ℃ and the OD value is between 0.6 and 0.8, adding IPTG (isopropyl-beta-thiogalactoside) with the final concentration of 0.5mM, and performing shake culture at 30 ℃ for 6 hours. Then, centrifuging for 10min at the temperature of 12,000rpm and 4 ℃, collecting thalli, washing the thalli for 2 times by using buffer solution, removing residual culture medium, and suspending in 10mL of buffer solution for later use;

(4) Carrying out ultrasonic disruption on ferritin and enzyme, then centrifuging at 12,000rpm and 4 ℃ for 30min, collecting supernatant, and obtaining purified ferritin and enzyme by utilizing affinity chromatography and size exclusion chromatography;

(5) Taking 2.0mg ferritin dissolved in 50mM Tris-HCl with pH of 8.5 for degassing treatment, preparing a degassed ammonium ferrous sulfate solution, a hydrogen peroxide solution, a sodium hydroxide solution and a sodium citrate solution, carrying out reaction in a water bath at 65 ℃, slowly dropwise adding the ammonium ferrous sulfate solution and the hydrogen peroxide solution into a reaction device, dropwise adding a proper amount of the sodium hydroxide solution to control the pH value of the reaction to be 8.5, waiting for 5min after 50min of reaction is finished, adding 200 mu L of the sodium citrate solution to chelate redundant iron, centrifuging the sample for 10,000rpm and 10min, collecting supernatant, and verifying the iron loading effect of the ferritin by volume exclusion chromatography;

(6) The effect of the ferritin surface display enzyme was analyzed by affinity chromatography, polyacrylamide gel electrophoresis and non-denaturing gel electrophoresis. The operation steps are as follows: firstly, cleaning and activating Ni-NTA resin filler; then, the purified protein sample of Kcoil-beta-glucosidase containing His tag is contacted with affinity Ni ²⁺ Column mixing; subsequently, the purified His-tag-free magnetic Ecoil-ferritin sample was mixed with the treated affinity Ni ²⁺ Placing the column (saturated with Kcoil-beta-glucosidase containing His tag) at 4 deg.C, rotating, mixing, maintaining for 1h, and removing supernatant; finally, eluting the immobilized enzyme by 250mM imidazole, reserving the supernatant, and detecting the result by SDS-PAGE and Native-PAGE electrophoresis;

(7) After detection, determining the enzyme activities of free enzyme and immobilized enzyme by a pNPG method, and comparing;

(8) The magnetic immobilized enzyme is recovered by a magnet, and the operation is as follows: attaching a centrifugal tube filled with magnetic immobilized enzyme to a magnet, standing at 4 ℃ for 24 hours, then directly absorbing the clear liquid of the adsorbed solution to determine enzyme activity, comparing the result with the result of enzyme activity determination of the original immobilized enzyme, calculating adsorption efficiency, then taking the enzyme liquid off the magnet, and repeating the magnet adsorption operation twice after heavy suspension.

The invention utilizes the property that Ecoil and Kcoil can be specifically combined in a solution, takes ferritin as a carrier, tries to construct a ferritin surface display system, namely, beta-glucosidase in a cellulase system is fixed on the surface of a shell structure of synthesized magnetic human H chain ferritin (human H chain ferricin), thereby realizing the preliminary research of the magnetic ferritin immobilized enzyme.

Example 1:

transforming the constructed HE/KG-containing recombinant plasmid (figure 2) into a TOP10 strain of escherichia coli competent cell, and standing overnight for culture in a constant-temperature incubator at 37 ℃; then, selecting a single colony, inoculating the single colony in 200mL LB culture medium, adding antibiotic ampicillin to make the final concentration of the ampicillin 50 mug/mL, carrying out shake culture in a constant-temperature incubator at 37 ℃ with OD value of 0.6-0.8, adding IPTG with the final concentration of 0.5mM, and carrying out shake culture in a shaking table at 30 ℃ for 6 hours; then, centrifuging at 12,000rpm and 4 ℃ for 10min, collecting thalli, washing the thalli for 2 times by using a buffer solution, removing residual culture medium, and finally suspending in 10mL of the buffer solution; then, the heavy suspension is subjected to ultrasonic crushing, is centrifuged for 30min at the temperature of 12,000rpm and 4 ℃, and the supernatant is collected; purified ferritin and enzyme were obtained by affinity chromatography and size exclusion chromatography (FIG. 3, FIG. 4, FIG. 5).

Example 2:

synthesis of magnetic ferritin: 2.0mg ferritin dissolved in 10mL 50mM 50mM Tris-HCl and with the pH value of 8.5 is degassed for 30min and then placed in a sealed reaction device, 12.5mM ammonium ferrous sulfate solution, 4.17mM hydrogen peroxide solution, 100mM sodium hydroxide solution and 300mM sodium citrate solution are degassed, the device is placed in a water bath with the magnetic stirring at the temperature of 65 ℃, ammonium ferrous sulfate solution and hydrogen peroxide solution are slowly dripped into the device (the total volume of the dripped reaction solution is 1.566 mL), meanwhile, a proper amount of sodium hydroxide solution is dripped to adjust the pH value to be 8.5, after the 50min reaction is finished, 5min is waited, 200 mu L of sodium citrate solution is added into the device to chelate redundant iron, and finally, the reaction solution is centrifuged for 10min at the speed of 10,000rpm, and the supernatant is collected.

The related reaction formula:

3Fe ²⁺ +H ₂ O ₂ +H ₂ O→Fe ₃ O ₄ +6H ⁺

the effect of the synthesis of magnetic ferritin was confirmed by size exclusion chromatography (figure 6).

Example 3:

cleaning andactivating Ni-NTA resin filler, and allowing purified protein sample containing His-tagged Kcoil-beta-glucosidase to have affinity to Ni ²⁺ Mixing the column, mixing at 4 deg.C for 30min, and removing supernatant; next, a magnetic Ecoil-ferritin sample without His tag was added to affinity Ni that had been saturated with Kcoil- β -glucosidase containing His tag ²⁺ Placing the column at 4 ℃, rotating and uniformly mixing for 1h, and removing the supernatant; finally, the binding to Ni is eluted with 250mM imidazole ²⁺ The magnetic ferritin immobilized enzyme on the supernatant is reserved; analyzing the effect of the ferritin surface display enzyme by affinity chromatography, polyacrylamide gel electrophoresis and non-denaturing gel electrophoresis (FIG. 7, FIG. 8); then, the enzymatic activities of the free enzyme and the immobilized enzyme were measured by the pNPG method and compared (FIG. 9).

Example 4:

attaching a centrifugal tube filled with the magnetic iron immobilized enzyme to a magnet, standing at 4 ℃ for 24 hours, then sucking clear liquid from the tube, measuring enzyme activity according to a pNPG method, and calculating the adsorbed amount and the adsorption efficiency. The centrifuge tube was then removed from the magnet, the adsorption operation was repeated twice after the enzyme solution was resuspended, the enzyme activity of the supernatant was measured, and the adsorption efficiency was calculated (fig. 10).

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Claims

1. A method for realizing enzyme immobilization through magnetic ferritin surface display is characterized by comprising the following steps:

(1) Constructing ferritin recombinant plasmid containing Ecoil short peptide and enzyme recombinant plasmid containing Kcoil short peptide: designing PCR primers to carry gene sequences coding the Ecoi short peptide and the Kcoil short peptide respectively, and fusing the gene sequences to the C end of a ferritin subunit and the N end of a beta-glucosidase subunit respectively through PCR to construct a ferritin recombinant plasmid containing the Ecoi short peptide and an enzyme recombinant plasmid containing the Kcoil short peptide;

(2) Transforming the constructed recombinant plasmid into an escherichia coli expression strain TOP10 competent cell, and carrying out constant temperature overnight culture at 37 ℃ to obtain a corresponding genetic engineering strain;

(3) Inoculating the genetically engineered bacteria into an LB culture medium for induction culture, and obtaining purified ferritin and enzyme by utilizing affinity chromatography and size exclusion chromatography; synthesis of Fe in the internal cavity of purified ferritin ₃ O ₄ Obtaining magnetic ferritin;

(4) Mixing the obtained magnetic ferritin with enzyme, and forming magnetic ferritin immobilized enzyme through the interaction of Ecoil helical peptide and Kcoil helical peptide, wherein the amino acid sequence of the Ecoil short peptide is shown as SEQ ID NO.1, and the amino acid sequence of the Kcoil short peptide is shown as SEQ ID NO. 2;

(5) The magnetic ferritin immobilized enzyme is adsorbed by a magnetic field, and the enzyme activity and the recovery efficiency are measured.

2. The method for immobilizing enzyme by magnetic ferritin surface display according to claim 1, wherein the PCR primers encoding the Ecoil short peptide are shown in SEQ ID No.3 and 4.

3. The method for immobilizing enzyme by magnetic ferritin surface display according to claim 1 wherein the PCR primers encoding the Kcoil short peptide are shown in SEQ ID No.5 and 6.

4. The method of claim 1, wherein Fe is synthesized in the internal cavity of purified ferritin ₃ O ₄ Comprises the following steps: 2.0mg of ferritin dissolved in 50mM Tris-HCl with the pH value of 8.5 is taken for degassing treatment, 12.5mM ammonium ferrous sulfate solution, 4.17mM hydrogen peroxide solution, 100mM sodium hydroxide solution and 300mM sodium citrate solution which are subjected to degassing treatment are prepared, the reaction is carried out in a water bath with the temperature of 65 ℃, 1.57mL of ammonium ferrous sulfate solution and 1.57mL of hydrogen peroxide solution are dropwise added into a reaction device, the pH value of the reaction is controlled to be 8.5 by dropwise adding the sodium hydroxide solution, 5min is waited after the 50min reaction is finished, 200 mu L of sodium citrate solution is added for chelating redundant iron, then a sample is centrifuged at 10,000rpm for 10min, and a supernatant is collected.

5. The method of claim 1, wherein the immobilized magnetic ferritin surface enzyme is β -glucosidase.