CN116328840B - A heme-based chimeric oligopeptide-DNA mimetic enzyme and its application - Google Patents

Abstract

The invention provides a chimeric oligopeptide-DNA mimic enzyme based on heme and application thereof, which are formed by covalently assembling three biological units, namely heme, a special DNA structure called G-quadruplex and oligopeptide. G-quadruplex nucleic acid sequences with different modifications at both ends are coupled to the surface of the magnetic microsphere using one of the modification groups at one end and one of the two carboxyl groups of heme using the remaining group at the other end. Chimeric oligopeptide-DNA mimic enzymes show great practical advantages over horseradish peroxidase (HRP) because they can be used under harsh experimental conditions (mixtures of organic solvents, high temperature, high/low pH environments, etc.). Examples were validated by catalytic degradation of common contaminant dyes (BB 9, AB74, BR 2) in a range of textile industry wastewater. In addition, the catalyst can be recycled without losing the catalytic efficiency (more than 90% of catalytic capacity is maintained after 10 times of recycling), and has the characteristics of recycling.

Description

A heme-based chimeric oligopeptide-DNA mimetic enzyme and its application

Technical Field

The invention relates to the technical field of application of artificial enzyme mimics, and in particular to a chimeric oligopeptide-DNA enzyme mimic based on double covalently coupled heme.

Background Art

Enzymes are biological catalysts that speed up chemical reactions in vivo and can be transferred to chemical processes for use as biocatalysts due to their high activity and specificity under mild conditions (buffered solutions, narrow temperature ranges, etc.). However, the application of these catalysts to real industrial conditions remains a challenging task due to the intrinsic fragility of natural enzymes. Therefore, scientists are designing strategies to circumvent this stability issue, for example through directed evolution (obtaining new and more powerful enzymes) or through immobilization (refolding the proteins). Currently, another strategy is the use of artificial enzymes, whose design is considered to meet industrial catalytic requirements.

A classic example that is currently under intensive investigation is the development of alternatives to horseradish peroxidase (HRP). HRP is a well-established tool for ELISA assays, immunohistochemical investigations, and immunoblot analysis. HRP is widely used in these biochemical manipulations because it catalyzes the oxidation of a wide range of organic substrates, including common reagents such as 2,2′-azidobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3,3′,5,5′-tetramethylbenzidine sulfate (TMB), luminol, nicotinamide adenine dinucleotide (NADH), and dopamine (DPA). Its catalytic activity is dependent on its cofactor heme (protoporphyrin IX iron(II)), which coordinates the oxidation step when it is precisely bound to the catalytic site of HRP. The structure of the active site of HRP has been studied in great detail.

Therefore, researchers have made efforts to artificially reconstruct this active site in the field of artificial enzyme mimics. The typical enzyme mimics of HRP are G-quadruplex/hemin (G4/Hemin) enzyme mimics. G-quadruplex is a DNA sequence rich in G bases that can form a stable secondary nucleic acid structure as shown in Figure 1 under certain conditions. Its ππ stacking complex with heme, G4/Hemin, has been regarded as a substitute for HRP and widely used in biochemical analysis and other fields since its discovery. Nevertheless, compared with natural enzymes, G4/Hemin enzyme mimics have problems with low conversion frequency and catalytic efficiency (catalytic constant k _cat = 0.3s ^-1 ), which limits its further application.

Summary of the invention

The present invention provides a chimeric oligopeptide-DNA mimetic enzyme based on double covalent coupling of heme, which is covalently assembled from three biological units, namely heme, G-quadruplex and short peptide. It can be used to replace horseradish peroxidase (HRP) to degrade pollutants such as industrial dyes and biochemical tests such as ELISA detection, immunohistochemical investigation and immunoblotting analysis involving HRP. The present invention uses self-assembling peptides to provide a suitable binding point for Hemin, encapsulating the catalytic center from the external water environment, thereby improving the catalytic activity of the system. The chimeric oligopeptide-DNA mimetic enzyme uniquely utilizes the possible dual chemical modification of Hemin (on its two carboxyl arms), and sequentially connects G4 on one side (benefiting from the recognized G4-mediated Hemin catalytic activation) and oligopeptides on the other side (taking into account the arrangement of specific amino acids in the natural enzyme). We gradually optimized the chimeric oligopeptide-DNA mimetic enzyme by changing the length and properties of its peptide arm, and finally constructed two sets of synergistic catalytic oligopeptide combinations, namely G4-Hemin-KHRRH, and found that its catalytic constant k _cat = 784.2s ^-1 (as shown in Figure 5B) exceeded that of HRP (k _cat = 520.9s ^-1 ). The superiority of this new system over native HRP was further demonstrated by the excellent catalytic reaction of the chimeric oligopeptide-DNA mimetic enzyme under non-friendly conditions (high temperature (95°C), extreme pH values (from 2 to 10) and non-aqueous media).

The present invention proposes a heme-based chimeric oligopeptide-DNA mimetic enzyme, which is covalently assembled from three biological units, namely heme (a natural cofactor of horseradish peroxidase HRP), a special DNA structure called G-quadruplex (known to show high affinity for heme and activate its catalytic properties) and a short peptide (recreating the microenvironment of the HRP active site). The G-quadruplex nucleic acid sequence with different modifications at both ends is coupled to the surface of a magnetic microsphere using a modified group at one end, and one of the two carboxyl groups of heme is coupled with the remaining group at the other end; then, the remaining carboxyl group of heme is coupled with an oligopeptide containing a combination of arginine and histidine to prepare a magnetically separable chimeric oligopeptide-DNA mimetic enzyme. The resulting mimetic enzyme system is gradually optimized, and the catalytic constant (k _cat ) of the optimal mimetic enzyme (G4-Hemin-KHRRH) under this design exceeds HRP in terms of counting a single catalytic site (based on the catalytic center heme). Importantly, chimeric oligopeptide-DNA mimetic enzymes show great practical advantages over HRP because they can be used under harsh experimental conditions, such as mixtures of organic solvents, high temperatures, high/low pH values, etc. Examples have been verified by catalytic degradation of a series of common pollutant dyes (BB9, AB74, BR2) in textile wastewater. In addition, the catalytic efficiency can be maintained during recycling (more than 90% of the catalytic capacity is maintained after 10 cycles), and it has the characteristics of being recyclable and reusable.

The present invention focuses on a dual covalent framework-chimeric oligopeptide-DNA mimetic enzyme that relies on oligopeptide, Hemin and G4. The double chemical modification (using carboxyl-amino connection) on the two carboxyl arms of Hemin is utilized to connect three arbitrary G4 chain body structures (3' terminal modified amino group, 5' modified group B) to one side of Hemin and an oligopeptide containing histidine or arginine (coupled with the amino group on the α-amino group or the lysine R group) on the other side. The three covalently coupled complex is modified to the surface of a magnetic microsphere-A (A and B are a group that can be covalently coupled) by the 5' modified group B of DNA, so as to use magnetic separation to optimize the preparation and recovery, and the group B is Biotin.

A chimeric oligopeptide-DNA mimetic enzyme based on heme, wherein the mimetic enzyme is coupled to an oligopeptide and a nucleic acid respectively through two carboxyl functional groups of heme;

The nucleic acid sequence coupled to heme is rich in base G and can form a G-quadruplex, wherein the 3' end of the nucleic acid sequence is modified with an amino group and the 5' end is modified with biotin;

The oligopeptide coupled to heme is connected to one of the carboxyl groups of heme via the amino group at the nitrogen (N) terminal or the amino group on the lysine R group;

The sequence of the oligopeptide coupled to heme can be any of the following general formulas:

Sequence 11: K _(0-1) H _(0-4) R _(0-4) X _(0-1)

The second oligopeptide to the seventh oligopeptide are any combination of H, R and X, the order of combination of H, R and X can be arbitrarily reversed, and X is selected from any amino acid of N, F and W.

Among them, H is histidine, R is arginine, K is lysine, N is asparagine, F is phenylalanine, and W is tryptophan.

The general formula of the nucleic acid sequence that preferably forms a G-quadruplex structure is as follows:

Sequence 12: H _(0-20) G _(2-4) H _(1-5) G _(2-4) H _(1-5) G _(2-4) H _(1-5) G _(2-4) H _(0-2)

In the above general formula, H is selected from the nucleic acid bases A, T, and C. When H is two or more nucleic acid bases, they may be the same or different.

The general formula of the nucleic acid sequence that preferably forms a G-quadruplex structure is as follows:

Sequence 13: H _(0-5) G ₃ H _(1-5) G ₃ H _(1-5) G ₃ H _(1-5) G ₃ H _(0-2)

In the above general formula, H is selected from the nucleic acid bases A, T, and C. When H is two or more nucleic acid bases, they may be the same or different.

The general formula of the nucleic acid sequence that preferably forms a G-quadruplex structure is as follows:

Sequence 14: H _(0-5) G ₃ H _(1-3) G ₃ H _(1-3) G ₃ H _(1-3) G ₃ H _(0-2)

In the above general formula, H is selected from the nucleic acid bases A, T, and C. When H is two or more nucleic acid bases, they may be the same or different.

The characteristic of G-quadruplex is H _(0-20) G _(2-4) H _(1-5) G _(2-4) H _(1-5) G _(2-4) H _(1-5) G _(2-4) H _(0-2) (H＝A, T, C), wherein four consecutive G bases form G4 chain through hootsent hydrogen bonds (Figure 1), H is a non-G spacer base, and three types of G4 structures can be formed (as shown in the circular dichroism spectrum of parallel, antiparallel and hybrid G4 structure characteristic spectral lines in Figure 2).

The sequence of the oligopeptide coupled to heme is as follows:

Sequence 15: KH _(0-4) R _(0-4) X _(0-1)

The second oligopeptide to the seventh oligopeptide are any combination of H, R and X, the order of combination of H, R and X can be arbitrarily reversed, and X is selected from any amino acid of N, F and W.

The chimeric oligopeptide-DNA mimetic enzyme is G4-Hemin-KHRRH.

The oligopeptide features are K _(0-1) +H _(0-4) +R _(0-4) +X _(0-1) (X＝N, F, W), among which the most important are the side chains of imidazole (histidine) and guanidine (arginine) with strong basicity (high charge density), and His and Arg also have synergistic catalytic effects. The N-terminal K has two advantages as a connecting chain (Figure 3): First, the side chain amino group of K is more likely to react with Hemin than its α-amino group, which has obvious advantages in improving the coupling efficiency. Second, the side chain of K is more flexible and can provide spatial freedom of C ₄ length, which is conducive to the rotation and folding of the synergistic catalytic block.

At the preparation and separation end, we used the easy-to-modify characteristics of nucleic acid sequences (Table 1) to couple magnetic microspheres and Hemin at both ends, and used the double carboxyl end of Hemin to covalently connect an oligopeptide (see Table 1 for the specific sequence, the α-amino group at the N-terminus of the oligopeptide or the amino group on the lysine R group was coupled. As shown in Figure 3). A chimeric oligopeptide-DNA mimetic enzyme with recycling characteristics was constructed.

The present invention provides a chimeric oligopeptide-DNA mimetic enzyme that can be separated magnetically (the mass spectra of three representative mimetic enzymes are shown in FIG4 ), which can achieve the degradation of various industrial dyes under high temperature, high organic phase, strong acid and strong base conditions, and has good recovery characteristics. Therefore, the invention can expand the application scope and utilization value of peroxidase mimetic enzymes in the field of industrial catalysis.

Table 1. Some nucleic acid and amino acid sequences listed in the present invention

The superscripts 123 correspond to the parallel, antiparallel and hybrid G4 structures, respectively.

Beneficial effects:

The present invention combines the characteristics of magnetic microsphere separation and enrichment with the advantages of high catalytic activity of chimeric oligopeptide-DNA mimetic enzymes to design a peroxidase mimetic enzyme solution with recycling, high temperature resistance, high organic resistance, acid and alkali resistance. Compared with the existing G4/Hemin mimetic enzyme, it has the following characteristics:

(1) The chimeric oligopeptide-DNA mimetic enzyme scheme involves a double covalent coupling system. Compared with the traditional π-π stacking G4/Hemin system, the chimeric G4-Hemin-KHRRH mimetic enzyme optimized by double covalent coupling and oligopeptide combination can improve the catalytic activity by 228 times (as shown in Figure 5). As shown in Figure 6, based on the optimal oligopeptide KHRRH, the three oligopeptide-DNA mimetic enzymes with different G4 structures all have high catalytic activity, and the parallel G4 group has the best catalytic ability. The Michaelis parameters of the representative combinations are shown in Figure 7, where the catalytic constant (k _cat ) of the optimal combination G4-Hemin-KHRRH is 2610 times higher than that of the existing G4/Hemin mimetic enzyme (784.25/0.3=2614.17), and exceeds HRP (k _cat =520.9s ^-1 );

(2) The chimeric oligopeptide-DNA mimetic enzyme has good stability compared to G4/Hemin, and can still maintain 93.8% activity after ten recycling (Figure 8A). As shown in Figure 8B, the recycling process only takes 30 seconds, and the magnetic mimetic enzyme is attracted to the wall by the magnet, showing a good rapid separation effect;

(3) Industrial dye degradation is often accompanied by a large amount of organic solvents. As shown in Figure 9, we listed five organic phase-aqueous phase systems. The chimeric G4-Hemin-KHRRH mimetic enzyme still maintained a higher level of catalytic activity than HRP;

(4) In order to adapt to the high temperature environment that may occur in industrial catalysis, as shown in Figure 10, the chimeric G4-Hemin-KHRRH mimetic enzyme maintains high stability and high catalytic activity at a high temperature of 95°C for a long time;

(5) The pH in the environment often has a significant effect on the catalyst. As shown in Figure 11, the chimeric G4-Hemin-KHRRH mimetic enzyme maintains high catalytic ability in the pH range of 2-10;

(6) When we compared the ability of different catalysts to degrade dyes under four different environments, we found that the chimeric G4-Hemin-KHRRH mimetic enzyme had great advantages over the protease HRP in degrading industrial dyes under high temperature, high organic phase, and acidic and alkaline conditions ( Figure 12 ).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of a G-plane and a G-quadruplex assembled therefrom;

Figure 2 is a circular dichroism spectrum of three different G4 structures (A: the characteristic peaks of the parallel structure are a positive peak at 265 nm and a negative peak at 245 nm; B: the characteristic peaks of the antiparallel structure are 245 nm, positive peaks at 195 nm and a negative peak at 265 nm; C: the characteristic peaks of the hybrid structure are a positive peak at 295 nm and a shoulder peak at 265 nm);

Figure 3 shows the structural formula of Arg, His, and dipeptides formed by Lys-Arg and Lys-His. The circle on the left represents the α-amino group, and the circle on the right represents the amino group on the R group in Lys. The amino group in the circle is coupled to the carboxyl group on Hemin.

Figure 4 shows the mass spectra of (A) G4-Hemin, (B) G4-Hemin-KHR and (C) G4-Hemin-KHRRH. The theoretical calculated mass of G4-Hemin is 5917.2, and the actual measured value is 5917.5; the theoretical calculated mass of G4-Hemin-KHR is 6338.7, and the actual measured value is 6338.4; the theoretical calculated mass of G4-Hemin-KHRRH is 6632.01,

The actual measured value is 6632.4;

FIG. 5 is a performance comparison of the activity of the chimeric oligopeptide-DNA mimetic enzyme composed of a parallel G-quadruplex structure and a G4/Hemin mimetic enzyme.

FIG6 shows the catalytic activity of chimeric oligopeptide-DNA mimetic enzymes composed of three different G-quadruplex structures.

Figure 7 shows the saturation curves of the oxidation rates of ABTS corresponding to the representative chimeric mimetic enzymes at different concentrations of H ₂ O ₂ , which were fitted according to the Michaelis-Menten model;

Figure 8A shows the catalytic performance of the chimeric G4-Hemin-KHRRH mimetic enzyme after ten consecutive recyclings. Figure 8B shows the effect of the chimeric oligopeptide-DNA mimetic enzyme after 30s of magnetic separation;

FIG9 is a comparison of the catalytic efficiency of HRP and chimeric G4-Hemin-KHRRH mimetic enzyme in conventional aqueous buffer and different organic water mixed solutions (each accounting for 50% by volume);

FIG10 is a histogram of the catalytic activity of H ₂ O ₂ on ABTS oxidation at different times in a high temperature (95° C.) environment with the chimeric G4-Hemin-KHRRH mimetic enzyme as a catalyst;

FIG11 is a histogram of the catalytic activity of ABTS oxidized by H ₂ O ₂ using the chimeric G4-Hemin-KHRRH mimetic enzyme as a catalyst at different pH values;

FIG. 12 is a comparison of HRP and chimeric G4-Hemin-KHRRH mimetic enzymes under extreme conditions. (AD) Absorbance changes of BB9 degradation by HRP and G4-Hemin-KHRRH in the presence of 50% methanol (v/v) for 20 minutes (A and B) and at high temperature (95°C) for 10 minutes (C and D). (EH) Absorbance changes of AB74 degradation by HRP and G4-Hemin-KHRRH in an acidic environment (pH 2) (E and F) or BR2 in an alkaline environment (pH 10) (G and H) for 20 minutes. H ₂ O ₂ was used as an oxidant in all experiments.

FIG13 is a schematic diagram of the preparation process of the chimeric oligopeptide-DNA mimetic enzyme and the optimal combined structure of the chimeric oligopeptide-DNA mimetic enzyme

DETAILED DESCRIPTION

In order to facilitate those skilled in the art to understand and implement the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

Example

1. Preparation of chimeric oligopeptide-DNA mimetic enzyme (MB-A and DNA-B, AB uses STV-biotin coupling combination)

1. Hemin amidation

Take 500 μL of 1 mg/mL Hemin (dissolved in DMSO), add 0.8 mg of EDC and 0.6 mg of NHS, mix well, and shake at room temperature for 30 minutes. Add a certain amount of 2-mercaptoethanol (2-ME) to extinguish EDC. The NHS-Hemin-NHS product is dispersed in DMSO.

2.1MB-STV coupled with bio-G4-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-G4-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-G4-NH ₂ .

2.2NHS-Hemin-NHS coupling MB-DNA and amino acids (H/R)

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), amino acids (H/R) and 0.2 mg/ml of DMAP were added to the MB-G4-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept at 0°C overnight. Then, magnetic separation was performed to obtain the sample MB-G4-Hemin-H (R), which was resuspended in 10 mM Tris-HCl buffer (pH = 7.0).

2.3G4 chain structure formation

100 mM potassium ions were added to the MB-G4-Hemin-H(R) sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH=7.0, K ⁺ =100 mM).

3.1MB-STV coupled with bio-G4-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-G4-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-G4-NH ₂ .

3.2NHS-Hemin-NHS coupling MB-DNA and dipeptide (KH/KR/KN)

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), dipeptide (KH/KR/KN) and 0.2 mg/ml of DMAP were added to the MB-G4-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept at 0°C overnight. Then, magnetic separation was performed to obtain the sample MB-G4-Hemin-KH (KR/KN), which was resuspended in 10 mM Tris-HCl buffer (pH = 7.0).

3.3G4 chain structure formation

100 mM potassium ions were added to the MB-G4-Hemin-KH (KR/KN) sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH=7.0, K+=100 mM).

4.1MB-STV coupled with bio-G4-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-G4-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-G4-NH ₂ .

4.2NHS-Hemin-NHS coupled MB-DNA and tripeptide (KRH/KHR/KHH/KHN/KNH)

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), tripeptide (KRH/KHR/KHH/KHN/KNH) and 0.2 mg/ml DMAP were added to the MB-G4-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept at 0°C overnight. Then, magnetic separation was performed to obtain the sample MB-G4-Hemin-KRH (KHR/KHH/KHN/KNH), which was resuspended in 10 mM Tris-HCl buffer (pH = 7.0).

4.3G4 chain structure formation

100 mM potassium ions were added to the MB-G4-Hemin-KRH (KHR/KHH/KHN/KNH) sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH=7.0, K ⁺ =100 mM).

5.1MB-STV coupled with bio-G4-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-G4-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-G4-NH ₂ .

5.2NHS-Hemin-NHS coupling MB-DNA and tetrapeptide (KNRH/KNHR…/KRRH) (specific sequence see Table 1)

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), tetrapeptide (KNRH/KNHR.../KRRH) and 0.2 mg/ml DMAP were added to the MB-G4-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept at 0°C overnight. Then, magnetic separation was performed to obtain the sample MB-G4-Hemin-KNRH (KNHR.../KRRH), which was resuspended in 10 mM Tris-HCl buffer (pH=7.0).

5.3G4 chain structure formation

100 mM potassium ions were added to the MB-G4-Hemin-KNRH (KNHR.../KRRH) sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH=7.0, K ⁺ =100 mM).

6.1MB-STV coupled with bio-G4-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-G4-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-G4-NH ₂ .

6.2NHS-Hemin-NHS coupling MB-DNA and pentapeptide (KRHHH/KHRHH…/KRHHR) (specific sequence see Table 1)

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), pentapeptide (KRHHH/KHRHH .../KRHHR) and 0.2 mg/ml of DMAP were added to the MB-G4-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept overnight at 0° C. Then, magnetic separation was performed to obtain the sample MB-G4-Hemin-KRHHH (KHRHH .../KRHHR), which was resuspended in 10 mM Tris-HCl buffer (pH=7.0).

6.3G4 chain structure formation

100 mM potassium ions were added to the MB-G4-Hemin-KRHHH (KHRHH ... / KRHHR) sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH = 7.0, K ⁺ = 100 mM).

7.1MB-STV coupled with bio-G4-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-G4-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-G4-NH ₂ .

7.2NHS-Hemin-NHS coupled MB-DNA and hexapeptide (KHRRHR/KHRRHH…/KHRHRH)

(See Table 1 for specific sequences)

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), hexapeptide (KHRRHR/KHRRHH ... / KHRHRH) and 0.2 mg/ml of DMAP were added to the MB-G4-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept at 0° C. overnight. Then, magnetic separation was performed to obtain the sample MB-G4-Hemin-KHRRHR (KHRRHH ... / KHRHRH), which was resuspended in 10 mM Tris-HCl buffer (pH = 7.0).

7.3G4 chain structure formation

100 mM potassium ions are added to the MB-G4-Hemin-KHRRHR (KHRRHH…/KHRHRH) sample after magnetic separation, and a chimeric oligopeptide-DNA mimetic enzyme can be obtained in Tris-HCl buffer (pH=7.0, K+=100 mM).

8.1MB-STV coupled with bio-G4-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-G4-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-G4-NH ₂ .

8.2 NHS-Hemin-NHS coupling of MB-DNA and heptapeptide

(KHRRHHH/KHRRHRR…/KHRHRHR) (See Table 1 for specific sequence)

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), hexapeptide (KHRRHHH/KHRRHRR .../KHRHRHR) and 0.2 mg/ml of DMAP were added to the MB-G4-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept at 0°C overnight. Then, magnetic separation was performed to obtain the sample MB-G4-Hemin-KHRRHHH (KHRRHRR .../KHRHRHR), which was resuspended in 10 mM Tris-HCl buffer (pH = 7.0).

8.3G4 chain structure formation

100 mM potassium ions were added to the MB-G4-Hemin-KHRRHHH (KHRRHRR ... / KHRHRHR) sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH = 7.0, K ⁺ = 100 mM).

9.1MB-STV coupled with bio-N-myc-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-N-myc-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-N-myc-NH ₂ .

9.2 NHS-Hemin-NHS coupling of MB-DNA and KHRRH

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), KHRRH and 0.2 mg/ml DMAP was added to the MB-N-myc-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept overnight at 0° C. Then, magnetic separation was performed to obtain the sample MB-N-myc-Hemin-KHRRH, which was resuspended in 10 mM Tris-HCl buffer (pH=7.0).

9.3G4 chain structure formation

100 mM potassium ions were added to the MB-N-myc-Hemin-KHRRH sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH=7.0, K ⁺ =100 mM).

10.1MB-STV coupled with bio-Myc-2345-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-Myc-2345-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-Myc-2345-NH ₂ .

10.2 NHS-Hemin-NHS coupling of MB-DNA and KHRRH

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), KHRRH and 0.2 mg/ml DMAP was added to the MB-Myc-2345-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept overnight at 0° C. Then, magnetic separation was performed to obtain the sample MB-Myc-2345-Hemin-KHRRH, which was resuspended in 10 mM Tris-HCl buffer (pH=7.0).

10.3G4 chain structure formation

100 mM potassium ions were added to the MB-Myc-2345-Hemin-KHRRH sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH=7.0, K ⁺ =100 mM).

11.1MB-STV coupled with bio-c-kit2-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-c-kit2-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-c-kit2-NH ₂ .

11.2 NHS-Hemin-NHS coupling of MB-DNA and KHRRH

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), KHRRH and 0.2 mg/ml DMAP was added to the MB-c-kit2-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept overnight at 0° C. Then, magnetic separation was performed to obtain the sample MB-c-kit2-Hemin-KHRRH, which was resuspended in 10 mM Tris-HCl buffer (pH=7.0).

11.3G4 chain structure formation

100 mM potassium ions were added to the MB-c-kit2-Hemin-KHRRH sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH=7.0, K ⁺ =100 mM).

12.1MB-STV coupled with bio-S-CTA-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-S-CTA-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-S-CTA-NH ₂ .

12.2 NHS-Hemin-NHS coupling of MB-DNA and KHRRH

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), KHRRH and 0.2 mg/ml DMAP was added to the MB-S-CTA-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept overnight at 0° C. Then, magnetic separation was performed to obtain the sample MB-S-CTA-Hemin-KHRRH, which was resuspended in 10 mM Tris-HCl buffer (pH=7.0).

12.3G4 chain structure formation

100 mM potassium ions were added to the MB-S-CTA-Hemin-KHRRH sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH=7.0, K ⁺ =100 mM).

13.1MB-STV coupled with bio-C9off72-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-C9off72-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-C9off72-NH ₂ .

13.2 NHS-Hemin-NHS coupling of MB-DNA and KHRRH

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), KHRRH and 0.2 mg/ml DMAP was added to the MB-C9off72-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept overnight at 0° C. Then, magnetic separation was performed to obtain the sample MB-C9off72-Hemin-KHRRH, which was resuspended in 10 mM Tris-HCl buffer (pH=7.0).

13.3G4 chain structure formation

100 mM potassium ions were added to the MB-C9off72-Hemin-KHRRH sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH=7.0, K ⁺ =100 mM).

14.1MB-STV coupled with bio-TBA-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-TBA-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-TBA-NH ₂ .

14.2 NHS-Hemin-NHS coupling of MB-DNA and KHRRH

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), KHRRH and 0.2 mg/ml DMAP was added to the MB-TBA-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept overnight at 0° C. Then, magnetic separation was performed to obtain the sample MB-TBA-Hemin-KHRRH, which was resuspended in 10 mM Tris-HCl buffer (pH=7.0).

14.3G4 chain structure formation

100 mM potassium ions were added to the MB-TBA-Hemin-KHRRH sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH=7.0, K ⁺ =100 mM).

15.1MB-STV coupled with bio-22AG-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-22AG-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-22AG-NH ₂ .

15.2 NHS-Hemin-NHS coupling of MB-DNA and KHRRH

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), KHRRH and 0.2 mg/ml DMAP was added to the MB-22AG-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept overnight at 0° C. Then, magnetic separation was performed to obtain the sample MB-22AG-Hemin-KHRRH, which was resuspended in 10 mM Tris-HCl buffer (pH=7.0).

15.3G4 chain structure formation

100 mM potassium ions were added to the MB-22AG-Hemin-KHRRH sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH=7.0, K ⁺ =100 mM).

16.1MB-STV coupled with bio-HTG-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-HTG-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, repeated three times, and then the sample was resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-HTG-NH ₂ .

16.2 NHS-Hemin-NHS coupling of MB-DNA and KHRRH

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), KHRRH and 0.2 mg/ml DMAP was added to the MB-HTG-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept overnight at 0° C. Then, magnetic separation was performed to obtain the sample MB-HTG-Hemin-KHRRH, which was resuspended in 10 mM Tris-HCl buffer (pH=7.0).

16.3G4 chain structure formation

100 mM potassium ions were added to the MB-HTG-Hemin-KHRRH sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH=7.0, K ⁺ =100 mM).

17.1MB-STV coupled with bio-JP-NH ₂

Streptavidin-modified magnetic microspheres (MB-STV) and 10-fold excess of 5'-terminal biotin-modified DNA chains (bio-JP-NH ₂ ) were mixed in 10 mM Tris-HCl buffer (pH=7.0) and shaken at 37° C. for 3 hours. After the reaction was completed, the supernatant was removed by magnetic separation, and the process was repeated three times. The sample was then resuspended in 10 mM Tris-HCl buffer (pH=7.0) to obtain the pure product MB-JP-NH ₂ .

17.2 NHS-Hemin-NHS coupling of MB-DNA and KHRRH

A 100-fold excess of premixed NHS-Hemin-NHS (DMSO), KHRRH and 0.2 mg/ml DMAP was added to the MB-JP-NH ₂ aqueous solution to ensure that the volume of the aqueous phase and the organic phase each accounted for 50%, and the reaction was kept overnight at 0° C. Then, magnetic separation was performed to obtain the sample MB-JP-Hemin-KHRRH, which was resuspended in 10 mM Tris-HCl buffer (pH=7.0).

17.3G4 chain structure formation

100 mM potassium ions were added to the MB-JP-Hemin-KHRRH sample after magnetic separation, and the chimeric oligopeptide-DNA mimetic enzyme was obtained in Tris-HCl buffer (pH=7.0, K ⁺ =100 mM).

2. Measurement of the catalytic activity (V ₀ ) of the chimeric oligopeptide-DNA mimetic enzyme

Chimeric oligopeptide-DNA mimetic enzyme (60 nM), ABTS (1 mM) and H ₂ O ₂ (1 mM) were mixed in 10 mM Tris-HCl buffer (pH 7.0, 100 mM K ⁺ ). The catalytic activity was measured by monitoring the absorbance of oxidized ABTS (ABTS· ⁺ ) at 25°C using a Cary100 (Agilent) spectrophotometer, which had a typical UV-visible characteristic at 420 nm and was recorded for 60 seconds. The extinction coefficient of ABTS· ⁺ at 420 nm is 36,000 M ^-1 cm ^-1 ; the initial rate of the oxidation reaction (V ₀ , nM/s) was obtained from the slope of the initial linear part (first 10 s) of the graph of the relationship between absorbance and reaction time. All kinetic results were obtained from triplicate experiments.

3. Kinetic Analysis

The oxidation reaction kinetics were established using a steady-state assay, with the initial reaction rate varying with the concentration of H ₂ O _2. The kinetic parameters were calculated according to the Michaelis-Menten equation. V ₀ =(V _max *[S])/(K _m +[S]), where V ₀ is the initial reaction rate, V _max is the maximum reaction rate, [S] is the concentration of the substrate (H ₂ O ₂ ), and K _m is the Michaelis constant. k _cat =V _max /[E ₀ ] is the catalytic number, where [E ₀ ] is the concentration of the catalyst.

IV. Industrial dye degradation steps of chimeric oligopeptide-DNA mimic enzyme

The degradation rate of dye under different conditions was determined by using UV dynamic scanning mode (20s/time interval) with a wavelength range of 400-800nm.

1. Organic solvent tolerance experiment 12 μM BB9, 0.5 mM H ₂ O ₂ and 100 nM enzyme were added to a 10 mM BR buffer solution (pH 7.0) containing 50% methanol (v/v) and 100 mM KCl, and UV spectra were collected at 25° C. for 20 minutes.

2. High temperature resistance experiment: 12 μM BB9, 0.5 mM H ₂ O ₂ and 100 nM enzyme were added to a 10 mM BR solution (pH 7.0) containing 100 mM KCl, and the UV spectrum was recorded at 95°C for 10 minutes.

3. Acid and alkali resistance test. Acid and alkali resistance test was performed with 12 μM AB74 or BR2, respectively. They were mixed with 0.5 mM H ₂ O ₂ and 100 nM enzyme in BR buffer, and the test was measured at 25°C for 20 minutes at pH 2.0 or 10.0.

The above is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (9)

1. A heme-based chimeric oligopeptide-DNA mimic enzyme, wherein said mimic enzyme is coupled to an oligopeptide and a nucleic acid, respectively, via two carboxyl functionalities of heme;

the nucleic acid sequence coupled with heme is rich in base G and forms a G-quadruplex, wherein the 3 '-end of the nucleic acid sequence is modified with amino group and the 5' -end is modified with biotin;

the oligopeptide coupled with heme is connected with one carboxyl of the heme through an amino group at the N end or an amino group on a lysine R group;

the sequence formula of the heme-coupled oligopeptide is as follows:

Sequence 11:K _(0-1)H_(0-4)R_(0-4)X_(0-1)

Wherein, the number of K, H and R is not 0 at the same time, the second oligopeptide to at most the seventh oligopeptide are any combination of H, R and X, the combination sequence of H, R and X can be arbitrarily reversed, and X is selected from any amino acid in N, F, W.

2. The heme-based chimeric oligopeptide-DNA mimic enzyme according to claim 1, wherein the nucleic acid sequence forming the G-quadruplex structure is of the general formula:

Sequence 12:H _(0-20)G_(2-4)H_(1-5)G_(2-4)H_(1-5)G_(2-4)H_(1-5)G_(2-4)H_(0-2)

In the above general formula, H is selected from nucleic acid bases A, T, C, and when H is 2 or more nucleic acid bases, H may be the same or different.

3. Heme-based chimeric oligopeptide-DNA mimic enzyme according to claim 1 or 2, wherein the nucleic acid sequence forming the G-quadruplex structure is of the general formula:

sequence 13:H _(0-5)G₃H_(1-5)G₃H_(1-5)G₃H_(1-5)G₃H_(0-2)

In the above general formula, H is selected from nucleic acid bases A, T, C, and when H is 2 or more nucleic acid bases, H may be the same or different.

4. Heme-based chimeric oligopeptide-DNA mimic enzyme according to claim 1 or 2, wherein the nucleic acid sequence forming the G-quadruplex structure is of the general formula:

sequence 14:H _(0-5)G₃H_(1-3)G₃H_(1-3)G₃H_(1-3)G₃H_(0-2)

In the above general formula, H is selected from nucleic acid bases A, T, C, and when H is 2 or more nucleic acid bases, H may be the same or different.

5. The heme-based chimeric oligopeptide-DNA mimic enzyme according to claim 1, wherein the nucleic acid sequence forming the G-quadruplex structure is selected from any one of the following general formulas, and the 5 'end modifies biotin, the 3' end modifies amino groups:

sequence 1: TTTTTTTTTGGGTGGGTGGGTGGG A

Sequence 2 TAGGGGCGGGAGGGAGGGAA

Sequence 3 TAAGGGTGGGGAGGGGGTGGGGAA

Sequence 4 CGGGCGGGCTCTCGGGAGGGG

Sequence 5 GGGCTAGGCTAGGGGCTAGGG

Sequence 6 GGGGCGGGGCCGGGGCGGGGCC

Sequence 7 GGTTGGTATGGGTTGG

Sequence 8 AGGGTTAGGGTTAGGGTTAGGGTTAGGG

Sequence 9 GGGTTAGGTTAGGGTTAGGGTTAGGG

Sequence 10 GGGATGGGACACACAGGGACGGG.

6. The heme-based chimeric oligopeptide-DNA mimic enzyme according to claim 1, wherein the sequence of the heme-coupled oligopeptide is of the general formula:

Sequence 15 KH _(0-3)R_(0-3)X_(0-1)

Wherein the number of H and R is not 0 at the same time, the second oligopeptide to at most the seventh oligopeptide are any combination of H, R and X, the combination sequence of H, R and X can be arbitrarily reversed, and X is selected from any amino acid in N, F, W.

7. The heme-based chimeric oligopeptide-DNA mimic enzyme according to claim 1, wherein the sequence of the heme-coupled oligopeptide is H, R, KH, KR, KN; KRH, KHR, KHH, KHN, KNH;

KNRH，KNHR，KHRN，KHNR，KRHN，KRNH，KFRH，KRFH，KRHF，

KFHR，KHFR，KHRF，KWHR，KWRH，KHWR，KRWH，KHRW，KRHW，

KRHH，KHRR，KHRH，KRHR，KHHR，KRRH，KRHHH，KHRHH，KHHRH，

KHHHR，KHRRR，KRHRR，KRRHR，KRRRH，KHHRR，KRRHH，KHRHR，

KRHRH，KHRRH，KRHHR，KHRRHR，KHRRHH，KRHHRR，KRHHRH，

KHRHRR，KHRHRH，KHRRHHH，KHRRHRR，KHRRHHR，KHRRHRH，

KHRHRHR, KRHRHRH.

8. The heme-based chimeric oligopeptide-DNA mimic enzyme according to claim 1, wherein the chimeric oligopeptide-DNA mimic enzyme is G4-Hemin-KHRRH.

9. Use of a heme-based chimeric oligopeptide-DNA mimic enzyme according to any one of claims 1-8 in the degradation of a dye.