Horseradish peroxidase mutant with high catalytic activity, construction method and application thereof

Abstract

The present invention discloses a horseradish peroxidase mutant with high catalytic activity, its construction method and application. This horseradish peroxidase mutant is obtained by mutating the histidine at position 40 of the wild-type horseradish peroxidase amino acid sequence shown in SEQ ID NO.1 into tryptophan. In the present invention, the histidine at position 40 of the amino acid sequence of horseradish peroxidase (PDB: 1HCH) derived from horseradish plants is mutated into tryptophan through site-directed mutagenesis technology. The activity of the obtained mutant (H40W) in catalyzing the generation of resorufin from hydrogen peroxide and ADHP is significantly higher than that of the wild type. It can be seen that the horseradish peroxidase disclosed in the present invention has high catalytic activity for ADHP and has great application potential.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and particularly relates to a mutant of horseradish peroxidase with high catalytic activity, a construction method thereof, and an application thereof. Background Art

[0002] Horseradish peroxidase (HRP, EC 1.11.1.7) was initially discovered from the roots of horseradish plants, and thus was named horseradish peroxidase. HRP is a secreted plant peroxidase and is an enzyme with high market demand, having high stability and water solubility. It is widely used in many fields such as medical diagnosis, detection and analysis, and bioremediation. For example: in immunology, it binds to specific antibodies to form enzyme-labeled antibodies for immunoassay (ELISA); in clinical chemistry, it is usually used as a histochemical tracer for light and electron microscopy; in medicine, it can be used for enzyme prodrug therapy for cancer treatment, etc. At the same time, HRP can also be used in food processing; degrading phenol in environmental wastewater, etc., so it has broad application value.

[0003] By increasing the catalytic activity of HRP towards ADHP (hydrogen peroxide fluorescence probe), the intensity of the detection signal can be significantly enhanced, the detection limit can be reduced, thereby improving the sensitivity and reliability of the analysis method. In addition, improving the catalytic efficiency of HRP also helps to reduce the amount of enzyme used, lower the detection cost, and shorten the reaction time, which is of great significance for high-throughput detection and point-of-care testing (POCT). In clinical diagnosis, HRP with high catalytic activity can detect low-concentration biomarkers more quickly, providing support for the early diagnosis and treatment of diseases. In the field of environmental monitoring, enhancing the catalytic ability of HRP towards ADHP can improve the detection sensitivity of pollutants (such as phenolic compounds), thereby more effectively monitoring environmental quality. From the perspective of scientific research, improving the catalytic activity of HRP also helps to deeply understand the interaction mechanism between enzymes and substrates, providing a theoretical basis for the rational design and modification of enzymes. Optimizing the active site or substrate-binding region of HRP through protein engineering or directed evolution technology can not only improve its catalytic performance, but also may expand its substrate spectrum, enabling it to play a role in more fields. Therefore, increasing the catalytic activity of HRP towards ADHP not only has important practical application value, but also provides a new direction for research in the fields of enzyme engineering and biocatalysis. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a mutant of horseradish peroxidase with high catalytic activity, a construction method thereof, and an application thereof in view of the deficiencies in the above-mentioned prior art.

[0005] To solve the above technical problems, the technical solution adopted by the present invention is as follows: In the first aspect of the present invention, a horseradish peroxidase mutant with high catalytic activity is provided, which is obtained by mutating the histidine at position 40 of the wild-type horseradish peroxidase amino acid sequence shown in SEQ ID NO.1 to tryptophan.

[0006] In the second aspect of the present invention, a gene encoding the horseradish peroxidase mutant as described above is provided.

[0007] Preferably, the nucleotide sequence encoding the wild-type horseradish peroxidase shown in SEQ ID NO.1 is SEQ ID NO.3.

[0008] Preferably, the gene encoding the horseradish peroxidase mutant is obtained by site-directed mutagenesis based on the nucleotide sequence shown in SEQ ID NO.3.

[0009] Preferably, the amino acid sequence of the horseradish peroxidase mutant is SEQ ID NO.2.

[0010] Preferably, the nucleotide sequence encoding the horseradish peroxidase mutant shown in SEQ ID NO.2 is SEQ ID NO.4.

[0011] In the third aspect of the present invention, a recombinant plasmid containing the gene as described above is provided.

[0012] In the fourth aspect of the present invention, a host cell containing the gene or recombinant plasmid as described above is provided.

[0013] In the fifth aspect of the present invention, an application of the horseradish peroxidase mutant with high catalytic activity as described above in catalyzing the generation of resorufin from hydrogen peroxide and ADHP is provided.

[0014] In the sixth aspect of the present invention, an application of the horseradish peroxidase mutant with high catalytic activity as described above in the detection of hydrogen peroxide is provided.

[0015] The beneficial effects of the present invention are as follows:

[0016] In the present invention, the histidine at position 40 of the horseradish peroxidase (PDB: 1HCH) amino acid sequence derived from horseradish plants is mutated to tryptophan by site-directed mutagenesis. The activity of the obtained mutant (H40W) in catalyzing the generation of resorufin from hydrogen peroxide and ADHP is significantly higher than that of the wild type. It can be seen that the horseradish peroxidase disclosed in the present invention has high catalytic activity for ADHP and has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1It is the structural model of horseradish peroxidase (HRP) and the molecular docking results with ADHP, where H40 is the target residue for mutation;

[0018] Figure 2 It is the schematic diagram of the method for detecting the activity of horseradish peroxidase (HRP) used in the present invention;

[0019] Figure 3 It is the comparison result of the enzyme activities of wild-type horseradish peroxidase (HRP) and H40W mutant. Detailed implementation manners

[0020] The following further elaborates on the present invention in conjunction with embodiments, so that those skilled in the art can implement it with reference to the text of the specification.

[0021] It should be understood that terms such as "having", "comprising", and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0022] Unless otherwise specified, the test methods used in the following examples are all conventional methods. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained through commercial channels. For those not specifying specific conditions in the following examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be purchased commercially.

[0023] I. The culture media and reagents involved in the following examples are as follows:

[0024] 1. YPD medium: 1% yeast extract, 2% peptone, 2% glucose. If preparing solid medium, add 2% agar powder; glucose is prepared separately, 20% glucose mother liquor.

[0025] 2. SD + CAA medium: Yeast nitrogen base medium (6.7 g / L), disodium hydrogen phosphate (5.4 g / L), sodium dihydrogen phosphate (8.56 g / L), glucose (20 g / L), tyrosine (CAA, 5 g / L).

[0026] 3. SG + CAA medium: Yeast nitrogen base medium (6.7 g / L), disodium hydrogen phosphate (5.4 g / L), sodium dihydrogen phosphate (8.56 g / L), galactose (20 g / L), tyrosine (CAA, 5 g / L).

[0027] 4. PBS buffer: Sodium chloride 8 g / L, KCl 0.2 g / L, disodium hydrogen phosphate (1.44 g / L), potassium dihydrogen phosphate (0.24 g / L), pH = 7.4.

[0028] II. The principle of the horseradish peroxidase activity detection method involved in the following examples is as follows Figure 2 as shown below, and the specific scheme is as follows:

[0029] Using hydrogen peroxide and 10-acetyl-3,7-dihydroxyphenazine (ADHP) as substrates, the fluorescence intensity of the product resorufin in the catalytic reaction is measured by an enzyme-linked immunosorbent assay (ELISA) reader.

[0030] Enzyme activity detection system (100 μL): 1 mM hydrogen peroxide, 100 μM ADHP, 1 mM PBS buffer (pH 7.4), wild-type or mutant horseradish peroxidase.

[0031] React at 37 °C and detect fluorescence in real time. Excitation wavelength: 550 nm, emission wavelength: 595 nm, gain: 60, continuously shake the plate for 5 s, detect fluorescence every 1 min, and continuously detect for 1 hour.

[0032] Example 1 Construction of a plasmid with site-directed mutants of horseradish peroxidase HRP

[0033] According to the amino acid sequence SEQ ID NO.1 of horseradish peroxidase HRP, the structure of horseradish peroxidase HRP (PDB: 1HCH) was downloaded from the Protein Data Bank (PDB). Then, the structure was docked with the ADHP substrate (using DiscoveryStudio2019 software), and its substrate-binding pocket was analyzed. Twenty-two sites in the HRP gene, namely R31, A34, L37, R38, H40, F41, H42, P139, P141, F152, L166, S167, G169, H170, F172, G173, K174, N175, F221, I244, F277, and M284, were subjected to site-directed saturation mutagenesis.

[0034] Using the primers shown in Table 1, site-directed mutagenesis PCR of the entire plasmid was performed with the wild-type plasmid pCTcon2-HRP Cwt as the template to construct site-directed mutant recombinant plasmids containing genes in which the sites of R31, A34, L37, R38, H40, F41, H42, P139, P141, F152, L166, S167, G169, H170, F172, G173, K174, N175, F221, I244, F277, and M284 were mutated into the coding genes of other amino acids. After correct sequencing, the plasmids were extracted and stored.

[0035] Table 1 Primers for constructing site-directed mutants of HRP C

[0036]

[0037] The HRP C site-directed mutagenesis PCR system is shown in Table 2.

[0038] Table 2 PCR reaction system

[0039]

[0040] The PCR program for whole plasmid single-point mutagenesis is set as follows:

[0041] (1) Denature at 98 °C for 30 seconds, cycle once; (2) Denature at 98 °C for 10 seconds; (3) Anneal at 55 °C for 5 seconds; (4) Extend at 72 °C for 40 seconds; (5) Repeat steps (2) - (4), cycle 35 times; (6) Extend fully at 72 °C for 5 minutes; (7) Store the amplification product at 4 °C.

[0042] Example 2 Comparison of enzyme activities of wild-type horseradish peroxidase and H40W mutant

[0043] The mutant plasmids obtained in Example 1 were respectively transformed into the competent cells of Saccharomyces cerevisiae EBY100, and the plates were coated and grown overnight; then single colonies were respectively picked and placed in 3 mL of SD + CAA liquid medium and cultured overnight; subsequently, they were transferred into 50 mL of SD + CAA liquid medium at a ratio of 1%, and when cultured at 30 °C for 24 h, they were replaced into 50 mL of SG + CAA liquid medium and cultured at 30 °C and 250 rpm for 24 hours to surface-display the target protein.

[0044] Take an appropriate amount of the above-obtained bacterial liquid and detect its activity respectively. The results are as Figure 3 shown. Compared with wild-type HRP, the H40W mutant showed a significant increase in activity, and the increase amplitude reached 3 times. H40W represents a mutant in which the 40th histidine of the wild-type horseradish peroxidase amino acid sequence shown in SEQ ID NO.1 is mutated to tryptophan.

[0045] Although the embodiments of the present invention have been disclosed as above, it is not limited to only the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details.

Claims

1. A horseradish peroxidase mutant with high catalytic activity, characterized in that: The horseradish peroxidase is obtained by mutating the 40th histidine in the amino acid sequence of the wild-type horseradish peroxidase shown in SEQ ID NO.1 into tryptophan.

2. A gene encoding the horseradish peroxidase mutant according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence encoding the wild-type horseradish peroxidase shown in SEQ ID NO.1 is SEQ ID NO.

3.

4. The gene according to claim 3, characterized in that The gene encoding the horseradish peroxidase mutant is obtained by site-directed mutagenesis based on the nucleotide sequence shown in SEQ ID NO.

3.

5. The gene according to claim 4, characterized in that The amino acid sequence of the horseradish peroxidase mutant is SEQ ID NO.2, and the nucleotide sequence encoding the horseradish peroxidase mutant as shown in SEQ ID NO.2 is SEQ ID NO.

4.

6. A recombinant plasmid comprising the gene according to any one of claims 2 to 5.

7. A host cell comprising the gene according to any one of claims 2 to 5 or the recombinant plasmid according to claim 6.

8. Use of the highly catalytically active horseradish peroxidase mutant according to claim 1 in catalyzing the production of resorufin from hydrogen peroxide and ADHP.

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