Superoxide dismutase mutant and its application

Abstract

The present invention discloses superoxide dismutase mutants and applications thereof. The superoxide dismutase mutants are E47S, G109H, and D120L mutants of the superoxide dismutase shown in SEQ ID NO: 1. The enzyme activities of the three mutants are all improved compared with the original enzyme. Among them, the activity of the G109H mutant is improved by 145.9%, and the thermal stability of the E47S mutant is improved by 22.5%, further enhancing the industrial application value of SOD, enabling it to be widely used in cosmetics, food, medicine, environmental protection and other fields.

Description

Technical Field

[0001] The present invention relates to the field of biological genes, and in particular to a superoxide dismutase mutant and application thereof. Background Art

[0002] Superoxide dismutase (SOD) is an important antioxidant enzyme in living organisms, widely distributed in various organisms, including animals, plants, and microorganisms. SOD possesses unique physiological activities and is the primary free radical scavenger in living organisms. SOD levels in an organism are a direct indicator of aging and mortality. Over 60 diseases have been shown to be caused by oxygen free radicals. SOD can counteract and block the damage caused by oxygen free radicals to cells, repairing damaged cells and restoring the cellular damage caused by free radicals. Due to the stress of modern life, environmental pollution, various radiation sources, and excessive exercise, which all contribute to the massive formation of oxygen free radicals, SOD plays an increasingly important role in the biological antioxidant mechanism.

[0003] Superoxide dismutase (SOD) can be divided into three types based on the metal cofactors they contain. The first type, Cu-Zn-SOD, is the most common SOD enzyme, containing copper and zinc cofactors. It is green and primarily found in the cytoplasm of the body. The second type, Mn-SOD, is purple and contains manganese cofactors. It is found in the mitochondria of eukaryotic cells and prokaryotes. The third type, Fe-SOD, is yellow-brown and contains iron cofactors.

[0004] SOD has anti-aging, immune regulation, blood lipid regulation, radiation resistance, and beauty-enhancing functions, and is widely used in cosmetics, food, medicine, environmental protection, and other fields. However, the application of SOD in the prior art is greatly limited by its shortcomings in heat resistance and stability.

[0005] The main methods for producing SOD include natural extraction (primarily from plant and animal tissues) and microbial fermentation. Currently, due to resource limitations, unstable supply chains, and ethical concerns, microbial fermentation has rapidly developed. The use of genetically engineered microorganisms has made it possible to stabilize the production process, reduce production costs, and expand the scope of experimental procedures.

[0006] Research on microbial production of superoxide dismutase (SOD) is increasing both domestically and internationally, but a key challenge is that the SOD produced cannot meet practical application requirements. Limited thermal stability and catalytic efficiency remain significant obstacles to the wider industrial application of SOD. For example, the SOD enzyme from Lactobacillus thermocyclus (Frontiers in Microbiology 12 (2021):577001) retains only 30% activity after incubation at 80°C for 60 minutes. Summary of the Invention

[0007] The first purpose of the present invention is to overcome the deficiencies of the prior art and provide a superoxide dismutase mutant having both good heat resistance and activity.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A superoxide dismutase mutant, wherein the 47th amino acid residue of superoxide dismutase is mutated to serine;

[0010] or mutating the 109th amino acid residue of superoxide dismutase to histidine;

[0011] or by mutating the 120th amino acid residue of superoxide dismutase to leucine;

[0012] Wherein, the amino acid sequence of the superoxide dismutase is shown as SEQ ID NO: 1.

[0013] A second object of the present invention is to provide a gene encoding the above-mentioned superoxide dismutase mutant.

[0014] The third object of the present invention is to provide a recombinant vector comprising the above gene. Preferably, the gene is cloned into a pET-22b plasmid to construct the recombinant vector.

[0015] The fourth object of the present invention is to provide a recombinant strain comprising the above gene. Preferably, the recombinant strain uses Escherichia coli as a host bacterium.

[0016] The fifth object of the present invention is to provide a method for expressing the mutant, specifically, using an E. coli expression system to express the enzyme protein.

[0017] As a preferred embodiment, the expression method comprises: cloning the gene sequence of the mutant into a vector plasmid, constructing a recombinant expression vector, and transforming the recombinant expression vector into Escherichia coli for induced expression.

[0018] As a preferred embodiment, the recombinant Escherichia coli bacterial solution was added to LB medium and cultured at 37°C and 200 rpm until the bacterial solution OD600 = 0.3~0.5, add IPTG inducer to induce expression

[0019] A seventh objective of the present invention is to provide the use of the aforementioned mutant in the preparation of antioxidant products. The superoxide dismutase mutant can counteract and block cellular damage caused by oxygen free radicals generated by environmental pollution, various radiation sources, and excessive exercise, and promptly repair damaged cells. It can be used in cosmetics, fruit and vegetable preservation, and pharmaceutical products.

[0020] This study purified and expressed the SOD enzyme from Deinococcus urumqiensis R12 (abbreviated as R12-SOD). Using semi-rational enzyme engineering techniques such as site-directed mutagenesis, the SOD enzyme was mutated to obtain three mutants with improved thermostability and activity: E47S, G109H, and D120L. The enzyme activity of all three mutants was enhanced compared to the original enzyme. The G109H mutant showed a 145.9% increase in activity, while the E47S mutant exhibited a 22.5% increase in thermostability. This further enhances the industrial application value of SOD, enabling its widespread application in cosmetics, food, medicine, environmental protection, and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Graph showing the thermal stability test results of R12-SOD at 80°C, 90°C, and 100°C in Example 2.

[0022] Figure 2 This is a graph showing the results of the enzyme activity test of the wild type R12-SOD and three mutants in Example 4.

[0023] Figure 3 Graph showing the thermal stability test results of the wild type R12-SOD and three mutants in Example 4 after incubation at 100°C for 60 minutes. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to the accompanying drawings and examples. The experimental methods below, where no specific conditions are specified, are based on conventional experimental conditions in the art or conditions recommended by the manufacturer.

[0025] The SOD gene of the present invention is derived from Deinococcus wulumuqiensis R12, and its amino acid sequence is shown in SEQ ID NO: 1.

[0026] Example 1

[0027] The protein expression vector of SOD gene was constructed by molecular cloning technology.

[0028] 1. Codon optimization and plasmid construction

[0029] The nucleotide sequence of the Mn-SOD gene encoding Deinococcus urumqiensis-R12 was codon-optimized, a His-tag was added at the 5' end, and then artificially synthesized and ligated into the pET-22b vector (manufactured by General Motors) through the cloning site NdeI-BlpI.

[0030] 2. Conversion

[0031] Take out the E. coli BL21 competent cells and plasmid from the -80°C and -20°C freezers, respectively, and thaw them on ice.

[0032] Add 100µL of thawed competent cells and 10µL of plasmid to the tube, gently stir with the pipette tip, and place on ice for 30 minutes.

[0033] Heat stimulate at 42°C for 90 seconds and quickly return to ice for 2 minutes.

[0034] Add 700 µL of antibiotic-free LB liquid medium to the competent cells containing the plasmid and incubate on a shaker at 37°C for 1 h.

[0035] Take 100µL of bacterial solution and spread it on LB solid medium containing corresponding antibiotics.

[0036] The culture dish was inverted and incubated at 37°C overnight.

[0037] 3. Sequencing verification and construction of expression strains

[0038] Multiple single colonies were selected from the culture dishes cultured overnight after transformation, inoculated into liquid LB medium with ampicillin resistance, cultured overnight, and verified by bacterial liquid sequencing. Sequencing was performed using T7 upstream and downstream universal sequencing primers, and the results showed that the sequence was correct.

[0039] Example 2

[0040] Protein induction expression, activity and thermal stability testing of R12-SOD

[0041] 1. Inducible expression

[0042] Take 300 μL of the bacterial solution with the correct sequence and overnight culture in step 3 of Example 1, add it to 50 mL of LB medium, add ampicillin, and culture at 37°C and 200 rpm.

[0043] When the OD value reached 0.5 (0.3-0.5), IPTG was added to a final concentration of 0.1 mM, and then cultured at 37°C, 200 rpm for 12 h.

[0044] 2. Ultrasonic cell disruption (30 mL bacterial solution)

[0045] Pour 30 mL of bacterial suspension into a 50 mL centrifuge tube, balance, and centrifuge at 8000 rpm for 5 minutes. Discard the supernatant and continue adding bacterial suspension to the tube. Balance with RO water, continue centrifugation, and discard the supernatant. Finally, add 5 mL of PBS and resuspend by pipetting. Centrifuge again and discard the supernatant.

[0046] After all the cells are collected, add 2 mL of PBS to dissolve the cells and transfer them to the corresponding 5 mL centrifuge tube. Then add 1 mL of PBS to wash the centrifuge tube and transfer it to the 5 mL centrifuge tube.

[0047] Ultrasonic disruption was performed in an ice bath at a power of 300 W, with a disruption time of 3 s followed by a rest time of 5 s for a total of 10 min.

[0048] The disrupted bacterial solution was centrifuged at 5000 rpm for 20 min at 4°C.

[0049] The supernatant and precipitate were separated and set aside. The precipitate was resuspended in 2 mL of PBS and sampled for subsequent protein gel running. The supernatant was used for purification and stored in a -20°C refrigerator.

[0050] The protein sequence of the SOD recombinantly expressed by genetic engineering in the supernatant is shown in SEQ ID NO: 1.

[0051] 3. Protein purification

[0052] Drain the alcohol from the nickel column stored at -20°C and add 5 mL of 30 Mm imidazole buffer (Buffer A), 3 mL of 300 Mm imidazole buffer (Buffer B), and 5 mL of Buffer A to wash the column.

[0053] Add crude enzyme solution and wash twice with 5 mL of Buffer A to remove contaminants. Then add 3 mL of Buffer B and pour into a prepared, labeled beaker to obtain the target protein.

[0054] Add 3 mL of Buffer B and 5 mL of Buffer A to wash the column and seal it with 20% alcohol.

[0055] Pour the crude enzyme solution in the beaker into an ultrafiltration tube (sealed with ultrapure water) and centrifuge at -4°C and 6500 rpm for 30 min.

[0056] Afterwards, add 2 mL of ultrapure water and centrifuge at 6500 rpm for 30 min.

[0057] Add 2 mL of ultrapure water and centrifuge at 6500 rpm for 15 minutes to obtain the concentrated pure enzyme solution. Store the concentrated pure enzyme solution in a -4°C refrigerator.

[0058] 4. Activity Detection

[0059] At approximately 25°C, add 94 μL of 0.1 mol / L Tris-HCl (1 mmol / L EDTA•2Na), 80 μL of 1× PBS buffer, and 6 μL of a 4.5 mmol / L pyrogallol hydrochloric acid solution to a 96-well plate cuvette. Immediately mix thoroughly after adding the 4.5 mmol / L pyrogallol hydrochloric acid solution and pour into a cuvette. Measure the absorbance at 325 nm at 0, 30 s, 60 s, 90 s, 120 s, 150 s, and 180 s. Calculate the absorbance increment from 30 s to 150 s: the absorbance at 150 s minus the absorbance at 30 s divided by 2 to obtain the pyrogallol autoxidation rate, ΔA325 (min-1).

[0060] The sample solution was diluted to 0.05 mg / mL. The inhibition rate of pyrogallol auto-oxidation by the sample solution was determined by adding a certain amount of sample solution according to step 1 so that the inhibition rate of pyrogallol auto-oxidation was approximately 1 / 2ΔA325 (min-1), that is, ΔA'325 (min-1).

[0061] Total enzyme activity =

[0062] Specific enzyme activity = total enzyme activity

[0063] The test results showed that the specific enzyme activity of R12-SOD was 1351U / mg.

[0064] 5. Thermal stability test

[0065] The pure enzyme solution was kept at 80°C, 90°C, and 100°C, and the SOD enzyme activity was measured at 15 minutes, 30 minutes, 45 minutes, and 60 minutes, respectively. The thermal stability was calculated and expressed as the percentage of activity retention.

[0066] The results are as follows Figure 1 As shown:

[0067] The SOD enzyme of the present invention retained 76% of its activity after being incubated at 80°C for 60 minutes, 73% of its activity after being incubated at 90°C for 60 minutes, and 48% of its activity after being incubated at 100°C for 60 minutes. This indicates that the SOD enzyme of the present invention has good thermal stability.

[0068] Example 3

[0069] Site-directed mutagenesis of R12-SOD superoxide dismutase

[0070] The glutamic acid at position 47, glycine at position 109, and aspartic acid at position 120 in the amino acid sequence of the SOD enzyme shown in SEQ ID NO: 1 were mutated to serine, histidine, and leucine, respectively, to obtain three mutants: E47S, G109H, and D120L. Blunt-ended primers were designed using SnapGene 6.0.2, and site-directed mutagenesis was performed using PCR using the R12-SOD enzyme of the present invention as a template.

[0071] The base sequences of the primers are shown in Table 1:

[0072] Table 1 Primer sequences

[0073]

[0074] The PCR reaction system and cycle process are shown in Table 2 and Table 3 respectively:

[0075] Table 2 PCR reaction system

[0076]

[0077] Table 3 PCR cycle process

[0078]

[0079] After the PCR reaction, 1 μL of DpnI and 5 μL of 10× EC Buffer were added to a 50 μL system and incubated at 37°C for 1 hour and 30 minutes, followed by incubation at 80°C for 45 minutes to digest the template. Agarose gel electrophoresis was used to verify site-directed mutagenesis. If successful, the PCR product was recovered and cloned into an expression vector according to the methods of Examples 1 and 2. Expression of the target protein was induced and purified.

[0080] 1. Activity detection

[0081] The specific enzyme activities of the E47S, G109H, and D120L mutants were measured using the pyrogallol method.

[0082] like Figure 2 As shown, the specific enzyme activities of E47S, G109H, and D120L mutants were 1726.9 U / mg, 3322.5 U / mg, and 1442.1 U / mg, respectively.

[0083] 2. Thermal stability test

[0084] The pure enzyme solution was kept at 100°C, and the SOD enzyme activity was measured at 15 minutes, 30 minutes, 45 minutes and 60 minutes, and its thermal stability was calculated and expressed as the percentage of activity retention. Figure 3The results showed that the residual activities of the three mutants, E47S, G109H, and D120L, after incubation at 100°C for 60 min were 89.4%, 48.2%, and 87.2%, respectively. The specific enzyme activities of the wild type and the three mutants, E47S, G109H, and D120L, as well as the thermal stability data after incubation at 100°C for 60 min, are shown in Table 4.

[0085] Table 4 Activity and thermal stability of the wild type and three mutants: E47S, G109H, and D120L

[0086]

[0087] The results of wild-type superoxide dismutase assays in Example 2 showed that the specific enzyme activity and thermal stability of the three mutants, E47S, G109H, and D120L, were improved compared to the wild type. The G109H mutant achieved a specific enzyme activity of 3322.5 U / mg, a 145.9% increase. The E47S mutant retained 89.4% of its activity after incubation at 100°C for 60 minutes, a 22.5% increase. These enhanced mutant activities and thermal stability further broaden the industrial application value of SOD.

Claims

1. A superoxide dismutase mutant, characterized in that Mutating glutamic acid at position 47 of superoxide dismutase to serine; or mutating the glycine at position 109 of superoxide dismutase to histidine; Or it can be obtained by mutating the aspartic acid at position 120 of superoxide dismutase to leucine; Wherein, the amino acid sequence of the superoxide dismutase is shown as SEQ ID NO:

1.

2. A gene encoding the superoxide dismutase mutant according to claim 1.

3. A recombinant vector comprising the gene according to claim 2.

4. The recombinant vector according to claim 3, characterized in that The gene was cloned into pET-22b plasmid to construct the recombinant vector.

5. A recombinant strain comprising the gene according to claim 2.

6. The recombinant strain according to claim 5, characterized in that The recombinant strain uses Escherichia coli as a host bacterium.

7. The method for expressing the mutant according to claim 1, characterized in that: The mutant was expressed using an E. coli expression system.

8. The expression method according to claim 7, characterized in that The gene sequence of the mutant is cloned into a vector plasmid to construct a recombinant expression vector, which is then transformed into Escherichia coli for induction of expression and purification.

9. The expression method according to claim 8, characterized in that The induction expression conditions were as follows: the recombinant E. coli bacterial solution was added to LB medium and cultured at 37°C and 200 rpm until the bacterial solution OD 600 = 0.3~0.5, add IPTG inducer to induce expression.

10. Use of the mutant according to claim 1 in preparing antioxidant products.

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