CN117987384A - Cold-resistant and salt-resistant superoxide dismutase and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of superoxide dismutase, and discloses a cold and salt tolerant superoxide dismutase, a preparation method and application thereof, wherein the superoxide dismutase is encoded by a nucleotide sequence shown as SEQ ID NO. 1, and the superoxide dismutase not only has cold and salt tolerant properties, but also can regulate the activity through metal ions and chemical reagents, and has good application prospects in the fields of low-temperature transportation of transplanted organs, low-temperature tolerant skin care products and the like.

Description

A psychrophilic and salt-tolerant superoxide dismutase and its preparation method and application

Technical Field

The invention belongs to the field of biotechnology and relates to a psychrophilic and salt-tolerant superoxide dismutase and a preparation method and application thereof.

Background technique

Superoxide dismutase (EC1.15.1.1) is an important antioxidant enzyme in organisms and is widely distributed in various organisms, such as animals, plants, and microorganisms. Superoxide dismutase has special physiological activity and is the primary substance for removing free radicals in organisms. It is widely used in food, cosmetics, and health products.

The development of superoxide dismutase has always been an important research direction in the enzyme industry. People widely use biochemical methods to extract superoxide dismutase from the serum or liver of animals such as pigs, chickens, and cattle, and also extract it from plants such as vegetables, seeds, fruits or grains. However, the poor stability of superoxide dismutase, low content in animals and plants, and low extraction yield have always been factors that restrict the industrialization of superoxide dismutase and limit its large-scale promotion and application. In recent years, the use of genetic engineering technology to obtain superoxide dismutase recombinant protein has become a research focus, but there are still few reports on the large-scale industrial production of superoxide dismutase using microorganisms. In addition, the thermal stability, tolerance to acid-base environments, salt tolerance, resistance to chemical reagents and metal ions of existing superoxide dismutase are generally good, and its application in some special use environments is limited, such as in the polar regions or in the north in winter, where the ambient temperature is low and the activity of superoxide dismutase is significantly reduced. Its application in cosmetics does not play a corresponding role. Reactive oxygen damage is easily generated during low-temperature transportation of human organs. The addition of superoxide dismutase can remove superoxide anion free radicals. The existing superoxide dismutase has poor salt and low-temperature resistance, which limits its use in this field.

Summary of the invention

The object of the present invention is to provide a psychrophilic and salt-tolerant superoxide dismutase and a preparation method and application thereof. The superoxide dismutase is encoded by a nucleotide sequence as shown in SEQ ID NO: 1, and the superoxide dismutase is a copper-zinc superoxide dismutase.

Based on the above purpose, on the one hand, the present invention provides a method for preparing a psychrophilic and salt-tolerant superoxide dismutase, comprising: connecting the nucleotide sequence shown in SEQ ID NO: 1 to a vector to prepare an expression vector, introducing the expression vector into a host bacterium to prepare the recombinant bacterium, extracting and purifying the protein in the recombinant bacterium, and separating 19-22kD protein.

The copper-zinc superoxide dismutase has an activity retention rate of not less than 70% under the condition of pH 8-9.5; the copper-zinc superoxide dismutase has an activity retention rate of not less than 50% in 1.5M sodium chloride; and the reaction temperature of the copper-zinc superoxide dismutase is 10°C-20°C and the enzyme activity is greater than 545U/mg.

In a second aspect, the present invention provides a method for regulating the activity of superoxide dismutase, wherein the method uses DTT, Tween-80, Mn ²⁺ , Cu ²⁺ , and Zn ²⁺ to promote the activity of the copper-zinc superoxide dismutase; uses SDS, TritonX-100, Ca ²⁺ , and Mg ²⁺ to inhibit the activity of the copper-zinc superoxide dismutase; and uses Co ²⁺ to completely inhibit the copper-zinc superoxide dismutase.

In a third aspect, the present invention provides a Deinococcus strain isolated from Arctic sandy soil, labeled as N-S031, the N-S031 strain can produce superoxide dismutase, and the nucleotide sequence of the gene encoding the superoxide dismutase is shown in SEQ ID NO:1.

The N-S031 strain described in the present invention was isolated from sandy soil collected in Langyearbyen, Arctic (15.28 east longitude, 78.14 north latitude) in September 2019, and the preservation information is as follows:

Taxonomic nomenclature: Deinococcus sp.

Storage time: November 5, 2023

Depository: Guangdong Microbiological Culture Collection Center

Address of the preservation unit: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou

Deposit number: GDMCC NO:63982

In a fourth aspect, the present invention provides a method for preparing superoxide dismutase, comprising cloning a gene with a nucleotide sequence as shown in SEQ ID NO: 1 into a prokaryotic expression vector pET22b(+), transforming the above recombinant vector into BL21(DE3) competent cells to obtain recombinant bacteria, extracting and purifying the protein in the recombinant bacteria, and separating a 19-22 kD protein.

Specifically, the present invention picks a single colony of Escherichia coli and inoculates it into 10 mL of LB culture medium containing 100 mg/mL ampicillin, cultures it at 37°C in a shaking incubator overnight, transfers it to an auto-induction medium containing Amp the next day, cultures it at 37°C in a shaking incubator until the middle of logarithmic growth, lowers the culture temperature to 8°C-12°C, maintains the cold pressure for 4h-8h, raises the culture temperature to 37°C, continues to culture for 8h-14h, and collects the bacteria by centrifugation.

More specifically, the present invention collects the recombinant bacteria and then analyzes the collected recombinant bacteria by SDS-PAGE electrophoresis to obtain a new electrophoresis band, indicating that only one superoxide dismutase is produced, and the expression amount of the superoxide dismutase is analyzed by gel scanning, and the expression amount of the superoxide dismutase accounts for 60% of the total amount of the recombinant bacterial protein.

In a fifth aspect, the superoxide dismutase provided by the present invention is used in preparations for ensuring organ activity during low-temperature transportation of transplanted organs and in low-temperature resistant cosmetics.

Compared with the prior art, the present invention has the following beneficial effects or advantages:

The present invention provides a superoxide dismutase having good cold-loving and salt-tolerance properties as measured by enzyme activity. In particular, the protein exhibits good activity under alkaline conditions, with the highest activity at pH 8.5, and the activity is maintained at more than 70% of the highest activity when the pH is within the range of 8-9.5. The copper-zinc superoxide dismutase has an activity retention rate of not less than 50% in 1.5M sodium chloride. The superoxide dismutase exhibits the highest enzyme activity within the range of 10-20°C, and thermal stability analysis also shows that the activity retention rate of the superoxide dismutase decreases with increasing temperature, and the superoxide dismutase is unstable at high temperatures above 30°C, and has good low-temperature properties.

The DTT, Tween-80, Mn ²⁺ , Cu ²⁺ , and Zn ²⁺ provided by the present invention have a promoting effect on the activity of copper-zinc superoxide dismutase, SDS, TritonX-100, Ca ²⁺ , and Mg ²⁺ have an inhibitory effect on the activity of copper-zinc superoxide dismutase, and Co ²⁺ completely inhibits the activity of copper-zinc superoxide dismutase.

The invention provides a cold-loving and salt-tolerant superoxide dismutase-producing strain, which has the advantages of being easy to culture and easy to produce on a large scale. The invention extracts a superoxide dismutase gene from the strain, constructs an expression vector, and transforms the recombinant bacteria to obtain a high-yield superoxide dismutase, thereby realizing the large-scale production of the superoxide dismutase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the growth morphology of the N-S031 strain on a TSA medium plate.

FIG. 2 shows the morphology of the N-S031 strain under a 10×100 optical microscope.

FIG. 3 is a diagram showing the PCR amplification results of the recombinant expression vector.

FIG. 4 is a diagram showing the results of SDS-PAGE analysis of the recombinant expression product, in which M is a low molecular weight marker; 1 is uninduced; and 2 is the result of IPTG-induced superoxide dismutase.

Figure 5 is a diagram showing the results of superoxide dismutase lysis of bacteria. In the figure, M is a low molecular weight marker; 1 is the supernatant of superoxide dismutase lysis of bacteria; and 2 is the precipitate of superoxide dismutase lysis of bacteria.

Figure 6 is a diagram showing the results of superoxide dismutase protein purification, where M is a low molecular weight marker and 1 is the purified superoxide dismutase.

FIG. 7 is a diagram showing the results of superoxide dismutase classification.

FIG. 8 is a graph showing the effect of different pH values on the activity of superoxide dismutase obtained in the present invention.

FIG. 9 is a graph showing the salt tolerance test results of superoxide dismutase obtained in the present invention.

FIG. 10 is the result of the effect of temperature on the enzyme activity of superoxide dismutase and recombinant human superoxide dismutase (rhSOD) obtained in the present invention.

FIG. 11 is a result showing the effect of temperature on the stability of superoxide dismutase activity obtained in the present invention.

Detailed ways

The technical solution of the present invention is described below in conjunction with embodiments; however, the present invention is not limited to the following embodiments.

In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

The experimental methods and detection methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

Example 1

This example provides strain isolation and identification.

(1) Strain isolation

The strain used in the present invention was isolated from sandy soil collected in Langyearbyen, Arctic (15.28 east longitude, 78.14 north latitude) in ^September 2019. After the soil sample was collected, 1g was placed in a sterile centrifuge tube, and then 9mL of sterile 0.9% saline and an appropriate amount of glass beads were added to shake at room temperature for 30min. After the gradient dilution was ^10-1-10-5 , 0.1mL of dilution was drawn from each gradient and added to the TSA (tryptone soy agar medium) plate and spread. The medium formula was 15g of tryptone, 5g of soy peptone, 5g of sodium chloride, and 15g of agar; the pH value was 7.3±0.2. The TSA medium plate was placed at 15°C for constant temperature culture. After the colonies were grown, colonies of different shapes, sizes, colors, etc. were picked and inoculated on the TSA medium plate until there were no mixed colonies. A strain was selected from them, numbered as the N-S031 strain, and the deposit number was: GDMCC NO:63982.

The N-S031 strain selected above was inoculated into TSB liquid medium (the medium formula was 15g tryptone, 5g soy peptone, 5g sodium chloride; pH value was 7.3±0.2.) and cultured in a shaking incubator at 15°C for 5 days. The cultured bacteria were collected to extract the genomic DNA of the N-S031 strain. The target fragment was amplified using 16S rRNA specific primers (27F: 5'-AGAGTTTGATCCTGG CTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'), and the amplified product was connected to the pGMT-18 vector and transformed into the host bacteria DH5α competent cells, and then coated on LB/Amp+/X-Gal/IPTG plates for blue-white screening. After selecting white clones for colony PCR verification, the target fragment was recovered and sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing.

(2) Strain identification

Basic identification of strains The results of 16S rRNA gene sequencing and clone sequencing of strain N-S031 showed that the strain had the highest similarity with Deinococcus aeria of the genus Deinococcus.

The results of the study on the morphological characteristics of the N-S031 strain showed that the colony morphology on the TSA medium plate was: light orange, with protruding surface, smooth, sticky, and 1-2 mm in diameter, as shown in Figure 1. The morphology of the N-S031 strain was observed under a 10×100 optical microscope. After staining with ammonium oxalate-crystal violet, the morphology of the N-S031 strain was short and thick rod-shaped or nearly spherical, and the Gram staining result was Gram-positive bacteria G ⁺ , as shown in Figure 2.

Example 2

This example provides gene acquisition and expression system construction.

1 g of N-S031 wet bacteria was collected and sent to Beijing Biomarker Biotechnology Co., Ltd. for whole genome sequencing of the strain. According to the whole genome data analysis, the superoxide dismutase gene sequence was searched and the nucleotide sequence of the expressed superoxide dismutase gene was determined to be as shown in SEQ ID NO: 1.

(1) Prokaryotic expression

According to the gene sequence of superoxide dismutase, primers were designed, and NdeⅠ and XhoⅠ restriction sites (Fw: 5'-ATACAT ATGGCCCCGGCTACCACG-3', Rv: 5'-GTGCTCGAGCAGGGA ATAGTCCCG-3') were added to both ends of the superoxide dismutase gene. The superoxide dismutase gene was cloned into the prokaryotic expression vector pET22b(+) through the NdeⅠ restriction site and XhoⅠ restriction site. The above recombinant vector was transformed into BL21(DE3) competent cells, and a single clone was picked and cultured overnight; the plasmid was extracted to obtain the engineered bacteria. It was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. As shown in Figure 3, the expected insertion fragment of 522bp size was obtained, indicating that the above vector was constructed and transformed successfully.

(2) Identification of expression products

A single colony of the superoxide dismutase engineering bacteria of this example was picked and inoculated into 10 mL of LB culture solution containing 100 mg/mL ampicillin (Amp), and cultured overnight at 37° C. in a shaking incubator. The next day, the colony was transferred to an autoinduction medium containing Amp at a volume ratio of 1:100 (the formula of the autoinduction medium was 12 g/L tryptone, 24 g/L yeast extract, 30 mL/L glucose, 2 g/L lactose, 12.8 g/L Na ₂ HPO ₄ , 3.4 g/L KH ₂ PO ₄ , 2.7 g/L NH ₄ Cl, 15.8 g/L succinate, 0.7 g/L Na ₂ SO ₄ , pH=6.8), and cultured at 37° C. in a shaking incubator for 3.5 h until the mid-logarithmic growth phase (OD600 was 0.4-0.6), the culture temperature was reduced to 10° C., the cold pressure was maintained for 6 h, the culture temperature was increased to 37° C., and the culture was continued for about 12 h. Collect the cells by centrifugation.

Take a small amount of the above bacteria, add 50 μL of water and 50 μL of buffer (2X), and mix well. Boil in boiling water for 5 minutes, centrifuge at 12000rpm for 5 minutes, take 4.5 μL of supernatant for SDS-PAGE detection, voltage 160V, electrophoresis 1h. Use 0.5% Coomassie Brilliant Blue R-250 to oscillate and stain for 1h, decolorize until the background is clear, and then prepare dry gel for storage. The results are shown in Figure 4. Lane 2 has a new electrophoresis band near 20.1kD, indicating that the expression vector of the protein is effectively expressed. Gel scanning analysis of expression, the results show that the overall expression of superoxide dismutase accounts for 60% of the total bacterial protein.

(3) Recombinant protein purification

Take 1g of the fermented bacteria collected in Example (2) and add it to 10mL of lysis buffer, stir at 4°C to obtain a bacterial suspension. Then add 0.5mg of lysozyme, stir at 4°C for 20min, add 7mg of sodium deoxycholate, stir at room temperature until it becomes viscous, add _MgCl2 to a final concentration of 10mmol/L, add 5μL of DNaseⅠ, and stir until it is no longer viscous. Take 50μL of the solution after lysis, centrifuge at 4°C, 15000rpm for 30min, aspirate the supernatant, add 50μL of buffer (2X), and mix. Add 50μL of water to the lysis precipitate sample, suspend it, and then add 50μL of buffer (2X) and mix. Boil in boiling water for 5min, and centrifuge at 12000rpm for 5min. Take 4.5μL of the supernatant of the obtained sample for SDS-PAGE detection. Use 0.5% Coomassie Brilliant Blue R-250 for oscillation staining for 1h, decolorize until the background is clear, and then prepare a dry gel. The results are shown in 5. The target protein (superoxide dismutase) was distributed in both the supernatant and the precipitate of the lysate.

The ratio of the target protein (superoxide dismutase) in the supernatant and the precipitate was analyzed by SDS-PAGE gel scanning. The results showed that the ratio of superoxide dismutase in the supernatant was 80.5% of the total protein expression.

The protein was purified from the supernatant. Copper sulfate was added to the supernatant of superoxide dismutase to a final concentration of 1 mmol/L, and then dialyzed into 0.01 M Tris-HCl buffer at pH 8.0, and purified by DEAE column. The target protein was eluted with a gradient of 0.01 M Tris-HCl at 0-0.5 M NaCl and pH 8.0, and the elution peak was collected. The purified protein was subjected to SDS-PAGE and specific activity assay. The SDS-PAGE results are shown in Figure 6. The target protein peak shown in lane 1 is near 20.1 kD, and the purity is greater than 90%, which is consistent with the expected results. According to GB/T5009.171-2003, the activity of the purified protein, superoxide dismutase, was assayed using the modified Marklund method, and the result was 545 U/mg.

Protein activity assay:

Reagents: Solution A: pH 8.2, 0.1 mol/L Tris-HCl (containing 1 mmol/L EDTA·2Na). Weigh 1.2114 g Tris and 37.2 mg EDTA·2Na, dissolve in 62.4 mL 0.1 mol/L HCl solution, and dilute to 100 mL with distilled water;

Solution B: 4.5mmol/L pyrogallol solution. Weigh 56.7mg of pyrogallol (AR) and dissolve it in 10mmol/L hydrochloric acid solution, then dilute to 100mL with 10mmol/L hydrochloric acid solution.

At about 25°C, add 2.35 mL of solution A, 2.00 mL of distilled water, and 0.15 mL of solution B in a 10 mL colorimetric tube. Mix immediately after adding solution B, start timing, and measure the self-oxidation rate absorbance of pyrogallol at 325 nm after 1 minute, expressed as ΔA ₃₂₅ ;

At about 25°C, add 2.35mL of solution A, 1.8mL of distilled water, 20uL of superoxide dismutase solution to be tested, and 0.15mL of solution B in a 10mL colorimetric tube. Mix immediately after adding solution B and start timing. After 1 minute, measure the absorbance of the sample at 325nm. Take the absorbance value of about 1/2 of the self-oxidation rate of pyrogallol as the sample measurement value and substitute it into the following formula for calculation:

U/mL—superoxide dismutase activity unit;

ΔA ₃₂₅ —pyrogallol autoxidation rate;

ΔA′ ₃₂₅ —Superoxide dismutase enzyme solution inhibits the rate of pyrogallol autooxidation;

V—superoxide dismutase enzyme solution volume (mL);

D—dilution multiple of superoxide dismutase solution;

4.5—Total reaction volume (mL).

Example 3

This example provides a method for determining the enzymatic properties of superoxide dismutase.

(1) Identification of superoxide dismutase enzyme types

2ug of purified superoxide dismutase was pre-incubated with different concentrations of NaN ₃ (5, 10, 15, 20 and 25mM) and H ₂ O ₂ (5, 10, 15, 20 and 25mM) at 35°C for 1h, and then the residual activity of each sample was determined by the enzyme activity assay method. Superoxide dismutase was treated with 25mM NaN ₃ and 25mM H ₂ O ₂ for 1h, and the residual rates of superoxide dismutase were 91.6% and 25.0%, respectively. The results showed that as shown in Figure 7, NaN ₃ had no significant effect on superoxide dismutase, but the presence of H ₂ O ₂ led to the denaturation of superoxide dismutase. Therefore, it was further speculated that superoxide dismutase belongs to copper-zinc superoxide dismutase, which is consistent with sequence analysis.

(2) Effect of different pH on the activity of superoxide dismutase of the present invention

In order to determine the optimal pH of copper-zinc superoxide dismutase, the samples were placed in different buffers: 100mM citric acid-Na ₂ HPO ₄ buffer (pH 4.0-7.0), 100mM Tris-HCl buffer (pH 7.0-9.0), 100mM glycine sodium hydroxide buffer (pH9.0-10.0), Na ₂ CO ₃ -NaHCO ₃ (pH 10.0-11.0), incubated at 35°C for 2min, and then the residual activity was detected. The study under different pH conditions is shown in Figure 8. Superoxide dismutase has good adaptability to a wide pH range. The protein showed good activity under alkaline conditions, with the highest activity at pH 8.5. When the pH range was 8-9.5, the activity remained above 70% of the highest activity, and the enzyme activity was relatively stable.

(3) Effects of different salt concentrations on superoxide dismutase activity

In terms of its salt tolerance, the purified superoxide dismutase was dissolved in a 0-2.5M sodium chloride solution, pre-incubated at 25°C for 1 hour, and its residual enzyme activity was measured. The results are shown in Figure 9. Superoxide dismutase can maintain about 50% of the maximum activity in 1.5M sodium chloride, indicating that the protein has a certain resistance to salt stress.

(4) Effects of different metal ions and chemical reagents on superoxide dismutase activity

After various metal ions or chemical reagents were mixed with purified superoxide dismutase and incubated at 25℃ for 1h, the residual activity was detected. The results are shown in Table 1. 1mM DTT and 0.2% Tween-80 can maintain 102.5% and 108.7% activity; 1% SDS and 0.2% TritonX-100 have a slight inhibitory effect on superoxide dismutase activity; Co ²⁺ completely inhibits rDs superoxide dismutase activity; while Ca ²⁺ and Mg ²⁺ have a partial inhibitory effect on superoxide dismutase activity. After adding 2.5mM Ba ²⁺ , the activity of superoxide dismutase is slightly reduced, and the addition of 2.5mM Mn ²⁺ , Cu ²⁺ and Zn ²⁺ can increase the activity of superoxide dismutase.

Table 1 Effects of different metal ions and chemical reagents on superoxide dismutase activity

Reagents concentration Residual activity (%) Reagents concentration Residual activity (%) Blank control 2.5mM 100 Co ²⁺ 2.5mM 0 Mg ²⁺ 2.5mM 80.2 Ba ²⁺ 2.5mM 92.4 Mn ²⁺ 2.5mM 110 SDS 1% 67 Ca ²⁺ 2.5mM 50.2 DTT 1mM 102.5 Cu ²⁺ 2.5mM 105 TritonX-100 0.2% 79.5 Zn2 ⁺ 2.5mM 121 Tween-80 0.2% 108.7

(5) Effect of different temperatures on the enzyme activity of superoxide dismutase and recombinant human superoxide dismutase (rhSOD) obtained in the present invention

The superoxide dismutase obtained by the present invention and the recombinant human superoxide dismutase (rhSOD) developed by our center were dissolved in a buffer solution with a pH of 8.5, and the purified superoxide dismutase was incubated at 4°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C and 90°C for 10 minutes. Then the superoxide dismutase activity was immediately determined, the maximum enzyme activity was set to 100%, and the activity retention rate of superoxide dismutase at different temperatures was calculated. The superoxide dismutase of the present invention exhibits the highest enzyme activity in the range of 10-20°C, and the enzyme activity decreases with increasing temperature; the recombinant human superoxide dismutase (rhSOD) has the highest enzyme activity at 30°C, and the enzyme activity shows a downward trend with increasing temperature. The results are shown in Figure 10. The comparison results of the two show that the enzyme activity of the superoxide dismutase of the present invention is better than that of the recombinant human superoxide dismutase (rhSOD) at lower temperatures.

(6) Effect of Detection Temperature on the Stability of Superoxide Dismutase Activity of the Present Invention

The superoxide dismutase obtained by the present invention is dissolved in a buffer solution with a pH of 8.5, and the purified superoxide dismutase is incubated for 60 min at 4°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C and 90°C, respectively. The superoxide dismutase activity is then measured at room temperature (about 25°C), the maximum value of the enzyme activity is set as 100%, and the activity retention rate of the superoxide dismutase at different temperatures is calculated. The thermal stability analysis shows that the activity retention rate of the superoxide dismutase decreases with the increase of temperature, and the superoxide dismutase is unstable at high temperatures above 30°C, and has good low-temperature characteristics (the results are shown in Figure 11).

Example 4

This example provides a superoxide dismutase activity regulation experiment

A mixture of 2 mM Ca ²⁺ , 2 mM Zn ²⁺ and 0.2% Tween-80 was prepared and divided into three portions. The superoxide dismutase obtained in the present invention was added to one portion and mixed. The mixture was incubated at 25° C. for 1 hour. The residual activity was detected and the residual activity was 105.4%.

Another portion of the mixed solution was added with 0.2% TritonX-100, and then the superoxide dismutase obtained in the present invention was added and mixed, and then incubated at 25°C for 1 hour. The residual activity was detected, and the residual activity was 103.7%.

To the third mixed solution, 2 mM Mn ²⁺ was added, and the superoxide dismutase obtained in the present invention was added and mixed, and then incubated at 25° C. for 1 hour. The residual activity was detected, and the residual activity was 106.3%.

The above test shows that the detection reagent provided by Example 3 of the present invention can control the activity of superoxide dismutase by adjusting the components of the mixed solution, so that the activity of superoxide dismutase can be adjusted in time according to the needs in different use environments. For example, when better oxygen free radical scavenging ability is required, ions or substances that promote activity are added, and when certain oxygen free radicals are required to achieve the balance between oxidation and anti-oxidation of the body, inhibitory reagents are used for inhibition.

Example 5

This example provides a specific application experiment of superoxide dismutase

(1) Application in organ transplantation transportation

The commonly used tissue cell preservation fluid UW solution was purchased and divided into two groups. The temperature of the low-temperature preservation box was adjusted to 0-8°C, and the experimental sample was pig heart.

In one group of UW solutions, 1% of the superoxide dismutase obtained by the present invention was added, and then 2mM Zn ²⁺ was added to make the final concentration reach 50mg/L; in one group of UW solutions, the superoxide dismutase and Zn ²⁺ obtained by the present invention were not added. After the two groups of solutions were stored for 72 hours, the cell survival rate was detected using a fluorescent cell counter. The results showed that the cell survival rate of the group without the superoxide dismutase and Zn ²⁺ obtained by the present invention was 85%, and the cell survival rate of the group with the superoxide dismutase and Zn ²⁺ obtained by the present invention was 89%. Therefore, the superoxide dismutase of the present invention can be used in the transportation of transplanted organs to reduce the impact of the transportation link on the quality and safety of organ transplantation.

(2) Application in cosmetics for low temperature environments

In special environments such as northern regions or polar regions and high mountains, it is common for outdoor temperatures to be below zero in winter, and the temperature of the human skin surface will drop accordingly. These regions have strong winds and sandstorms and strong ultraviolet rays, and the demand for cosmetics is urgent. In this example, the superoxide dismutase obtained by the present invention and the common superoxide dismutase available on the market are added to cosmetics in the same amount to test the antioxidant activity of the two different superoxide dismutases at about 10°C.

The selected cosmetic ingredients contain aloe extract, oil, moisturizer, emulsifier, thickener, preservative, essence and deionized water, and do not contain superoxide dismutase. The control group uses commercially available S9697 superoxide dismutase; the experimental group uses the superoxide dismutase obtained by the present invention. 2.5 mL of experimental cosmetics and 1.8 mL of distilled water are added to a 10 mL colorimetric tube in sequence, and 120 uL of the superoxide dismutase solution of the control group and the superoxide dismutase solution obtained by the present invention are added respectively to obtain a control group and an experimental group. The mixture is stored at 10° C. for 12 hours, and the enzyme activity of the control group and the experimental group is tested. As a result, the enzyme activity of the control group is 42%, while the enzyme activity of the experimental group is 79%. It can be seen that the superoxide dismutase of the present invention can continue to play a skin care effect of scavenging free radicals in a low temperature environment, while the superoxide dismutase enzyme activity of the prior art is greatly reduced, and it is difficult to play a good skin care effect.

As described above, the present invention can be well implemented. The above embodiments are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the present invention.

Claims (10)

1. A superoxide dismutase, characterized in that the superoxide dismutase is encoded by the nucleotide sequence shown in SEQ ID NO: 1. 2 . The superoxide dismutase according to claim 1 , wherein the superoxide dismutase is copper-zinc superoxide dismutase. 3 . The superoxide dismutase according to claim 2 , wherein the reaction temperature of the copper-zinc superoxide dismutase is 10° C.-20° C. 4. A method for regulating the activity of superoxide dismutase according to claim 1, characterized in that the method uses DTT, Tween-80, Mn ²⁺ , Cu ²⁺ , and Zn ²⁺ to promote the activity of the copper-zinc superoxide dismutase; uses SDS, TritonX-100, Ca ²⁺ , and Mg ²⁺ to inhibit the activity of the copper-zinc superoxide dismutase; and uses Co ²⁺ to completely inhibit the copper-zinc superoxide dismutase. 5. An expression vector, characterized in that it comprises the nucleotide sequence shown in SEQ ID NO: 1. 6. The method for preparing superoxide dismutase according to claim 1, characterized in that it comprises: connecting the nucleotide sequence shown in SEQ ID NO: 1 to a vector to obtain the expression vector according to claim 5, introducing the expression vector into a host bacterium to obtain a recombinant bacterium, extracting and purifying the protein in the recombinant bacterium, and isolating the 19-22 kD protein. 7. The preparation method according to claim 6, characterized in that the vector is pET22b(+), and the host bacteria is Escherichia coli; Pick a single colony of Escherichia coli and inoculate it into 10 mL of LB culture medium containing 100 mg/mL ampicillin, culture it at 37°C in a shaking incubator overnight, transfer it to the auto-induction medium containing Amp the next day, culture it at 37°C in a shaking incubator until the mid-logarithmic growth phase, lower the culture temperature to 8°C-12°C, maintain the cold pressure for 4h-8h, then increase the culture temperature to 37°C, continue to culture for 8h-14h, and collect the bacteria by centrifugation. 8. A Deinococcus aeria, characterized in that the Deinococcus aeria is classified and named as Deinococcus aeria, and is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC NO: 63982, and the Deinococcus aeria produces the superoxide dismutase according to claim 1. 9. Use of the superoxide dismutase according to claim 1 in a preparation for ensuring organ activity during low-temperature transportation of transplanted organs. 10. Use of the superoxide dismutase according to claim 1 in low-temperature resistant cosmetics.