Preparation method and application of superoxide dismutase

Abstract

The invention relates to superoxide dismutase (SOD) as well as a preparation method and application thereof. By performing codon optimization on the SOD gene of the extreme microorganism geobacillus thermoalkane, a recombinant expression system is successfully constructed, high-efficiency expression of soluble protein is realized, and the concentration of protein in fermentation liquor reaches 200mg / L. The enzyme shows excellent stability under extreme conditions, the retention rate of enzyme activity exceeds 50% within the range of lasting at 80 DEG C for 2 h and pH 4.0-12, and the free radical scavenging capacity of the enzyme is improved by 20 times compared with that of vitamin C with the same concentration. The enzyme has a wide application prospect in the field related to cosmetics, the stability exceeds 95% in the environment of 60 DEG C, and the stability is higher than 75% in the pH range of 5.0-11.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for preparing and applying superoxide dismutase. Background Technology

[0002] Superoxide dismutase (SOD) is a core antioxidant enzyme in organisms that scavenge superoxide radicals, converting them into hydrogen peroxide and oxygen to maintain the dynamic balance of reactive oxygen species (ROS) within cells. Superoxide radicals are generated during physiological processes such as electron transport in the respiratory chain. Excessive accumulation under pathological conditions leads to oxidative stress, causing DNA damage, lipid peroxidation, and protein denaturation, accelerating the progression of diseases such as aging, inflammation, and tumors. SOD, with its anti-aging, anti-radiation, anti-inflammatory, and anti-tumor properties, has significant application value in the pharmaceutical, cosmetic, and food industries. However, traditional SOD mainly relies on plant and animal extraction, which has limitations such as low yield, high cost, and poor stability, restricting its widespread application. Natural SOD also has a significant drawback: poor thermal stability. Even short-term treatment at high temperatures leads to severe loss of enzyme activity, limiting its application in high-temperature environments such as industrial and clinical settings. Currently, no completely consistent description of this in publicly available literature has been found. However, related studies do indirectly indicate that natural Cu / Zn-SOD has poor thermal stability under high temperature conditions; acid and alkali sensitivity: Chinese invention patent CN112522221B discloses that most natural SOD undergoes irreversible denaturation at pH < 5.0, limiting its application in acidic industrial settings; insufficient stability and environmental adaptability: extreme temperatures, high salt or complex chemical environments can easily lead to loss of enzyme activity, affecting its actual performance.

[0003] Although genetic engineering and protein modification technologies have been used to optimize SOD, significant bottlenecks still exist. For example, Chinese invention patent application CN105112379A discloses the use of thermophilic bacteria SOD genes to improve heat resistance, but it does not solve the stability problem under high salt or acid conditions. Chinese invention patent CN100374554C improves heat resistance through site-directed mutagenesis, but the expression level is only 120 mg / L and the production cost is high, making it difficult to scale up production. Summary of the Invention

[0004] The first aspect of this invention provides a method for preparing superoxide dismutase, comprising the following steps:

[0005] S1. Obtain the GtSOD gene (superoxide dismutase of Bacillus) of Geobacillus thermoleovorans from the NCBI database. The amino acid sequence of the GtSOD gene is SEQ ID NO: 1.

[0006] S2, the codons of the GtSOD gene are optimized to obtain the optimized GtSOD gene; the amino acid sequence of the optimized GtSOD gene is SEQ ID NO: 2;

[0007] S3. The optimized GtSOD gene was cloned, a recombinant plasmid was constructed, and then transformed and induced to express the crude product. After purification, a fermentation broth containing superoxide dismutase was obtained.

[0008] The codon optimization includes controlling the GC content (the percentage of guanine and cytosine in DNA) of the encoded amino acid sequence of the GtSOD gene to 55%-60% and replacing rare codons in E. coli.

[0009] In S3, the clone was inserted into the pET28a vector; S3 was transformed into Escherichia coli BL21; and S3 was induced to express using isopropyl-β-D-thiogalactoside.

[0010] The concentration of isopropyl-β-D-thiogalactoside in the crude product is 0.3-0.8 M.

[0011] Optionally, the concentration of isopropyl-β-D-thiogalactoside in the crude product is 0.4-0.6 M.

[0012] The purification in S3 was performed using gradient imidazole buffer for elution.

[0013] Optionally, the concentration of imidazole in the imidazole buffer is 20-250 mM.

[0014] The concentration of superoxide dismutase in the fermentation broth is greater than 150 mg / L.

[0015] Optionally, the concentration of superoxide dismutase in the fermentation broth is greater than 180 mg / L.

[0016] Optionally, the concentration of superoxide dismutase in the fermentation broth is 200 mg / L.

[0017] The induction temperature is 10-25℃, and the time is 10-20h.

[0018] Optionally, the induction temperature is 15-20℃ and the time is 10-15h.

[0019] The second aspect of this invention provides an application of a method for preparing superoxide dismutase, which can be used in the preparation of pharmaceuticals, cosmetics and food.

[0020] Beneficial effects

[0021] 1. This invention accidentally discovered that using *Bacillus thermophilus* for cloning, expression, and purification is not only suitable for...

[0022] Producing it in bioengineered strains can also improve the thermostability and enzyme activity of the prepared SOD enzyme.

[0023] 2. By optimizing the codons of the SOD gene of *Bacillus thermophilus*, the S3 gene was cloned into the pET28a vector; the S3 gene was transformed into *Escherichia coli* BL21; and the S3 gene was induced to express soluble protein using isopropyl-β-D-thiogalactoside, achieving high-efficiency expression of soluble protein with a protein concentration of 200 mg / L in the fermentation broth.

[0024] 3. This invention combines modern microbial genetic engineering and synthetic biology techniques to achieve high-efficiency and low-cost SOD enzyme preparation, providing significant advantages for industrial applications.

[0025] 4. The SOD enzyme prepared by the method of the present invention can maintain more than 80% activity in most organic solvents, and its enzyme activity can be moderately enhanced in isopropanol, urea and Tween 20.

[0026] 5. The SOD enzyme prepared by the method of the present invention exhibits excellent stability under extreme conditions. The enzyme activity retention rate exceeds 50% when kept at 80°C for 2 hours and within the pH range of 4.0-12. Its free radical scavenging ability is 20 times higher than that of vitamin C at the same concentration.

[0027] 6. The SOD enzyme prepared by the method of this invention has broad application prospects in the cosmetic field, with a stability of over 95% at 60℃ and a stability of over 75% in the pH range of 5.0-11. Attached Figure Description

[0028] Figure 1 This is an SDS-PAGE electrophoresis image of GtSOD expression and purification in Example 1. Where M: protein molecular weight marker; 1: crude enzyme solution; 2: purified superoxide dismutase.

[0029] Figure 2 The effect of metal ions on the activity of GtSOD in Example 1.

[0030] Figure 3 The effect of temperature on GtSOD in Example 1.

[0031] Figure 4 The effect of pH on the stability of GtSOD in Example 1.

[0032] Figure 5 The effect of organic solvents on the stability of GtSOD in Example 1. Detailed Implementation

[0033] Example 1

[0034] A method for preparing superoxide dismutase includes the following steps:

[0035] The gene sequence of GtSOD from Geobacillus thermoleovorans was obtained from the NCBI database, and the encoded amino acid sequence is shown in SEQ ID NO: 1;

[0036] Using the GtSOD gene of Bacillus thermophilus as a template, the GtSOD gene sequence was optimized to improve protein expression level. The GC content was adjusted to 55%-60%, and rare codons of Escherichia coli were replaced. The optimized sequence is shown in SEQ ID NO: 2.

[0037] The GtSOD gene was obtained using gene recombination (PCR amplification). The GC content was adjusted to 55%-60%, and rare codons of E. coli were replaced. The primers were designed as follows: forward primer 5′-CCGGAATTC ATGGGCATGCCATTCGAACTGCCGG-3′ and reverse primer 5′-CCGCTCGAG CTCGAGTTTAGCTTTCGCTTCAGAG-3′, where the underlined bases are EcoRI and XhoI restriction endonuclease sites, respectively. The PCR product was ligated with the pET28-(a) vector via enzyme digestion to construct an N-terminal His-tag recombinant plasmid. The recombinant plasmid was introduced into *E. coli* BL21(DE3) competent cells via heat shock transformation, and the accuracy of the recombinant plasmid was verified by DNA sequencing. *E. coli* BL21(DE3) competent cells were transformed with the pET28a-sod plasmid. Single colonies were selected in 5 mL LB medium containing 0.1 wt% 50 mg / mL kanamycin and cultured at 37°C and 220 rpm for 12–16 h. Subsequently, the cells were transferred to 200 mL LB medium at a 1 wt% inoculum and cultured until the OD600 nm reached 0.6–0.8. Isopropyl-β-D-thiogalactoside (IPTG) and 1 mM manganese chloride were added to a final concentration of 0.5 mM, and the cells were induced to culture at 25°C and 180 rpm for 12 h. The bacterial cells were collected by centrifugation, resuspended in 50 mM Tris-HCl buffer (pH 8.0), and sonicated at 70% power for 15 min intermittently. The cells were then centrifuged at 10,000 rpm for 30 min at 4 °C, and the supernatant was used as the crude enzyme solution. Purification was performed using nickel affinity chromatography: the nickel column was equilibrated with deionized water and buffer A (50 mM Tris-HCl, 250 mM NaCl, and 20 mM imidazole, pH 8.0). The crude enzyme solution was added, followed by washing with buffer A, elution with buffer B (50 mM Tris-HCl, 250 mM NaCl, and 40 mM imidazole, pH 8.0) to remove contaminating proteins, and elution with buffer C (50 mM Tris-HCl, 250 mM NaCl, and 250 mM imidazole, pH 8.0) to remove the target protein, yielding purified superoxide dismutase (in fermentation broth form, with a superoxide dismutase concentration of 200 mg / L).

[0038] Where SEQ ID NO:1 is:

[0039] MPFELPALPYPYDALEPHIDKETMNIHHTKHHNTYVTNLNAALEGHPDLQNK

[0040] SLEELLSNLEALPESIRTAVRNNGGGHANHSLFWTILSPNGGGEPTGELAEAIN

[0041] KKFGSFTAFKDEFSKAAAGRFSPGWAWLVVNNGELEITSTPNQDSPIMEGKTPI

[0042] LGLDVWEHAYYLKYQNRRPEYIAAFWNIVNWDEVAKRYSEAKAK

[0043] SEQ ID NO: 2 is:

[0044] ATGGGCATGCCATTCGAACTGCCGGCGTTGCCATATCCGTACGACGCACTG

[0045] GAACCGCACATCGACAAAGAAACCATGAACATCCACCACACCAAACACCA

[0046] CAACACCTACGTTACTAACCTGAACGCTGCACTGGAAGGCCATCCGGACCT

[0047] GCAGAACAAGAGCCTGGAAGAACTGCTGTCTAACCTGGAAGCGCTGCCGG

[0048] AATCCATCCGTACCGCTGTTCGTAACAACGGTGGTGGTCATGCTAACCACT

[0049] CTCTGTTCTGGACCATCTTGTCTCCGAACGGTGGTGGTGAACCAACCGGTG

[0050] AACTGGCAGAAGCGATCAACAAGAAATTCGGTTCTTTCACCGCATTCAAA

[0051] GACGAGTTCAGCAAAGCTGCAGCGGGTCGTTTCTCTCCGGGCTGGGCGTG

[0052] GCTGGTTGTTAACAACGGCGAACTGGAAATCACCTCTACTCCGAACCAAG

[0053] ACAGTCCGATCATGGAAGGTAAGACTCCGATCTTGGGTCTGGACGTTTGGG

[0054] AACATGCTTACTACCTGAAATACCAGAACCGTCGTCCGGAATACATCGCGG

[0055] CGTTCTGGAACATCGTTAACTGGGACGAAGTTGCTAAACGTTACTCTGAAG

[0056] CGAAAGCTAAACTCGAGTGA

[0057] Performance testing methods and data

[0058] The product from Example 1 was subjected to performance testing.

[0059] 1. Expression and purification of protein (superoxide dismutase): SDS-PAGE (gel electrophoresis) was used to identify the expression and purification status of the protein, such as... Figure 1 As shown, the protein (superoxide dismutase) has high purity.

[0060] 2. Effects of metal ions on GtSOD activity

[0061] Different concentrations (1 mM, 5 mM, and 10 mM) of MgCl2, ZnSO4, CaCl2, NiSO4, and FeSO4 were used to prepare purified superoxide dismutase (SOD) solutions. After incubation at room temperature for 1 h, enzyme activity was measured using the pyrogallol method, with untreated SOD activity serving as a control. The relative enzyme activity of each treatment group was calculated. Figure 2 As shown, the research results indicate that Mn 2+ It significantly promoted the enzyme activity of GtSOD, increasing it by nearly 2.5 times.

[0062] 3. Thermal stability analysis of GtSOD

[0063] 100 μL of purified superoxide dismutase (SOD) solution was placed in temperature gradients of 60℃, 70℃, 80℃, and 90℃ for heat treatment at preset time points (10, 20, 30, 60, and 120 min). After heat treatment, the solution was rapidly cooled in an ice bath, and enzyme activity was determined using the pyrogallol colorimetric method. The relative enzyme activity changes under each temperature and time condition were calculated, using the untreated SOD activity at room temperature as a control. Figure 3 As shown, the results indicate that GtSOD exhibits significant thermal stability, retaining over 90% of its original activity after treatment at 60℃ and 70℃ for 2 hours; while after treatment at 80℃ for 2 hours, the enzyme activity still retains 60%.

[0064] 4. pH stability analysis of GtSOD

[0065] Take 100 μL of purified superoxide dismutase (SOD) solution and incubate it with 200 μL of buffer solutions of different pH values ​​(3.0-12.0) at room temperature for 1 h. The buffer series includes: 50 mM citrate-sodium citrate buffer (pH 3.0-5.0), 50 mM sodium phosphate buffer (pH 5.0-7.0), 50 mM Tris-HCl buffer (pH 7.0-9.0), and 50 mM NaOH-Na2HPO4 buffer (pH 10.0-12.0). Enzyme activity was determined using the pyrogallol method, with the enzyme activity of untreated SOD at room temperature as a control, and the relative enzyme activity of each treatment group was calculated. Figure 4 As shown, the results indicate that GtSOD maintains over 50% of its enzyme activity over a very wide pH range (3.0-12.0), especially in the pH range of 5.0-11.0, where the enzyme activity remains above 70%. Figure 4 ).

[0066] 5. Organic solvent tolerance analysis of GtSOD

[0067] Take 50 μL of purified superoxide dismutase (SOD) solution and add organic solvent to a final concentration of 5 vol%. After incubation at room temperature for 1 h, determine enzyme activity using the pyrogallol method, and calculate the relative enzyme activity of each treatment group using untreated SOD activity as a control. Figure 5 As shown, the experimental results indicate that GtSOD maintains more than 80% of its activity in most organic solvents, and isopropanol, urea and Tween 20 can moderately enhance its enzyme activity, demonstrating the excellent stability of GtSOD in various organic solvents.

[0068] The pyrogallol method was used to determine GtSOD enzyme activity. The principle is as follows: Under alkaline conditions, pyrogallol undergoes auto-oxidation to produce superoxide anion radicals and a yellow intermediate product. This intermediate product exhibits a characteristic absorption peak at 325 nm, and the absorbance change is linearly related to time within the initial 4-5 minutes of the reaction. SOD inhibits the auto-oxidation rate of pyrogallol by reacting with superoxide anions to generate hydrogen peroxide. The specific method for determining enzyme activity is as follows: 45 mM pyrogallol (dissolved in 10 mM hydrochloric acid) was added to a 3 mL reaction system in Tris-HCl buffer (pH 8.2). After rapid mixing, the absorbance at 325 nm was measured every 30 seconds over 4 minutes, and the linear slope of its change with time was calculated. Simultaneously, the auto-oxidation rate of pyrogallol was controlled to approximately 0.07 OD / min. The enzyme activity unit (U) is defined as the amount of SOD enzyme required per milliliter of reaction system per minute to inhibit the auto-oxidation rate of pyrogallol by 50% at 25°C.

Claims

1. A method for preparing superoxide dismutase, characterized in that, Includes the following steps: S1. Obtain the GtSOD gene of Bacillus thermophilus from the NCBI database. The encoded amino acid sequence of the GtSOD gene is SEQ ID NO:

1. S2, the codons of the GtSOD gene are optimized to obtain the optimized GtSOD gene; the amino acid sequence of the optimized GtSOD gene is SEQ ID NO: 2; S3. The optimized GtSOD gene was cloned, a recombinant plasmid was constructed, and then transformed and induced to express the crude product. After purification, a fermentation broth containing superoxide dismutase was obtained.

2. The method for preparing superoxide dismutase according to claim 1, characterized in that, The codon optimization includes: controlling the GC content of the encoded amino acid sequence of the GtSOD gene to 55%-60% and replacing rare codons in E. coli.

3. The method for preparing superoxide dismutase according to claim 1, characterized in that, The cloning in S3 includes cloning into the pET28a vector.

4. The method for preparing superoxide dismutase according to claim 1, characterized in that, The transformation in S3 includes transformation into Escherichia coli BL21.

5. The method for preparing superoxide dismutase according to claim 1, characterized in that, The induction of expression in S3 includes induction using isopropyl-β-D-thiogalactoside.

6. The method for preparing superoxide dismutase according to claim 5, characterized in that, The concentration of isopropyl-β-D-thiogalactoside in the crude product is 0.3-0.8 M.

7. The method for preparing superoxide dismutase according to claim 1, characterized in that, The purification in S3 was performed using gradient imidazole buffer for elution.

8. The method for preparing superoxide dismutase according to claim 1, characterized in that, The concentration of superoxide dismutase in the fermentation broth is greater than 150 mg / L.

9. The method for preparing superoxide dismutase according to claim 1, characterized in that, The induction temperature is 10-25℃, and the time is 10-20h.

10. An application of the method for preparing superoxide dismutase according to any one of claims 1-9, characterized in that, It is used in the preparation of pharmaceuticals, cosmetics and food.

Images