Mutants of superoxide dismutase, nucleic acid molecules and uses thereof

Abstract

This invention relates to the field of bioengineering, and more particularly to mutants of superoxide dismutase (SOD), nucleic acid molecules, and their applications. This invention provides mutants of SOD, comprising: a mutation at position 39 of the SOD from V to L; and / or a mutation at position 46 of the SOD from T to Y; and / or a mutation at position 57 of the SOD from V to L; and / or a mutation at position 70 of the SOD from P to A. This invention uses online prediction software to analyze and predict the amino acid sequence of wild-type SOD, obtaining a mutation library that can improve stability. By constructing and utilizing this mutation library, and through experimental screening and a strategy of iterative mutation, mutants with improved activity and stability were obtained.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and in particular to mutants of superoxide dismutase, nucleic acid molecules, and their applications. Background Technology

[0002] Superoxide dismutase (SOD) is a protease with a metal cofactor, whose main function is to eliminate harmful free radicals produced by normal metabolism in organisms. SOD is one of the most effective antioxidants in nature. It can prevent and alleviate oxidative stress by inhibiting damage induced by reactive free radicals, thereby improving cell repair and regeneration. It has enormous application potential and broad development prospects in the medical, daily chemical, food, and agricultural industries.

[0003] Superoxide dismutase (SOD) is mainly classified into four types according to its metal cofactor: copper-zinc superoxide dismutase (CuZn-SOD), manganese superoxide dismutase (Mn-SOD), iron superoxide dismutase (Fe-SOD), and nickel superoxide dismutase (Ni-SOD). Copper-zinc superoxide dismutase is found in the cytoplasm and chloroplasts of eukaryotes and in some prokaryotes. Manganese superoxide dismutase is mostly in dimer or tetramer form and is found in the mitochondrial matrix, chloroplasts, and cytoplasm. Iron superoxide dismutase is mainly found in protozoa, prokaryotes, a few algae, and plant chloroplasts. The basic catalytic process of different types of SOD involves electrostatic guidance from the superoxide substrate, altering the redox potential of metal ions to a suitable range for superoxide dismutation. This catalyzes the alternation of redox reactions of metal ions, promoting the dismutation reaction of superoxide anion free radicals to produce H₂O₂ and O₂.

[0004] The current heat resistance of SOD still cannot meet the requirements of high-temperature processes in industrial production, and its activity is greatly affected by pH, remaining stable only within the neutral range, which greatly limits the industrial production and application of SOD enzymes. Summary of the Invention

[0005] In view of this, the present invention provides mutants of superoxide dismutase, nucleic acid molecules, and their applications. The present invention uses online prediction software to analyze and predict the amino acid sequence of wild-type superoxide dismutase, obtaining a mutation library that can improve stability. By constructing and utilizing this mutation library, and through experimental screening and a strategy of iterative mutation, mutants with improved activity and stability were obtained.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides mutants of superoxide dismutase, comprising: a mutation at position 39 of the superoxide dismutase from V to L; and / or

[0008] The 46th position of the superoxide dismutase is mutated from T to Y; and / or

[0009] The 57th position of the superoxide dismutase is mutated from V to L; and / or

[0010] The 70th position of the superoxide dismutase is mutated from P to A.

[0011] In some embodiments of the present invention, the mutant includes: a mutation at position 39 of the superoxide dismutase from V to L, a mutation at position 46 from T to Y, and a mutation at position 70 from P to A; and / or

[0012] The superoxide dismutase has a V-to-L mutation at position 39 and a T-to-Y mutation at position 46; and / or

[0013] The superoxide dismutase has a T-to-Y mutation at position 46 and a P-to-A mutation at position 70; and / or

[0014] The superoxide dismutase has a V-to-L mutation at position 39 and a P-to-A mutation at position 70; and / or

[0015] The superoxide dismutase has a V-to-L mutation at position 39, a P-to-A mutation at position 70, and a V-to-L mutation at position 57.

[0016] In some embodiments of the present invention, the mutant includes: the superoxide dismutase having a V-to-L mutation at position 39, a T-to-Y mutation at position 46, and a P-to-A mutation at position 70.

[0017] In some embodiments of the present invention, the mutant includes:

[0018] (I) The superoxide dismutase is derived from Xanthomonas; and / or

[0019] (II) The superoxide dismutase has a specific amino acid sequence; and / or

[0020] (III) The gene for the superoxide dismutase has a specific nucleotide sequence;

[0021] The specific amino acid sequence has:

[0022] (1) An amino acid sequence as shown in SEQ ID NO:1; or

[0023] (2) A sequence based on the amino acid sequence shown in (1) by substitution, deletion, addition and / or replacement of one or more amino acids; or

[0024] (3) A sequence that is more than 80% homologous to the amino acid sequence shown in (1) or (2);

[0025] The specific nucleotide sequence has:

[0026] (4) A nucleotide sequence as shown in SEQ ID NO:2; or

[0027] (5) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (4), and which has the same or similar function as the nucleotide sequence shown in (4); or

[0028] (6) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (4) or (5).

[0029] In some embodiments of the present invention, the sequence of SEQ ID NO:1 is: MAYTLPQLPYAYDALEPNIDAQTMEIHHTKHHQTYINNVNAALEGTEYADLPIEELVSKLKSLPENLQGPVRNNGGGHANHSLFWTVLSPNGGGEPKGEVAKAIDKDLGGFEKFKEAFTKAAVSRFGSGWAWLSVTPDKKLVVESTANQDSPLFEGNTPILGLDVWEHAYYLKYQNRRPEYIGAFYNAVNWEEVERRYHAAIA*.

[0030] In some embodiments of the present invention, the sequence of SEQ ID NO:2 is: *.

[0031] In some embodiments of the present invention, the mutant has:

[0032] (7) Any amino acid sequence as shown in SEQ ID NO:3 to SEQ ID NO:10; or

[0033] (8) A sequence based on the amino acid sequence shown in (7) by substitution, deletion, addition and / or replacement of one or more amino acids; or

[0034] (9) A sequence that is more than 80% homologous to the amino acid sequence shown in (7) or (8).

[0035] In some embodiments of the present invention, the sequence of SEQ ID NO:3 is: MAYTLPQLPYAYDALEPNIDAQTMEIHHTKHHQTYINNLNAALEGTEYADLPIEELVSKLKSLPENLQGPVRNNGGGHANHSLFWTVLSPNGGGEPKGEVAKAIDKDLGGFEKFKEAFTKAAVSRFGSGWAWLSVTPDKKLVVESTANQDSPLFEGNTPILGLDVWEHAYYLKYQNRRPEYIGAFYNAVNWEEVERRYHAAIA. (V39L)

[0036] In some embodiments of the present invention, the sequence of SEQ ID NO:4 is: MAYTLPQLPYAYDALEPNIDAQTMEIHHTKHHQTYINNVNAALEGYEYADLPIEELVSKLKSLPENLQGPVRNNGGGHANHSLFWTVLSPNGGGEPKGEVAKAIDKDLGGFEKFKEAFTKAAVSRFGSGWAWLSVTPDKKLVVESTANQDSPLFEGNTPILGLDVWEHAYYLKYQNRRPEYIGAFYNAVNWEEVERRYHAAIA. (T46Y)

[0037] In some embodiments of the present invention, the sequence of SEQ ID NO:5 is: MAYTLPQLPY AYDALEPNIDAQTMEIHHTKHHQTYINNVNAALEGTEYADLPIEELVSKLKSLPENLQGAVRNNGGGHANHSLFWTVLSPNGGGEPKGEVAKAIDKDLGGFEKFKEAFTKAAVSRFGSGWAWLSVTPDKKLVVESTANQDSPLFEGNTPILGLDVWEHAYYLKYQNRRPEYIGAFYNAVNWEEVERRYHAAIA. (P70A)

[0038] In some embodiments of the present invention, the sequence of SEQ ID NO:6 is: MAYTLPQLPY AYDALEPNIDAQTMEIHHTKHHQTYINNLNAALEGYEYADLPIEELVSKLKSLPENLQGAVRNNGGGHANHSLFWTVLSPNGGGEPKGEVAKAIDKDLGGFEKFKEAFTKAAVSRFGSGWAWLSVTPDKKLVVESTANQDSPLFEGNTPILGLDVWEHAYYLKYQNRRPEYIGAFYNAVNWEEVERRYHAAIA. (V39L, T46Y, P70A)

[0039] In some embodiments of the present invention, the sequence of SEQ ID NO:7 is: MAYTLPQLPYAYDALEPNIDAQTMEIHHTKHHQTYINNLNAALEGYEYADLPIEELVSKLKSLPENLQGPVRNNGGGHANHSLFWTVLSPNGGGEPKGEVAKAIDKDLGGFEKFKEAFTKAAVSRFGSGWAWLSVTPDKKLVVESTANQDSPLFEGNTPILGLDVWEHAYYLKYQNRRPEYIGAFYNAVNWEEVERRYHAAIA. (V39L, T46Y)

[0040] In some embodiments of the present invention, the sequence of SEQ ID NO:8 is: MAYTLPQLPYAYDALEPNIDAQTMEIHHTKHHQTYINNVNAALEGYEYADLPIEELVSKLKSLPENLQGAVRNNGGGHANHSLFWTVLSPNGGGEPKGEVAKAIDKDLGGFEKFKEAFTKAAVSRFGSGWAWLSVTPDKKLVVESTANQDSPLFEGNTPILGLDVWEHAYYLKYQNRRPEYIGAFYNAVNWEEVERRYHAAIA. (T46Y, P70A)

[0041] In some embodiments of the present invention, the sequence of SEQ ID NO:9 is: MAYTLPQLPYAYDALEPNIDAQTMEIHHTKHHQTYINNLNAALEGTEYADLPIEELVSKLKSLPENLQGAVRNNGGGHANHSLFWTVLSPNGGGEPKGEVAKAIDKDLGGFEKFKEAFTKAAVSRFGSGWAWLSVTPDKKLVVESTANQDSPLFEGNTPILGLDVWEHAYYLKYQNRRPEYIGAFYNAVNWEEVERRYHAAIA. (V39L, P70A)

[0042] In some embodiments of the present invention, the sequence of SEQ ID NO:10 is: MAYTLPQLPYAYDALEPNIDAQTMEIHHTKHHQTYINNLNAALEGTEYADLPIEELLSKLKSLPENLQGAVRNNGGGHANHSLFWTVLSPNGGGEPKGEVAKAIDKDLGGFEKFKEAFTKAAVSRFGSGWAWLSVTPDKKLVVESTANQDSPLFEGNTPILGLDVWEHAYYLKYQNRRPEYIGAFYNAVNWEEVERRYHAAIA. (V39L, P70A, V57L)

[0043] The present invention also provides a nucleic acid molecule encoding the above-mentioned mutant, wherein the G at position 115 of the nucleic acid molecule encoding the superoxide dismutase is mutated to C; and / or

[0044] The nucleic acid molecule encoding the superoxide dismutase has an A mutation at position 136, changing it to T; and / or

[0045] The C at position 137 of the nucleic acid molecule encoding the superoxide dismutase is mutated to A; and / or

[0046] The C at position 138 of the nucleic acid molecule encoding the superoxide dismutase is mutated to T; and / or

[0047] The G at position 169 of the nucleic acid molecule encoding the superoxide dismutase is mutated to C; and / or

[0048] The C at position 208 of the nucleic acid molecule encoding the superoxide dismutase is mutated to G; and / or

[0049] The G at position 210 of the nucleic acid molecule encoding the superoxide dismutase is mutated to A.

[0050] In some embodiments of the present invention, the nucleic acid molecule has:

[0051] (10) A nucleotide sequence as shown in any of SEQ ID NO:11 to SEQ ID NO:18; or

[0052] (11) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (10), and which has the same or similar function as the nucleotide sequence shown in (10); or

[0053] (12) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (10) or (11).

[0054] In some embodiments of the present invention, the sequence of SEQ ID NO:11 is: (V39L)

[0055] In some embodiments of the present invention, the sequence of SEQ ID NO:12 is: Atggcgtataccctgccgcagctgccgtatgcgtatgatgcgctggaaccgaacattgatgcgcagaccatggaaattcatcataccaaacatcatcagacctatattaacaacgtgaacgcggcgctggaaggctatgaatatgcggatctgccgattgaagaactggtgagcaaactgaaaagcctgccggaaaacctgcagggcccggtgcgcaacaacggcggcggccatgcgaaccatagcctgttttggaccgtgctgagcccgaacggcggcggcgaaccgaaaggcgaagtggcgaaagcgattgataaagatctgggcggctttgaaaaatttaaagaagcgtttaccaaagcggcggtgagccgctttggcagcggctgggcgtggctgagcgtgaccccggataaaaaactggtggtggaaagcaccgcgaaccaggatagcccgctgtttgaaggcaacaccccgattctgggcctggatgtgtgggaacatgcgtattatctgaaatatcagaaccgccgcccggaatatattggcgcgttttataacgcggtgaactgggaagaagtggaacgccgctatcatgcggcgattgcg. (T46Y)

[0056] In some embodiments of the present invention, the sequence of SEQ ID NO:13 is: Atggcgtataccctgccgcagctgccgtatgcgtatgatgcgctggaaccgaacattgatgcgcagaccatggaaattcatcataccaaacatcatcagacctatattaacaacgtgaacgcggcgctggaaggcaccgaatatgcggatctgccgattgaagaactggtgagcaaactgaaaagcctgccggaaaacctgcagggcgcagtgcgcaacaacggcggcggccatgcgaaccatagcctgttttggaccgtgctgagcccgaacggcggcggcgaaccgaaaggcgaagtggcgaaagcgattgataaagatctgggcggctttgaaaaatttaaagaagcgtttaccaaagcggcggtgagccgctttggcagcggctgggcgtggctgagcgtgaccccggataaaaaactggtggtggaaagcaccgcgaaccaggatagcccgctgtttgaaggcaacaccccgattctgggcctggatgtgtgggaacatgcgtattatctgaaatatcagaaccgccgcccggaatatattggcgcgttttataacgcggtgaactgggaagaagtggaacgccgctatcatgcggcgattgcg. (P70A)

[0057] In some embodiments of the present invention, the sequence of SEQ ID NO:14 is: Atggcgtataccctgccgcagctgccgtatgcgtatgatgcgctggaaccgaacattgatgcgcagaccatggaaattcatcataccaaacatcatcagacctatattaacaacctgaacgcggcgctggaaggctatgaatatgcggatctgccgattgaagaactggtgagcaaactgaaaagcctgccggaaaacctgcagggcgcagtgcgcaacaacggcggcggccatgcgaaccatagcctgttttggaccgtgctgagcccgaacggcggcggcgaaccgaaaggcgaagtggcgaaagcgattgataaagatctgggcggctttgaaaaatttaaagaagcgtttaccaaagcggcggtgagccgctttggcagcggctgggcgtggctgagcgtgaccccggataaaaaactggtggtggaaagcaccgcgaaccaggatagcccgctgtttgaaggcaacaccccgattctgggcctggatgtgtgggaacatgcgtattatctgaaatatcagaaccgccgcccggaatatattggcgcgttttataacgcggtgaactgggaagaagtggaacgccgctatcatgcggcgattgcg. (V39L, T46Y, P70A)

[0058] In some embodiments of the present invention, the sequence of SEQ ID NO:15 is: Atggcgtataccctgccgcagctgccgtatgcgtatgatgcgctggaaccgaacattgatgcgcagaccatggaaattcatcataccaaacatcatcagacctatattaacaacctgaacgcggcgctggaaggctatgaatatgcggatctgccgattgaagaactggtgagcaaactgaaaagcctgccggaaaacctgcagggcccggtgcgcaacaacggcggcggccatgcgaaccatagcctgttttggaccgtgctgagcccgaacggcggcggcgaaccgaaaggcgaagtggcgaaagcgattgataaagatctgggcggctttgaaaaatttaaagaagcgtttaccaaagcggcggtgagccgctttggcagcggctgggcgtggctgagcgtgaccccggataaaaaactggtggtggaaagcaccgcgaaccaggatagcccgctgtttgaaggcaacaccccgattctgggcctggatgtgtgggaacatgcgtattatctgaaatatcagaaccgccgcccggaatatattggcgcgttttataacgcggtgaactgggaagaagtggaacgccgctatcatgcggcgattgcg. (V39L, T46Y)

[0059] In some embodiments of the present invention, the sequence of SEQ ID NO:16 is: Atggcgtataccctgccgcagctgccgtatgcgtatgatgcgctggaaccgaacattgatgcgcagaccatggaaattcatcataccaaacatcatcagacctatattaacaacgtgaacgcggcgctggaaggctatgaatatgcggatctgccgattgaagaactggtgagcaaactgaaaagcctgccggaaaacctgcagggcgcagtgcgcaacaacggcggcggccatgcgaaccatagcctgttttggaccgtgctgagcccgaacggcggcggcgaaccgaaaggcgaagtggcgaaagcgattgataaagatctgggcggctttgaaaaatttaaagaagcgtttaccaaagcggcggtgagccgctttggcagcggctgggcgtggctgagcgtgaccccggataaaaaactggtggtggaaagcaccgcgaaccaggatagcccgctgtttgaaggcaacaccccgattctgggcctggatgtgtgggaacatgcgtattatctgaaatatcagaaccgccgcccggaatatattggcgcgttttataacgcggtgaactgggaagaagtggaacgccgctatcatgcggcgattgcg. (T46Y, P70A)

[0060] In some embodiments of the present invention, the sequence of SEQ ID NO:17 is: Atggcgtataccctgccgcagctgccgtatgcgtatgatgcgctggaaccgaacattgatgcgcagaccatggaaattcatcataccaaacatcatcagacctatattaacaacctgaacgcggcgctggaaggcaccgaatatgcggatctgccgattgaagaactggtgagcaaactgaaaagcctgccggaaaacctgcagggcgcagtgcgcaacaacggcggcggccatgcgaaccatagcctgttttggaccgtgctgagcccgaacggcggcggcgaaccgaaaggcgaagtggcgaaagcgattgataaagatctgggcggctttgaaaaatttaaagaagcgtttaccaaagcggcggtgagccgctttggcagcggctgggcgtggctgagcgtgaccccggataaaaaactggtggtggaaagcaccgcgaaccaggatagcccgctgtttgaaggcaacaccccgattctgggcctggatgtgtgggaacatgcgtattatctgaaatatcagaaccgccgcccggaatatattggcgcgttttataacgcggtgaactgggaagaagtggaacgccgctatcatgcggcgattgcg. (V39L, P70A)

[0061] In some embodiments of the present invention, the sequence of SEQ ID NO:18 is: (V39L, P70A, V57L).

[0062] The present invention also provides primer sets for amplifying the above-mentioned nucleic acid molecules, including one or more of primer sets 1 to 4;

[0063] The primer set 1 has:

[0064] (13) Nucleotide sequences as shown in SEQ ID NO:19 and SEQ ID NO:20; or

[0065] (14) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (13), and which has the same or similar function as the nucleotide sequence shown in (13); or

[0066] (15) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (13) or (14); and / or

[0067] Primer set 2 has:

[0068] (16) Nucleotide sequences as shown in SEQ ID NO:21 and SEQ ID NO:22; or

[0069] (17) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (16), and which has the same or similar function as the nucleotide sequence shown in (16); or

[0070] (18) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (16) or (17); and / or

[0071] The primer set 3 has:

[0072] (19) Nucleotide sequences as shown in SEQ ID NO:23 and SEQ ID NO:24; or

[0073] (20) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (19), and which has the same or similar function as the nucleotide sequence shown in (19); or

[0074] (21) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (19) or (20); and / or

[0075] Primer set 4 has:

[0076] (22) Nucleotide sequences as shown in SEQ ID NO:25 and SEQ ID NO:26; or

[0077] (23) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (22), and which has the same or similar function as the nucleotide sequence shown in (22); or

[0078] (24) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (22) or (23).

[0079] The present invention also provides a recombinant expression vector comprising the above-described nucleic acid molecules and acceptable gene elements.

[0080] The present invention also provides a host, transformation and / or transfection of the above-mentioned recombinant expression vector.

[0081] The present invention also provides a method for preparing the above-mentioned mutant, wherein the mutant is prepared based on any of the following:

[0082] (I) The above-mentioned nucleic acid molecules; and / or

[0083] (II) The primer set described above; and / or

[0084] (III) The above-mentioned recombinant expression vectors; and / or

[0085] (IV) The aforementioned hosts.

[0086] In some embodiments of the present invention, the preparation method includes the following steps:

[0087] S1: Obtain the nucleic acid molecule encoding the mutant;

[0088] S2: The nucleic acid molecule is fused with the expression vector to construct a recombinant expression vector, which is then transformed and / or transfected into a host.

[0089] S3: Induce host expression containing the recombinant expression vector, and obtain the mutant after isolation and purification.

[0090] The present invention also provides the application of the above-mentioned mutant, the above-mentioned nucleic acid molecule, the above-mentioned primer set, the above-mentioned recombinant expression vector, the above-mentioned host and / or the mutant obtained by the above-mentioned preparation method in any of the following:

[0091] (I) Preparation of antioxidant products; and / or

[0092] (II) Preparation of anti-aging products; and / or

[0093] (III) Preparing products containing superoxide dismutase; and / or

[0094] (IV) Improve the thermal stability of superoxide dismutase; and / or

[0095] (V) Increase the activity of superoxide dismutase.

[0096] The present invention provides mutants of superoxide dismutase, including: the 39th position of the superoxide dismutase is mutated from V to L; and / or the 46th position of the superoxide dismutase is mutated from T to Y; and / or the 57th position of the superoxide dismutase is mutated from V to L; and / or the 70th position of the superoxide dismutase is mutated from P to A.

[0097] This invention uses online prediction software to analyze and predict the amino acid sequence of wild-type superoxide dismutase, obtaining a mutation library that can improve stability. By constructing and utilizing this mutation library, and through experimental screening and a strategy of iterative mutation, mutants with improved activity and stability were obtained. Attached Figure Description

[0098] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0099] Figure 1 The three-dimensional structure of the protein, RoseTTAFold2, is shown.

[0100] Figure 2 SDS-PAGE maps showing SOD expression in wild-type and mutant strains;

[0101] Figure 3 The enzyme activity of wild-type SOD and its mutants is shown;

[0102] Figure 4 This demonstrates the temperature tolerance of SOD wild-type and its mutants;

[0103] Figure 5 The pH tolerance of Xc-SOD and XcSOD-Mut1 is shown. Detailed Implementation

[0104] This invention discloses mutants of superoxide dismutase, nucleic acid molecules, and their applications.

[0105] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0106] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0107] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0108] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0109] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0110] English annotations in this invention:

[0111] SOD Superoxide dismutase XcSOD Xanthomonas superoxide dismutase WT wild type

[0112] In Examples 1 to 7 and Comparative Example 1 of this invention, all raw materials and reagents used can be purchased from the market.

[0113] The present invention will be further illustrated below with reference to the embodiments:

[0114] Example 1: Construction of recombinant expression plasmid pRSFDuet-1-XcSOD

[0115] The amino acid sequence of superoxide dismutase (XcSOD, Uniprot#Q8PJZ1) derived from *Homo spp.* (pv. citri (strain 306)) is shown in SEQ ID NO.1. It possesses all the characteristic amino acid residues of the Mn-SOD family dimer, including Met24, Gly76, Gly77, His78, Gln149, and Asp150. Only one literature report has described it (PLoS ONE 14(1):e0209988.). The encoding gene was optimized according to the codon preference of *E. coli* and sent to Shanghai Sangon Biotech to synthesize the target gene fragment (nucleotide sequence shown in SEQ ID NO.2). The synthesized sequence was ligated into the pRSFDuet-1 vector through EcoRI and NotI restriction sites to obtain the recombinant expression vector pRSFDuet-1-XcSOD.

[0116] Example 2: Protein structure prediction analysis of XcSOD enzyme

[0117] The amino acid sequence of wild-type superoxide dismutase was used for protein structure modeling using RoseTTAFold2 online prediction software (e.g., Figure 1 As shown in Table 1), the three-dimensional structure was subjected to site-directed mutation prediction analysis to improve thermal stability using the FireProt online prediction website. The mutation sites determined based on the binding energy change were selected as follows:

[0118] Table 1. Prediction of changes in binding free energy at mutation sites.

[0119]

[0120] Example 3: Construction of a site-directed mutant library for predicted sites

[0121] Based on the mutation sites described in Example 2, primers were designed to perform site-directed mutagenesis on pRSFDuet-1-XcSOD (as shown in Table 2).

[0122] Table 2 Primer sequences

[0123]

[0124]

[0125] Using pRSFDuet-1-XcSOD as a template, the PCR reaction systems shown in Table 3 were constructed using the forward and reverse primers for each of the above-mentioned amino acid mutation sites:

[0126] Table 3

[0127] Element volume 2x Phanta Max Master Mix 25μL template 100ng forward primer 1μL reverse primer 1μL <![CDATA[H2O]]> Make up the margin Total volume 50μL

[0128] Amplification was performed using the following PCR program: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for 30 cycles.

[0129] The PCR product was digested with Dpn1 enzyme at 37°C for 1 hour. The digested product was then transformed into E. coli DH4a, plated on kanamycin-resistant LB plates, and incubated overnight at 37°C.

[0130] Transformants were picked from LB plates, cultured overnight in liquid LB medium, and plasmids were extracted and sent to Sangon Biotech for sequencing. The sequencing results were consistent, and expression vectors for each amino acid mutant were obtained.

[0131] Example 4: Expression and purification of XcSOD and its mutants in Escherichia coli

[0132] (1) The point mutation recombinant expression vector and the wild-type XcSOD expression vector constructed in Example 3 were transformed into the expression host Escherichia coli BL21(DE3) to obtain recombinant bacteria.

[0133] (2) The recombinant bacteria obtained in step (1) were inoculated into LB liquid medium containing 50 μg / mL kanamycin (the medium was supplemented with MnSO4 to a final concentration of 1 mM, where manganese ions are the ligands of the superoxide dismutase active site), and cultured at 37°C with shaking at 220 rpm until the bacterial culture reached OD. 600nm=0.6~0.8, add IPTG to the bacterial culture to a final concentration of 0.1mM, and induce culture overnight at 16℃ and 220rpm. After induction, centrifuge at 8000rpm for 7min to collect the bacterial cells.

[0134] (3) The bacterial cells collected in step (2) were resuspended in 20 mM PB buffer at pH 7.5, and sonicated (power 40%, on for 3 seconds, off for 2 seconds, total disruption time 30 min). The disrupted whole-cell lysate was centrifuged at 12000 rpm for 30 min to obtain crude enzyme solution. 10 μL of the supernatant sample was loaded and analyzed by SDS-PAGE for expression. The results are as follows: Figure 2 As shown, both wild-type and mutant were expressed in the supernatant; finally, the crude enzyme solution was purified using a nickel ion affinity chromatography column to obtain pure enzyme.

[0135] Example 5 Enzyme activity assay of XcSOD and its single-point mutant

[0136] The protein concentration of the SOD protein solution obtained in Example 4 was determined using an ultra-micro spectrophotometer. The SOD enzyme activity in the purified SOD and its mutant protein solutions was determined using the pyrogallol autoxidation method (GB / T 5009.171-2003 Determination of Superoxide Dismutase (SOD) Activity in Health Foods). The results are as follows: Figure 3 As shown in Table 4 (specific data are shown), the mutant V39L has the highest specific activity at 2.56 million U / g; the enzyme activities of the other mutants V39L, T46Y, and P70A are all higher than those of the wild-type XcSOD.

[0137] Definition of SOD activity unit: The amount of SOD required to inhibit the auto-oxidation rate of pyrogallol by 50% at 25℃ is defined as one activity unit.

[0138] Table 4

[0139]

[0140]

[0141] Example 6: Multi-point combination mutation

[0142] To further improve the catalytic activity and thermostability of wild-type XcSOD, V39L, T46Y, P70A, and V57L were further combined with mutants. Pure enzymes were obtained according to the steps in Example 4, and the enzyme activity of the multi-site combined mutants was tested according to the method in Example 5. Combined mutants with increased enzyme activity were obtained and named XcSOD-Mut1, XcSOD-Mut2, XcSOD-Mut3, and XcSOD-Mut4, respectively. The results are shown in Table 5. The enzyme activity of the multi-site combined mutant XcSOD-Mut1 was 3.02 million U / g, approximately 1.5 times higher than that of the wild type.

[0143] Among them, XcSOD-Mut1 contains mutation sites V39L, T46Y, and P70A;

[0144] XcSOD-Mut2 contains the mutation sites V39L and T46Y;

[0145] XcSOD-Mut3 contains the mutation sites T46Y and P70A;

[0146] XcSOD-Mut4 contains mutation sites V39L and P70A;

[0147] XcSOD-Mut5 contains mutation sites V39L, P70A, and V57L;

[0148] Table 5 Relative enzyme activities of wild-type superoxide dismutase and combined mutants

[0149] mutation site Enzyme activity (10,000 U / g) wild type 198 XcSOD-Mut1 302 XcSOD-Mut2 245 XcSOD-Mut3 234 XcSOD-Mut4 249 XcSOD-Mut5 231

[0150] Example 7 Enzymatic properties of XcSOD and its mutant XcSOD-Mut1 - heat resistance, pH tolerance

[0151] (1) Temperature tolerance: The XcSOD and its mutant XcSOD-Mut1 protein solutions prepared in Examples 4 and 6 were incubated at 90℃ and 100℃ for 3 hours, respectively. The residual enzyme activity in the SOD protein solution was determined by the pyrogallol auto-oxidation method (with the enzyme activity before heating as 100%). The results are as follows: Figure 4 As shown, the results indicate that the enzyme activities of XcSOD and XcSOD-Mut1 did not change significantly after treatment at 90℃ for 3 hours, indicating that they are heat-resistant up to 90℃. After treatment at 100℃ for 3 hours, the XcSOD-Mut1 mutant still retained more than 90% of its enzyme activity.

[0152] Table 6. Statistical table of enzyme activity changes after different high-temperature treatments.

[0153] mutation site 90℃ 100℃ WT 70％ 51％ I36V 30％ 12％ V39L 92％ 74％ T46Y 71％ 49％ E47P 38％ 10％ I53V 36％ 9％ V57L 62％ 40％ P70A 92％ 72％ V100L 42％ 11％ XcSOD-Mut1 98％ 91％

[0154] (2) Acid and alkali resistance: 100 μL of the XcSOD and its XcSOD-Mut1 mutant protein solution prepared in Example 4 was added to 200 μL of buffer solutions with different pH values ​​(2-11), and incubated at room temperature for 1 h. The residual activity of the samples was determined using the pyrogallol method. Different buffer solutions were used for different pH ranges, in the following order: pH 1-2: KCl-HCl, 200 mM; pH 2-5: CH3COONa, 200 mM; pH 5-7: Na2HPO4-Na H2PO4, 200 mM; pH 7-9: TRIS-HCl, 200 mM; pH 9-11: glycine-NaOH, 200 mM.

[0155] The results are as follows Figure 5 As shown, the XcSOD-Mut1 mutant exhibits a residual enzyme activity greater than 70% within a pH range of 3 to 11, demonstrating greater tolerance to acidic and alkaline environments compared to Xc-SOD, with its activity being less affected by pH.

[0156] Table 7. Statistical table of enzyme activity changes after different pH treatments.

[0157]

[0158]

[0159] Comparative Example 1

[0160] In Example 2, all sites predicted by the three-dimensional structure to improve thermal stability were expressed and purified in E. coli according to Example 4, and enzyme activity was measured according to the method in Example 5. The results showed that the enzyme activity of mutants I36V, E47P, I53V, V57L, and V100L was not improved, but decreased by 1 to 2 times. The heat resistance and pH tolerance of mutants E47P and I53V were measured according to the method in Example 7. The results are shown in the table below. After being placed at 90°C for 3 hours, the enzyme activity of mutants E47P and I53V was only 38%. When the temperature was increased to 100°C, the enzyme activity was only 10% of that at room temperature of 25°C. Their heat resistance was lower than that of XcS OD-Mut. Furthermore, the enzyme activity was significantly affected by pH, with no activity or extremely low activity in both acidic and alkaline environments.

[0161] Table 8. Statistical table of enzyme activity changes under high-temperature treatment in comparative studies.

[0162] mutation site 90℃ 100℃ XcSOD-Mut1 98％ 91％ E47P 38％ 10％ I53V 36％ 9％

[0163] Table 9. Statistical table of enzyme activity changes under pH treatment in the comparative examples.

[0164]

[0165]

[0166] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutant of superoxide dismutase, characterized in that, The mutant sequence is an amino acid sequence as shown in SEQ ID NO:3 or any of SEQ ID NO:5 to SEQ ID NO:

10.

2. A nucleic acid molecule encoding the mutant as described in claim 1, characterized in that, The sequence of the nucleic acid molecule is as shown in SEQ ID NO:11 or any of the nucleotide sequences shown in SEQ ID NO:13 to SEQ ID NO:

18.

3. A recombinant expression vector, characterized in that, Includes nucleic acid molecules as described in claim 2 and acceptable gene elements.

4. A host cell, characterized in that, Transformation and / or transfection with the recombinant expression vector as described in claim 3.

5. The method for preparing the mutant as described in claim 1, characterized in that, The mutant was prepared based on any of the following: ( ), the nucleic acid molecule as described in claim 2; and / or ( ), the recombinant expression vector as described in claim 3; and / or ( (and) the host cell as described in claim 4.

6. The use of the mutant as described in claim 1, the nucleic acid molecule as described in claim 2, the recombinant expression vector as described in claim 3, the host cell as described in claim 4, and / or the mutant obtained by the preparation method as described in claim 5 in any of the following: ( ), preparation of antioxidant products; and / or ( ), and prepare anti-aging products; and / or ( ), and prepare products containing superoxide dismutase; and / or ( ), and prepare products that enhance the activity of superoxide dismutase.

Images