Mutant of anti-aging protein and raw material preparation method and application thereof

Abstract

The present application relates to the field of bioengineering, and particularly relates to a mutant of anti-aging protein and a raw material preparation method and application thereof. The present application provides a mutant of superoxide dismutase, wherein the 46th position of the superoxide dismutase is mutated from L to Y; and / or the 52nd position of the superoxide dismutase is mutated from L to V; and / or the 53rd position of the superoxide dismutase is mutated from S to E; and / or the 116th position of the superoxide dismutase is mutated from Q to K; and / or the 157th position of the superoxide dismutase is mutated from S to Q; and / or the 158th position of the superoxide dismutase is mutated from L to D. The present application firstly performs heat treatment (50 DEG C) screening on an SOD enzyme library, and then focuses on a mesophilic bacteria-derived SOD protein, wherein HaSOD is used as a template, and after multiple rounds of iterative mutation, a mutant with high heat resistance, good stability and good enzyme activity is obtained.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and in particular to mutants of anti-aging proteins, their raw material preparation methods, and applications. Background Technology

[0002] In 1969, McCord and Fridovich discovered a blood cell copper protein, which they named superoxide dismutase (SOD). Superoxide dismutase has antioxidant and anti-aging effects, and its mechanism of action is mainly to scavenge harmful superoxide anion free radicals (O₂O₃). 2- Superoxide anion free radicals produced by organisms are normal metabolic products. However, the accumulation of free radicals can cause lipid peroxidation of cell membranes, leading to membrane rupture, cell damage, and even death. The main function of SOD is to catalyze the dismutation of superoxide anion free radicals into hydrogen peroxide and oxygen, becoming the most important free radical scavenger in organisms and maintaining metabolic balance.

[0003] Superoxide dismutase (SOD) has shown great promise in disease treatment, the food industry, the daily chemical industry, and agriculture. This enzyme is extremely widely distributed and has been isolated from various organisms, including bacteria, fungi, algae, plants, protozoa, insects, fish, and mammals. However, natural extraction has significant limitations. With the development of genetic engineering and technology, engineered bacteria expressing recombinant proteins have shown greater advantages. To meet the increasing demand for SOD, the stability of SOD is crucial, requiring high stability (e.g., wide pH range adaptability, good heat resistance, etc.). Researchers have improved the performance of SOD through homologous recombination technology, enzyme screening, and modification, thereby obtaining highly stable SOD enzyme preparations. Summary of the Invention

[0004] In view of this, the present invention provides mutants of anti-aging proteins, methods for preparing their raw materials, and their applications. The present invention first screens an SOD enzyme library by heat treatment (50°C), focusing on an SOD protein derived from mesophilic bacteria: using HaSOD as a template, a HaSOD3179 mutant with high heat resistance, good stability, and good enzyme activity was obtained after multiple rounds of iterative mutation.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides mutants of superoxide dismutase, said mutants comprising: a mutation at position 46 of the superoxide dismutase from L to Y; and / or

[0007] The 52nd position of the superoxide dismutase is mutated from L to V; and / or

[0008] The 53rd position of the superoxide dismutase is mutated from S to E; and / or

[0009] The superoxide dismutase is mutated from Q to K at position 116; and / or

[0010] The superoxide dismutase is mutated from S to Q at position 157; and / or

[0011] The superoxide dismutase was mutated from L to D at position 158.

[0012] In some embodiments of the present invention, the above-mentioned mutant includes:

[0013] (I) The superoxide dismutase is derived from mesophilic bacteria; and / or

[0014] (II) The superoxide dismutase has a specific amino acid sequence; and / or

[0015] (III) The gene for the superoxide dismutase has a specific nucleotide sequence;

[0016] The specific amino acid sequence has:

[0017] (1) An amino acid sequence as shown in SEQ ID NO:1; or

[0018] (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

[0019] (3) A sequence that is more than 80% homologous to the amino acid sequence shown in (1) or (2);

[0020] The specific nucleotide sequence has:

[0021] (4) A nucleotide sequence as shown in SEQ ID NO:2; or

[0022] (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

[0023] (6) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (4) or (5).

[0024] In some embodiments of the present invention, the sequence of SEQ ID NO:1 is: MAYELPQLPYAYDALEPHFDKATMEIHHTKHHQTYVTNANAALEKLPEFASLSAEELVAKLDEVPEASCTAIRNNAGGHANHSLFWTILTPNSEGAPVGELKAAIDSTFGSFEQFQEQFETAAKGRFGSGWAWLTVKDGKLAVGSTANQDSPLSPKSLGGLEATPVLGLDVWEHAYYLNYQNRRPDYIKAFWNLVNWAEVEKRYQAAK.

[0025] In some embodiments of the present invention, the sequence of SEQ ID NO:2 is:

[0026] In some embodiments of the present invention, the mutant has:

[0027] (7) Any amino acid sequence as shown in SEQ ID NO:3 to SEQ ID NO:9; or

[0028] (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

[0029] (9) A sequence that is more than 80% homologous to the amino acid sequence shown in (7) or (8).

[0030] In some embodiments of the present invention, the sequence of SEQ ID NO:3 is: MAYELPQLPYAYDALEPHFDKATMEIHHTKHHQTYVTNANAALEKYPEFASLSAEELVAKLDEVPEASCTAIRNNAGGHANHSLFWTILTPNSEGAPVGELKAAIDSTFGSFEQFQEQFETAAKGRFGSGWAWLTVKDGKLAVGSTANQDSPLSPKSLGGLEATPVLGLDVWEHAYYLNYQNRRPDYIKAFWNLVNWAEVEKRYQAAK. (L46Y)

[0031] In some embodiments of the present invention, the sequence of SEQ ID NO:4 is: MAYELPQLPYAYDALEPHFDKATMEIHHTKHHQTYVTNANAALEKLPEFASVSAEELVAKLDEVPEASCTAIRNNAGGHANHSLFWTILTPNSEGAPVGELKAAIDSTFGSFEQFQEQFETAAKGRFGSGWAWLTVKDGKLAVGSTANQDSPLSPKSLGGLEATPVLGLDVWEHAYYLNYQNRRPDYIKAFWNLVNWAEVEKRYQAAK. (L52V)

[0032] In some embodiments of the present invention, the sequence of SEQ ID NO:5 is: MAYELPQLPY AYDALEPHFDKATMEIHHTKHHQTYVTNANAALEKLPEFASLEAEELVAKLDEVPEASCTAIRNNAGGHANHSLFWTILTPNSEGAPVGELKAAIDSTFGSFEQFQEQFETAAKGRFGSGWAWLTVKDGKLAVGSTANQDSPLSPKSLGGLEATPVLGLDVWEHAYYLNYQNRRPDYIKAFWNLVNWAEVEKRYQAAK.

[0033] (S53E)

[0034] In some embodiments of the present invention, the sequence of SEQ ID NO:6 is: MAYELPQLPY AYDALEPHFDKATMEIHHTKHHQTYVTNANAALEKLPEFASLSAEELVAKLDEVPEASCTAIRNNAGGHANHSLFWTILTPNSEGAPVGELKAAIDSTFGSFEQFKEQFETAAKGRFGSGWAWLTVKDGKLAVGSTANQDSPLSPKSLGGLEATPVLGLDVWEHAYYLNYQNRRPDYIKAFWNLVNWAEVEKRYQAAK.

[0035] (Q116K)

[0036] In some embodiments of the present invention, the sequence of SEQ ID NO:7 is: MAYELPQLPY AYDALEPHFDKATMEIHHTKHHQTYVTNANAALEKLPEFASLSAEELVAKLDEVPEASCTAIRNNAGGHANHSLFWTILTPNSEGAPVGELKAAIDSTFGSFEQFQEQFETAAKGRFGSGWAWLTVKDGKLAVGSTANQDSPLSPKQLGGLEATPVLGLDVWEHAYYLNYQNRRPDYIKAFWNLVNWAEVEKRYQAAK.

[0037] (S157Q)

[0038] In some embodiments of the present invention, the sequence of SEQ ID NO:8 is: MAYELPQLPY AYDALEPHFDKATMEIHHTKHHQTYVTNANAALEKLPEFASLSAEELVAKLDEVPEASCTAIRNNAGGHANHSLFWTILTPNSEGAPVGELKAAIDSTFGSFEQFQEQFETAAKGRFGSGWAWLTVKDGKLAVGSTANQDSPLSPKSDGGLEATPVLGLDVWEHAYYLNYQNRRPDYIKAFWNLVNWAEVEKRYQAAK.

[0039] (L158D)

[0040] In some embodiments of the present invention, the sequence of SEQ ID NO:9 is: MAYELPQLPY AYDALEPHFDKATMEIHHTKHHQTYVTNANAALEKYPEFASVEAEELVAKLDEVPEASCTAIRNNAGGHANHSLFWTILTPNSEGAPVGELKAAIDSTFGSFEQFKEQFETAAKGRFGSGWAWLTVKDGKLAVGSTANQDSPLSPKQDGGLEATPVLGLDVWEHAYYLNYQNRRPDYIKAFWNLVNWAEVEKRYQAAK. (L46Y, L52V, S53E, Q116K, S157Q, L158D)

[0041] The present invention also provides a nucleic acid molecule encoding the above-mentioned mutant, wherein the CTG at positions 136-138 of the nucleic acid molecule encoding the superoxide dismutase is mutated to TAC; and / or

[0042] The C at position 154 of the nucleic acid molecule encoding the superoxide dismutase is mutated to G; and / or

[0043] The CTC at positions 156-158 of the nucleic acid molecule encoding the superoxide dismutase is mutated to GAG; and / or

[0044] The C at position 346 of the nucleic acid molecule encoding the superoxide dismutase is mutated to A; and / or

[0045] The TCCCTG at positions 469-474 of the nucleic acid molecule encoding the superoxide dismutase is mutated to CAGGAC.

[0046] In some embodiments of the present invention, the nucleic acid molecule has:

[0047] (10) A nucleotide sequence as shown in any of SEQ ID NO:10 to SEQ ID NO:16; or

[0048] (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

[0049] (12) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (10) or (11).

[0050] In some embodiments of the present invention, the sequence of SEQ ID NO:10 is: ATGGCCTAC GAACTTCCGCAACTCCCCTACGCCTACGATGCGCTAGAGCCGCACTTCGACAAGGCGACGATGGAAATCCACCACACCAAGCACCATCAGACTTACGTCACCAATGCCAATGCCGCGCTCGAGAAGTACCCCGAGTTCGCCAGCCTCTCGGCCGAGGAGCTGGTGGCCAAGCTCGACGAAGTACCTGAAGCCAGCTGCACCGCGATCCGCAACAACGCAGGCGGTCACGCCAACCACTCGCTGTTCTGGACCATCCTCACGCCGAACTCCGAAGGCGCCCCGGTGGGTGAGCTGAAAGCTGCGATCGACAGCACCTTCGGCAGCTTCGAGCAGTTCCAGGAGCAGTTCGAGACCGCCGCCAAGGGGCGCTTCGGCTCCGGCTGGGCATGGCTTACGGTCAAGGATGGCAAGCTCGCGGTCGGCTCCACCGCCAACCAGGACAGCCCGCTGTCGCCGAAGTCCCTGGGCGGGCTCGAAGCCACCCCTGTGCTTGGCCTGGACGTGTGGGAACATGCCTACTACCTGAATTACCAGAACCGCCGCCCCGACTACATCAAGGCGTTCTGGAACCTGGTCAACTGGGCCGAAGTCGAAAAGCGCTACCAAGCCGCCAAGTAA。(L46Y) In some embodiments of the present invention, the sequence of SEQ ID NO:11 is: ATGGCCTAC GAACTTCCGCAACTCCCCTACGCCTACGATGCGCTAGAGCCGCACTTCGACAAGGCGACGATGGAAATCCACCACACCAAGCACCATCAGACTTACGTCACCAATGCCAATGCCGCGCTCGAGAAGCTGCCCGAGTTCGCCAGCGTATCGGCCGAGGAGCTGGTGGCCAAGCTCGACGAAGTACCTGAAGCCAGCTGCACCGCGATCCGCAACAACGCAGGCGGTCACGCCAACCACTCGCTGTTCTGGACCATCCTCACGCCGAACTCCGAAGGCGCCCCGGTGGGTGAGCTGAAAGCTGCGATCGACAGCACCTTCGGCAGCTTCGAGCAGTTCCAGGAGCAGTTCGAGACCGCCGCCAAGGGGCGCTTCGGCTCCGGCTGGGCATGGCTTACGGTCAAGGATGGCAAGCTCGCGGTCGGCTCCACCGCCAACCAGGACAGCCCGCTGTCGCCGAAGTCCCTGGGCGGGCTCGAAGCCACCCCTGTGCTTGGCCTGGACGTGTGGGAACATGCCTACTACCTGAATTACCAGAACCGCCGCCCCGACTACATCAAGGCGTTCTGGAACCTGGTCAACTGGGCCGAAGTCGAAAAGCGCTACCAAGCCGCCAAGTAA。(L52V) In some embodiments of the present invention, the sequence of SEQ ID NO:12 is: ATGGCCTAC GAACTTCCGCAACTCCCCTACGCCTACGATGCGCTAGAGCCGCACTTCGACAAGGCGACGATGGAAATCCACCACACCAAGCACCATCAGACTTACGTCACCAATGCCAATGCCGCGCTCGAGAAGCTGCCCGAGTTCGCCAGCCTCGAGGCCGAGGAGCTGGTGGCCAAGCTCGACGAAGTACCTGAAGCCAGCTGCACCGCGATCCGCAACAACGCAGGCGGTCACGCCAACCACTCGCTGTTCTGGACCATCCTCACGCCGAACTCCGAAGGCGCCCCGGTGGGTGAGCTGAAAGCTGCGATCGACAGCACCTTCGGCAGCTTCGAGCAGTTCCAGGAGCAGTTCGAGACCGCCGCCAAGGGGCGCTTCGGCTCCGGCTGGGCATGGCTTACGGTCAAGGATGGCAAGCTCGCGGTCGGCTCCACCGCCAACCAGGACAGCCCGCTGTCGCCGAAGTCCCTGGGCGGGCTCGAAGCCACCCCTGTGCTTGGCCTGGACGTGTGGGAACATGCCTACTACCTGAATTACCAGAACCGCCGCCCCGACTACATCAAGGCGTTCTGGAACCTGGTCAACTGGGCCGAAGTCGAAAAGCGCTACCAAGCCGCCAAGTAA。(S53E) In some embodiments of the present invention, the sequence of SEQ ID NO:13 is: ATGGCCTAC GAACTTCCGCAACTCCCCTACGCCTACGATGCGCTAGAGCCGCACTTCGACAAGGCGACGATGGAAATCCACCACACCAAGCACCATCAGACTTACGTCACCAATGCCAATGCCGCGCTCGAGAAGCTGCCCGAGTTCGCCAGCCTCTCGGCCGAGGAGCTGGTGGCCAAGCTCGACGAAGTACCTGAAGCCAGCTGCACCGCGATCCGCAACAACGCAGGCGGTCACGCCAACCACTCGCTGTTCTGGACCATCCTCACGCCGAACTCCGAAGGCGCCCCGGTGGGTGAGCTGAAAGCTGCGATCGACAGCACCTTCGGCAGCTTCGAGCAGTTCAAGGAGCAGTTCGAGACCGCCGCCAAGGGGCGCTTCGGCTCCGGCTGGGCATGGCTTACGGTCAAGGATGGCAAGCTCGCGGTCGGCTCCACCGCCAACCAGGACAGCCCGCTGTCGCCGAAGTCCCTGGGCGGGCTCGAAGCCACCCCTGTGCTTGGCCTGGACGTGTGGGAACATGCCTACTACCTGAATTACCAGAACCGCCGCCCCGACTACATCAAGGCGTTCTGGAACCTGGTCAACTGGGCCGAAGTCGAAAAGCGCTACCAAGCCGCCAAGTAA。(Q116K) In some embodiments of the present invention, the sequence of SEQ ID NO:14 is: ATGGCCTAC GAACTTCCGCAACTCCCCTACGCCTACGATGCGCTAGAGCCGCACTTCGACAAGGCGACGATGGAAATCCACCACACCAAGCACCATCAGACTTACGTCACCAATGCCAATGCCGCGCTCGAGAAGCTGCCCGAGTTCGCCAGCCTCTCGGCCGAGGAGCTGGTGGCCAAGCTCGACGAAGTACCTGAAGCCAGCTGCACCGCGATCCGCAACAACGCAGGCGGTCACGCCAACCACTCGCTGTTCTGGACCATCCTCACGCCGAACTCCGAAGGCGCCCCGGTGGGTGAGCTGAAAGCTGCGATCGACAGCACCTTCGGCAGCTTCGAGCAGTTCCAGGAGCAGTTCGAGACCGCCGCCAAGGGGCGCTTCGGCTCCGGCTGGGCATGGCTTACGGTCAAGGATGGCAAGCTCGCGGTCGGCTCCACCGCCAACCAGGACAGCCCGCTGTCGCCGAAGCAGCTGGGCGGGCTCGAAGCCACCCCTGTGCTTGGCCTGGACGTGTGGGAACATGCCTACTACCTGAATTACCAGAACCGCCGCCCCGACTACATCAAGGCGTTCTGGAACCTGGTCAACTGGGCCGAAGTCGAAAAGCGCTACCAAGCCGCCAAGTAA。(S157Q) In some embodiments of the present invention, the sequence of SEQ ID NO:15 is: ATGGCCTAC GAACTTCCGCAACTCCCCTACGCCTACGATGCGCTAGAGCCGCACTTCGACAAGGCGACGATGGAAATCCACCACACCAAGCACCATCAGACTTACGTCACCAATGCCAATGCCGCGCTCGAGAAGCTGCCCGAGTTCGCCAGCCTCTCGGCCGAGGAGCTGGTGGCCAAGCTCGACGAAGTACCTGAAGCCAGCTGCACCGCGATCCGCAACAACGCAGGCGGTCACGCCAACCACTCGCTGTTCTGGACCATCCTCACGCCGAACTCCGAAGGCGCCCCGGTGGGTGAGCTGAAAGCTGCGATCGACAGCACCTTCGGCAGCTTCGAGCAGTTCCAGGAGCAGTTCGAGACCGCCGCCAAGGGGCGCTTCGGCTCCGGCTGGGCATGGCTTACGGTCAAGGATGGCAAGCTCGCGGTCGGCTCCACCGCCAACCAGGACAGCCCGCTGTCGCCGAAGTCCGACGGCGGGCTCGAAGCCACCCCTGTGCTTGGCCTGGACGTGTGGGAACATGCCTACTACCTGAATTACCAGAACCGCCGCCCCGACTACATCAAGGCGTTCTGGAACCTGGTCAACTGGGCCGAAGTCGAAAAGCGCTACCAAGCCGCCAAGTAA。(L158D) In ​​some embodiments of the present invention, the sequence of SEQ ID NO:16 is: ATGGCCTAC. (L46Y, L52V, S53E, Q116K, S157Q, L158D).

[0051] The present invention also provides a recombinant expression vector comprising the above-described nucleic acid molecules and acceptable gene elements.

[0052] The present invention also provides a host, transformation and / or transfection of the above-mentioned recombinant expression vector.

[0053] In some embodiments of the present invention, the host includes Corynebacterium glutamicum strain ATCC13032.

[0054] The present invention also provides a method for preparing the above-mentioned mutant, comprising the following steps:

[0055] S1: 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.

[0056] S2: Induce host expression containing the recombinant expression vector, and after purification, obtain the mutant.

[0057] In some embodiments of the present invention, the purification in the above preparation method includes a thermal activation step; the thermal activation temperature is 75-100°C and the time is 30-120 min.

[0058] In some embodiments of the present invention, the purification in the above preparation method includes a thermal activation step; the thermal activation temperature is 100°C and the time is 30 min.

[0059] In some embodiments of the present invention, in the above preparation method, the thermally activated activator includes: 50–300 μM Mn 2+ .

[0060] The present invention also provides primer sets, including one or more of primer sets 1 to 6;

[0061] The primer set 1 has:

[0062] (13) Nucleotide sequences as shown in SEQ ID NO:17 and SEQ ID NO:18; or

[0063] (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

[0064] (15) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (13) or (14); and / or

[0065] Primer set 2 has:

[0066] (16) Nucleotide sequences as shown in SEQ ID NO:19 and SEQ ID NO:20; or

[0067] (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

[0068] (18) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (16) or (17); and / or

[0069] Primer set 3 has:

[0070] (19) Nucleotide sequences as shown in SEQ ID NO:21 and SEQ ID NO:22; or

[0071] (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

[0072] (21) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (19) or (20); and / or

[0073] Primer set 4 has:

[0074] (22) Nucleotide sequences as shown in SEQ ID NO:23 and SEQ ID NO:24; or

[0075] (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

[0076] (24) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (22) or (23); and / or

[0077] The primer set 5 has:

[0078] (25) Nucleotide sequences as shown in SEQ ID NO:25 and SEQ ID NO:26; or

[0079] (26) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (25), and which has the same or similar function as the nucleotide sequence shown in (25); or

[0080] (27) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (25) or (26); and / or

[0081] Primer set 6 has:

[0082] (28) Nucleotide sequences as shown in SEQ ID NO:27 and SEQ ID NO:28; or

[0083] (29) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (28), and which has the same or similar function as the nucleotide sequence shown in (28); or

[0084] (30) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (28) or (29).

[0085] In some embodiments of the present invention, the sequence of SEQ ID NO:17 is: GAGAAGtacCCCGAGTTCGCCAGC.

[0086] In some embodiments of the present invention, the sequence of SEQ ID NO:18 is: GCTGGCGAACTCGGGgtaCTTCTC.

[0087] In some embodiments of the present invention, the sequence of SEQ ID NO:19 is: GCCAGGCGTAtcgGCCGAGGAGCTGGTGGCCAAGC.

[0088] In some embodiments of the present invention, the sequence of SEQ ID NO:20 is: GCCGAGGAGCTGGTGGCCAAGC.

[0089] In some embodiments of the present invention, the sequence of SEQ ID NO:21 is: GCCAGCctcGAGGCCGAGGAGCTGGTGGCCAAGC.

[0090] In some embodiments of the present invention, the sequence of SEQ ID NO:22 is: GCCGAGGAGCTGGTGGCCAAGC.

[0091] In some embodiments of the present invention, the sequence of SEQ ID NO:23 is: CAGTTCaagGAGCAGTTCGAGACCGCCGCCAAG.

[0092] In some embodiments of the present invention, the sequence of SEQ ID NO:24 is: CTTGGCGGCGGTCTCGAACTGCTC.

[0093] In some embodiments of the present invention, the sequence of SEQ ID NO:25 is: CCGAAGCAGctgGGCGGGCTCGAAGCCACCCCT.

[0094] In some embodiments of the present invention, the sequence of SEQ ID NO:26 is: AGGGGTGGC TTCGAGCCCGCC.

[0095] In some embodiments of the present invention, the sequence of SEQ ID NO:27 is: CCGAAGtccGACGGCGGGCTCGAAGCCACCCCT.

[0096] In some embodiments of the present invention, the sequence of SEQ ID NO:28 is: AGGGGTGGC TTCGAGCCCGCC.

[0097] The present invention also provides the use of the mutant, the nucleic acid molecule, the recombinant expression vector, the host, and / or the mutant obtained by the preparation method in any of the following:

[0098] (I) Preparation of antioxidant products; and / or

[0099] (II) Preparation of anti-aging products; and / or

[0100] (III) Preparing products containing superoxide dismutase; and / or

[0101] (IV) Improve the thermal stability of superoxide dismutase; and / or

[0102] (V) Increase the activity of superoxide dismutase.

[0103] This invention provides mutants of superoxide dismutase, the mutants comprising: a mutation of L to Y at position 46 of the superoxide dismutase; and / or a mutation of L to V at position 52 of the superoxide dismutase; and / or a mutation of S to E at position 53 of the superoxide dismutase; and / or a mutation of Q to K at position 116 of the superoxide dismutase; and / or a mutation of S to Q at position 157 of the superoxide dismutase; and / or a mutation of L to D at position 158 of the superoxide dismutase.

[0104] The beneficial effects of this invention include:

[0105] (1) Experimental data on protein expression and activity of Uniprot#:A0A172YBF5 were disclosed for the first time.

[0106] (2) The protein expression system of Corynebacterium glutamicum does not contain endotoxin, and the application of SOD products can be more extensive.

[0107] (3) The selected mutant HaSOD6-3179 has high thermal stability and high enzyme activity.

[0108] (4) The preparation method of this invention uses thermal activation followed by Q-column purification. The process is simple and can be scaled up industrially. Attached Figure Description

[0109] 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.

[0110] Figure 1As shown, SOD01, 02, 03, 04, 05, 06, 07, 08, 09 and HaSOD represent different strain sequence numbers;

[0111] Figure 2 The DNA Marker is 5000bp, and the full length of the target gene for cloning is 627bp.

[0112] Figure 3 The diagram shows different amino acid mutation sites (single point mutation, multiple site mutation) and mutants and their corresponding crude enzyme activities.

[0113] Figure 4 The results show that mutants with different amino acid mutation sites exhibit varying enzyme activities under optimal treatment conditions.

[0114] Figure 5 The results of protein expression in crude enzyme solution after induction of different mutants are shown in 12.5% ​​SDS-PAGE, lanes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0115] Figure 6 As shown, 12.5% ​​SDS-PAGE, lanes 1, 2, 3, 4, 5, 6, 7, and 8 indicate the numbering of different collection tubes during protein collection;

[0116] Figure 7 The results of different treatment methods for the target protein under non-denaturing conditions are shown in the 12.5% ​​SDS-PAGE. Lanes 1, 2, 3, 4, 5, and 6 represent the results obtained by different treatment methods for the target protein under non-denaturing conditions.

[0117] Figure 8 The figure 1 indicates that no metal activator Mn is added. 2+ The enzyme activity after treatment at 75℃ for 30 min; 2 indicates the enzyme activity after adding 50 μM Mn. 2+ The enzyme activity after treatment at 75℃ for 30 min; 3 indicates the enzyme activity after adding 50 μM N. 2+ The enzyme activity after treatment at 90℃ for 30 min; 4 indicates the enzyme activity after adding 50 μM Mn. 2+ The enzyme activity after treatment at 100℃ for 30 min;

[0118] Figure 9 The values ​​0, 30, 60, 90, and 120 min represent the enzyme activity results of the target protein after treatment at 100°C for different times. Detailed Implementation

[0119] This invention discloses mutants of anti-aging proteins, their raw material preparation methods, and applications.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] This invention provides a highly stable HaSOD6-3179 mutant obtained by using HaSOD, a SOD protein derived from mesophilic bacteria, as a template and undergoing multiple rounds of iterative mutation.

[0126] Specifically, the protein expression system of Corynebacterium glutamicum is used, and a unique purification process is employed: a thermal activation reaction (on the one hand, the thermal activation reaction increases enzyme activity; on the other hand, the target protein is initially crudely purified through heating treatment), followed by Q-column purification to obtain the protein.

[0127] In Examples 1 to 8 of this invention, all raw materials and reagents used can be purchased from the market.

[0128] The present invention will be further illustrated below with reference to the embodiments:

[0129] Example 1: Obtaining the target gene

[0130] Through database searching, screening experiments were conducted on the heat resistance (50℃) of various SOD enzymes from different sources (e.g., Figure 1 As shown in the figure, a SOD protein (Uniprot#:A0A172YBF5) derived from mesophilic bacteria was obtained. The full-length gene is 627 bp, and its sequence (as shown in SEQ ID NO:1) encodes 208 amino acids. It was named HaSOD, and its sequence (as shown in SEQ ID NO:2) is shown in the figure.

[0131] Example 2: Construction of the carrier

[0132] HaSOD was synthesized into the pUC19 vector via gene synthesis. Further homologous recombination was used to design the upstream primer HaSOD-F (ctcggtacccggggatcctctATGGCCTACGAACTTCCGCAACT) (as shown in SEQ ID NO:29) and the downstream primer HaSOD-R (gcctgcaggtcgactctTACTTGGCGGCTTGGTAGCGC) (as shown in SEQ ID NO:30) to obtain the target gene (e.g., Figure 2 (As shown). The pECXK99E vector was then double-digested (BamHI / SalI) to clone HaSOD into the pECXK99E vector via homologous recombination. This clone was then transformed into DH5α recipient cells (verified by sequencing) and stored at -80°C. Subsequently, the HaSOD / pECXK99E was transformed into Corynebacterium glutamicum ATCC13032 strain (purchased from the China Microbial Culture Collection), yielding positive clones.

[0133] Example 3 Enzyme modification and induction of recombinant protein expression

[0134] A 96-well plate enzyme activity screening system was constructed by iterative saturation mutagenesis at the active site. Different mutants were induced to express overnight, and then Mn metal was added. 2+ The enzyme was treated at 100℃ for 30 min, and its crude enzyme activity was measured to screen for (e.g.) Figure 3 (As shown) 24 single-clone bacterial cells. After purification, under a tolerance condition of 100℃ for 30 min, HaSOD3179 mutants (L46Y, L52V, S53E, Q116K, S157Q, L158D) were screened out, and those with the highest specific enzyme activity (e.g.) Figure 4 (As shown).

[0135] Different mutant strains of HaSOD enzyme were inoculated into 10 mL of LBG kanamycin liquid medium and cultured at 30℃ and 180 rpm for 12 h to obtain seed culture. Then, 1 mL of seed culture was inoculated into 50 mL of LBG kanamycin liquid medium and cultured at 30℃ and 180 rpm for 3 h. 0.5 mmol / L IPTG was then added, and the culture was continued at 25℃ and 180 rpm for 7 h to induce SOD enzyme expression. The bacterial cells were then collected for cell disruption and protein electrophoresis analysis (e.g., ...). Figure 5 (As shown).

[0136] Example 4 Purification Process

[0137] Protein purification of HaSOD3179 enzyme:

[0138] (1) Weigh the bacterial cells, add 20mM Tris-HCl loading solution (pH 8.0) to resuspend the bacterial cells, and shake to mix well;

[0139] (2) After homogenization, the material was subjected to thermal activation purification by adding 300 μM MnCl₂. 2+ Then, treat at 100℃ for 0.5 hours;

[0140] (3) After centrifugation at 8000 rpm for 10 min, the product was purified by elution on a Q Bestarose HP column with 10%-100% linear elution buffer (20 mM Tris-HCl, 500 mM NaCl) for 5-10 CV each.

[0141] (4) Perform SDS-PAGE testing (e.g.) Figure 6 (As shown).

[0142] Example 5 Enzyme Activity Analysis

[0143] The enzyme activity of the sample was determined according to the detection method GB / T 41906-2022. 50 μM N was added. 2+ After being thermally activated at 100℃ for 30 minutes, the highest enzyme activity was measured to be 2433.2 U / mg.

[0144] Example 6 Metal ion Mn 2+ Stability analysis of HaSOD3179

[0145] Simultaneously add metal ions Mn 2+ After heating (100℃ for 30 min), HaSOD3179 showed no degradation; after heating alone (100℃ for 30 min), HaSOD3179 showed significant degradation; and no metal ion Mn was added. 2+After heating (75℃ for 30 min), the HaSOD3179 protein showed slight degradation. This indicates that the enzyme exists in a polymeric form under non-denaturing conditions, and the addition of metal ions (Mn) further contributes to its degradation. 2+ This contributes to the stability of the enzyme's multimeric protein structure and to greater stability at high temperatures (100°C), such as... Figure 7 As shown.

[0146] Example 7 Thermal stability

[0147] The specific enzyme activity of SOD pure enzyme was determined by subjecting cell lysate (1 part cell mass to 9 parts buffer mass) to heat activation at different temperatures for 30 min, followed by measurement of its specific enzyme activity. The table below shows that the enzyme activity of HaSOD3179 increases with increasing heat treatment temperature. Figure 8 As shown, this indicates that the enzyme's activity is related to Mn. 2+ Related to, and metal ion Mn 2+ It serves as the activator for the thermal activation of this enzyme. Furthermore, it exhibits excellent thermal stability; even after treatment at 100°C for 30 minutes, the protein retains high enzyme activity.

[0148] Example 8 Heat resistance stability

[0149] HaSOD3179 enzyme, add 50 μM N 2+ Subsequently, the enzyme was heated to 100℃ (sampling time points of 30 min, 60 min, 90 min, and 120 min), and its enzyme activity was measured. The results showed that it exhibited high heat resistance; after treatment at 100℃ for 120 min, its enzyme activity did not change significantly, demonstrating extremely high thermal stability. Figure 9 As shown.

[0150] 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 is one of mutant 1 to mutant 24, and the amino acid sequence of the superoxide dismutase is shown in SEQ ID NO:1; The mutant 1 is characterized by a mutation where position 46 of the superoxide dismutase is changed from L to Y. The mutant 2 is characterized by a mutation at position 52 of the superoxide dismutase, where L is mutated to V. The mutant 3 is characterized by a mutation where position 53 of the superoxide dismutase is changed from S to E. The mutant 4 is characterized by a mutation where position 116 of the superoxide dismutase is changed from Q to K. The mutant 5 is characterized by a mutation where position 157 of the superoxide dismutase is changed from S to Q. The mutant 6 is characterized by a mutation at position 158 of the superoxide dismutase, where L is mutated to D. The mutant 7 is characterized by a mutation where position 46 of the superoxide dismutase is changed from L to Y and position 53 is changed from S to E. The mutant 8 is characterized by a mutation where position 52 of the superoxide dismutase is changed from L to V and position 53 is changed from S to E. The mutant 9 is characterized by a mutation where position 46 of the superoxide dismutase is changed from L to Y and position 158 is changed from L to D. The mutant 10 is: the 53rd position of the superoxide dismutase is mutated from S to E and the 158th position is mutated from L to D; The mutant 11 is: the superoxide dismutase has a Q-to-K mutation at position 116 and a S-to-Q mutation at position 157; The mutant 12 is: the 157th position of the superoxide dismutase is mutated from S to Q and the 158th position is mutated from L to D; The mutant 13 is characterized by a mutation in the superoxide dismutase where position 46 is changed from L to Y, position 53 from S to E, and position 158 from L to D. The mutant 14 is characterized by a mutation in the superoxide dismutase where L is mutated to V at position 52, S is mutated to E at position 53, and Q is mutated to K at position 116. The mutant 15 is characterized by a mutation in the superoxide dismutase where L is mutated to V at position 52, S is mutated to E at position 53, and S is mutated to Q at position 157. The mutant 16 is: the 46th position of the superoxide dismutase is mutated from L to Y, the 52nd position is mutated from L to V, and the 158th position is mutated from L to D; The mutant 17 is characterized by a mutation where position 46 of the superoxide dismutase is changed from L to Y, position 116 from Q to K, and position 158 from L to D. The mutant 18 is: the 53rd position of the superoxide dismutase is mutated from S to E, the 157th position is mutated from S to Q, and the 158th position is mutated from L to D; The mutant 19 is: the 46th position of the superoxide dismutase is mutated from L to Y, the 53rd position from S to E, the 116th position from Q to K, and the 158th position from L to D; The mutant 20 is: the 52nd position of the superoxide dismutase is mutated from L to V, the 53rd position from S to E, the 116th position from Q to K, and the 158th position from L to D; The mutant 21 is: the 46th position of the superoxide dismutase is mutated from L to Y, the 52nd position from L to V, the 53rd position from S to E, and the 116th position from Q to K; The mutant 22 is: the 46th position of the superoxide dismutase is mutated from L to Y, the 52nd position from L to V, the 53rd position from S to E, the 116th position from Q to K, and the 157th position from S to Q; The mutant 23 is: the 46th position of the superoxide dismutase is mutated from L to Y, the 52nd position from L to V, the 53rd position from S to E, the 116th position from Q to K, and the 158th position from L to D; The mutant 24 is: the 46th position of the superoxide dismutase is mutated from L to Y, the 52nd position from L to V, the 53rd position from S to E, the 116th position from Q to K, the 157th position from S to Q, and the 158th position from L to D.

2. The mutant as described in claim 1, characterized in that, The sequence of the mutant is any of the amino acid sequences shown in SEQ ID NO:3 to SEQ ID NO:

9.

3. A nucleic acid molecule encoding the mutant as described in claim 1 or 2, characterized in that, The sequence of the nucleic acid molecule is any of the nucleotide sequences shown in SEQ ID NO:10 to SEQ ID NO:

16.

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

5. The host, characterized in that, Transformation and / or transfection with the recombinant expression vector as described in claim 4; the host includes: Corynebacterium glutamicum ATCC13032 strain.

6. The method for preparing the mutant as described in claim 1 or 2, characterized in that, Includes the following steps: S1: Take the nucleic acid molecule as described in claim 3 and fuse it with the expression vector to construct the recombinant expression vector as described in claim 4, and transform and / or transfect it into the host as described in claim 5; S2: Induce host expression containing the recombinant expression vector, and after purification, obtain the mutant.

7. The preparation method according to claim 6, characterized in that, The purification includes a thermal activation step; the thermal activation temperature is 75~100℃ and the time is 30~120 min.

8. The preparation method according to claim 7, characterized in that, The thermally activated activator includes: 50~300 μMn 2+ .

9. The use of the mutant as described in claim 1 or 2, the nucleic acid molecule as described in claim 3, the recombinant expression vector as described in claim 4, the host as described in claim 5, and / or the mutant obtained by the preparation method as described in any one of claims 6 to 8 in the preparation of antioxidant products.

10. The use of the mutant as described in claim 1 or 2, the nucleic acid molecule as described in claim 3, the recombinant expression vector as described in claim 4, the host as described in claim 5, and / or the mutant obtained by the preparation method as described in any one of claims 6 to 8 in the preparation of anti-aging products.

11. The use of the mutant of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant expression vector of claim 4, the host of claim 5, and / or the mutant obtained by any one of the preparation methods of claims 6 to 8 in the preparation of a product containing superoxide dismutase.

12. The use of the mutant of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant expression vector of claim 4, the host of claim 5, and / or the mutant obtained by the preparation method of any one of claims 6 to 8 in improving the activity of superoxide dismutase.

Images