Preparation method and application of grease-resistant low-temperature superoxide dismutase SOD1 derived from abyssal

Abstract

The invention relates to the technical field of protein, in particular to a preparation method and application of deep-deep-derived grease-resistant low-temperature superoxide dismutase SOD1. Through rational design or high-throughput screening, the SOD1 variant which keeps high activity in a grease environment and is sensitive to temperature is obtained, and the sequence is as shown in SEQ ID NO: 6. The enzyme can be widely applied to the technical fields of food preservation, skin care products, grease oxidation resistance and low-temperature biology, and the key defects of existing SOD1 in industrial application are overcome.

Description

Technical Field

[0001] The present invention relates to the field of protein technology, and in particular to a preparation method and application of an abyss-derived, oil-resistant, low-temperature superoxide dismutase SOD1. Background Art

[0002] Superoxide dismutase (SOD) is a kind of antioxidant enzyme widely present in organisms, which can catalyze superoxide free radicals (O 2 • - ) is dismutated into hydrogen peroxide (H2O2) and oxygen (O2), thereby protecting cells from oxidative damage. Among them, SOD1 (Cu / Zn-SOD) is mainly distributed in the cytoplasm and cell nucleus, and its activity depends on copper (Cu² + ) and zinc (Zn² + ) ions, which play a key role in maintaining redox balance in cells.

[0003] However, natural SOD1 has many limitations in industrial applications.

[0004] Poor oil resistance: SOD1 is usually stable in an aqueous environment, but is easily inactivated in oil or organic solvent systems, limiting its application in food, cosmetics and oil products.

[0005] Temperature sensitivity: The optimum temperature of SOD1 is usually between 25 and 37°C. High temperatures (>50°C) can easily lead to conformational destruction and loss of enzyme activity, making it difficult to adapt to high-temperature processing or extreme environmental requirements.

[0006] Insufficient stability: Natural SOD1 is easily degraded in complex environments (such as high salt and extreme pH), affecting its long-term storage and practical application effects.

[0007] Currently, some studies have been conducted to improve the stability of SOD through protein engineering (such as site-directed mutagenesis and directed evolution) or chemical modification (such as PEGylation and cross-linking immobilization), but most modifications are still focused on heat resistance or antioxidant capacity, and there are few studies on the synergistic optimization of oil resistance and low-temperature adaptability. Summary of the Invention

[0008] In view of this, the present invention aims to provide a superoxide dismutase SOD1 mutant that is resistant to oil and fat and has strong low-temperature adaptability, and a preparation method thereof, so as to solve the problems existing in the industrial application of natural SOD1.

[0009] The purpose of this invention is to improve the stability of SOD1 in oily environments: through protein engineering, SOD1 can maintain high enzyme activity in oily or organic solvent systems, expanding its application in lipid-containing products (such as food and cosmetics);

[0010] Another objective of the present invention is to optimize the temperature sensitivity of SOD1: to design a SOD1 variant that is highly active at low temperatures (0-25°C) and controllably inactivated at high temperatures (>50°C), making it suitable for low-temperature storage or industrial scenarios requiring temperature regulation.

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0012] The present invention provides a mutant of superoxide dismutase having the following mutations:

[0013] (i) His at position 68 was mutated to Leu;

[0014] (ii) Asn 93 mutated to Asp;

[0015] (iii) Gly is inserted between positions 93 and 94.

[0016] In some specific embodiments of the present invention, the wild type of the mutant is a superoxide dismutase from lionfish (Pseudoliparis swirei), and the sequence may be:

[0017] (1) the amino acid sequence shown in SEQ ID NO: 4; or

[0018] (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0019] (3) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (1) or (2).

[0020] In some specific embodiments of the present invention, the amino acid sequence of the mutant is:

[0021] (4) the amino acid sequence shown in SEQ ID NO: 6; or

[0022] (5) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (4), and having the same or similar function as (4); or

[0023] (6) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (4) or (5).

[0024] The present invention also provides a nucleic acid molecule having a gene encoding the mutant.

[0025] In some specific embodiments of the present invention, the nucleic acid molecule has:

[0026] (1) the nucleotide sequence shown in SEQ ID NO: 5 or 6; or

[0027] (2) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (1), and having the same or similar function as (1); or

[0028] (3) A nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (1) or (2).

[0029] The present invention also provides a recombinant expression vector comprising the above nucleic acid molecule.

[0030] The present invention also provides a method for constructing the above-mentioned recombinant expression vector, comprising:

[0031] Primers are used to introduce restriction sites into the nucleic acid molecule to obtain an amplified product, which is then subjected to restriction enzyme digestion and ligated to a linearized expression vector backbone to obtain a recombinant expression vector.

[0032] In some specific embodiments of the present invention, the sequences of the primers in the above construction method are shown as SEQ ID NOs: 1 and 2.

[0033] The present invention also provides a host cell, which comprises the above nucleic acid molecule or the above recombinant expression vector;

[0034] The host cells do not include totipotent cells.

[0035] The present invention also provides a method for constructing the host cell, comprising: introducing the recombinant expression vector into a cell to obtain the host cell;

[0036] The cell may be a eukaryotic or prokaryotic organism;

[0037] The eukaryotic organism may be yeast;

[0038] The prokaryotic organism may be Escherichia coli.

[0039] The present invention also provides a method for preparing the above mutant, which is prepared based on any of the following:

[0040] (I) the above-mentioned nucleic acid molecule;

[0041] (II) the above-mentioned recombinant expression vector;

[0042] (III) The above-mentioned host cell.

[0043] In some specific embodiments of the present invention, the preparation method comprises:

[0044] (a) connecting the nucleic acid molecule to an expression vector backbone to obtain a recombinant expression vector, introducing the recombinant expression vector into cells, culturing, inducing, and obtaining a mutant; or

[0045] (b) introducing the above-mentioned recombinant expression vector into cells, culturing and inducing cells to obtain mutants; or

[0046] (c) Cultivating and inducing the above host cells to obtain mutants.

[0047] In some specific embodiments of the present invention, the preparation method comprises:

[0048] (A) connecting the nucleic acid molecule to an expression vector backbone to obtain a recombinant expression vector, introducing the recombinant expression vector into cells, culturing, inducing, collecting, disrupting, and purifying the cells to obtain a mutant; or

[0049] (B) introducing the above-mentioned recombinant expression vector into cells, culturing, inducing, collecting, disrupting, and purifying the cells to obtain mutants; or

[0050] (C) Cultivating and inducing the above host cells, collecting the cells, disrupting them, purifying them, and obtaining mutants.

[0051] The present invention also provides the use of the mutant in preparing food, medicine or cosmetic products.

[0052] The present invention also provides food comprising the mutant.

[0053] The present invention also provides a drug comprising the above mutant.

[0054] The present invention also provides cosmetic products comprising the mutant.

[0055] In some specific embodiments of the present invention, the above-mentioned food includes health food, and the mutant can play an anti-oxidation role in the health food.

[0056] In some specific embodiments of the present invention, the above-mentioned medicine includes a lipid-containing medicine, and the mutant can play a role in improving the stability of the medicine itself and can also play a role in improving the health status of the user.

[0057] In some specific embodiments of the present invention, the above-mentioned cosmetic products include cosmetics or skin care products, and may contain emulsions. The mutants can improve the stability of the cosmetic products themselves and improve the skin health of users.

[0058] The oil-resistant and low-temperature-adaptable superoxide dismutase SOD1 mutant provided by the present invention and its preparation method have the following significant advantages over the existing technology:

[0059] 1. Excellent oil and fat resistance: Through amino acid substitution or surface modification, the stability of SOD1 in oils and organic solvents is significantly enhanced, allowing it to maintain high activity in lipid-containing products (such as edible oils, dairy products, and cosmetics), solving the problem of natural SOD1 being easily inactivated in non-aqueous environments;

[0060] 2. Unique temperature adaptability: This SOD1 mutant exhibits high activity at low temperatures (0-25°C), making it suitable for applications such as refrigerated foods and low-temperature biological preparations. Furthermore, it is controllably inactivated at high temperatures (>50°C), preventing residual enzyme activity from affecting product quality during high-temperature processing.

[0061] 3. High-efficiency expression and low-cost production: Using optimized recombinant expression systems (such as E. coli or yeast) combined with efficient purification processes can significantly increase yields, reduce industrial production costs, and facilitate large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0063] Figure 1 Shows the optimum temperature of SOD1 enzyme;

[0064] Figure 2 Shows the optimal pH for SOD1 enzyme;

[0065] Figure 3 Shows the effect of oil concentration on SOD1 enzyme activity;

[0066] Figure 4 The results show the effect of different treatment time of homemade emulsion on SOD1 enzyme activity. DETAILED DESCRIPTION

[0067] The present invention discloses a method for preparing and applying a deep-oil-resistant, low-temperature superoxide dismutase (SOD1). Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications obvious to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is clear that those skilled in the art can modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0068] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0069] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0070] It should be understood that the order of steps or the order in which certain actions are performed are not important as long as the present invention remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0071] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present invention and does not limit the scope of the present invention. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the present invention.

[0072] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values ​​in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, amounts, values, and percentages used in this disclosure are modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0073] The present invention uses the SOD1 of the abyssal lionfish (Pseudoliparis swirei) originating from the extreme deep-sea environment as the mother parent, provides an oil-resistant and low-temperature superoxide dismutase SOD1 and its application, and also provides a preparation method thereof. The specific steps are as follows.

[0074] 1. Vector construction (cloning the SOD1 gene into pCold I)

[0075] (1) SOD1 gene: The SOD1 gene sequence was synthesized at Shanghai Bioengineering using PrimeSTAR ® HS DNA Polymerase (Cat. No. R010) was used to amplify the SOD1 fragment. The amplification primers are as follows.

[0076] SOD1F:GGAATTC CATATGGTTATGAAAGCTGTCTGTGTGC (SEQ ID NO: 1, restriction enzyme cleavage site: NdeI);

[0077] SOD1R:CTAG TCTAGA TTACTGGGCGATGCCAATGACTCCACA (SEQ ID NO: 2, restriction enzyme cleavage site: XbaI).

[0078] The PCR product was recovered and purified using a DNA gel recovery kit.

[0079] (2) Enzyme digestion and ligation: Double digest the pCold I plasmid and the SOD1 gene fragment with NdeI and XbaI. Recover the target fragment from the gel and ligate with T4 ligase at 16°C overnight.

[0080] (3) Transformation and screening: The ligation product was transformed into DH5α competent cells, coated with Amp (100 μg / mL) plates, and cultured at 37°C for 12-16 hours. Single colonies were selected for PCR verification, and plasmid (pCold-Sod1) was extracted after sequencing confirmation.

[0081] 2. Transformation and induction of expression strains

[0082] (1) Transformation into BL21(DE3): Transform the pCold-Sod1 plasmid into E. coli BL21(DE3) competent cells, spread on Amp plates, and culture at 37°C for 12-16 hours.

[0083] (2) Pick a single colony and inoculate it into 300 mL LB (Amp+), shake and culture at 37℃ until OD 600 ≈0.6. Cold shock induction: Transfer the culture to 15°C and let it rest for 30 minutes. Add 0.3 mM IPTG and induce at 15°C with shaking for 24 hours (low temperature slows inclusion body formation).

[0084] 3. Cell Lysis and Crude Extraction

[0085] (1) Collect the cells: Centrifuge at 6000 × g for 10 minutes at 4°C, discard the supernatant, and resuspend the cells in PBS (containing 1 mM PMSF).

[0086] (2) Ultrasonic disruption: Ultrasonication in an ice bath (power 30%, 3 seconds on / 5 seconds off, total duration 10 minutes). Centrifuge at 12,000 × g for 30 minutes at 4°C, and collect the supernatant (soluble protein) and precipitate (inclusion bodies).

[0087] 4. Ni-NTA Affinity Purification

[0088] (1) Column equilibration: Equilibrate the Ni-NTA column with binding buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0).

[0089] (2) Loading and washing: Slowly pass the lysate supernatant through the column and use washing buffer (same as binding buffer, with imidazole increased to 50 mM) to remove impurities.

[0090] (3) Elution: Use elution buffer (containing 250-500 mM imidazole) to collect the elution peaks in segments and detect the target bands by SDS-PAGE.

[0091] 5. Dialysis and Concentration

[0092] (1) Removal of imidazole: Place the eluate into a dialysis bag (MWCO 10 kDa) and dialyze against PBS at 4°C for 12 hours (change the solution three times).

[0093] (2) Concentration: Use ultrafiltration centrifuge tubes (10 kDa cutoff) to concentrate the protein to 1-5 mg / mL and quantify by Bradford method.

[0094] 6. Activity Detection and Storage

[0095] (1) SOD activity determination: Use the nitroblue tetrazolium (NBT) photoreduction method or a commercial kit to verify the enzyme activity (unit: U / mg).

[0096] (2) Storage: Add 10% glycerol and store at -80℃ after aliquoting.

[0097] The wild-type SOD1 nucleic acid sequence involved in the examples is:

[0098] ATGGTTATGAAAGCTGTCTGTGTGCTGAAAGGAGCTGGAGAGACCAGCGGGACTGTTTATTTTGAGCAGGAGAGTGATGCAGCAGCTGTGAAGCTGACCGGTGAAATCAAAGGCCTTGCTACTGGAGAGCATGGTTTCCATGTCCATGCTTTTGGAGACAATACAAACGGGTGCATCAGTGCAGGCCCTCACTTCAATCCCCACAACAAGACTCATGCAGGTCCTAATGATGA AGACAGGCATGTTGGAGACTTGGGGAATGTGACAGCTGAAGATAATGTTGCCAACATTGACATCACAGACGAGATGATCACCCTCACTGGCCCCCACTCAATCATTGGCAGAACCATGGTGATCCATGAGAAGGCCGACGACCTGGGAAAAGGAGGCAATGATGAGAGTCTGAAGACGGGCAATGCTGGCGCTCGTCTGGCGTGTGGAGTCATTGGCATCGCCCAGTAA (SEQ ID NO: 3);

[0099] The wild-type SOD1 amino acid sequence involved in the examples is:

[0100] MVMKAVCVLKGAGETSGTVYFEQESDAAAVKLTGEIKGLATGEHGFHVHAFGDNTNGCISAGPHFNPHNKTHAGPNDEDRHVGDLGNVTAEDNVANIDITDEMITLTGPHSIIGRTMVIHEKADDLGKGGNDESLKTGNAGARLACGVIGIAQ (SEQ ID NO: 4);

[0101] The mutant SOD1 nucleic acid sequence involved in the example (lowercase letters represent mutant bases):

[0102] ATGGTTATGAAAGCTGTCTGTGTGCTGAAAGGAGCTGGAGAGACCAGCGGGACTGTTTATTTTGAGCAGGAGAGTGATGCAGCAGCTGTGAAGCTGACCGGTGAAATCAAAGGCCTTGCTACTGGAGAGCATGGTTTCCATGTCCATGCTTTTGGAGACAATACAAACGGGTGCATCAGTGCAGGCCCTCACTTCAATCCCCtCAACAAGACTCATGCAGGTCCTAATGATGAAGACAGGCATGTTGGAGACTTGGGGAATGTGACAGCTGAAGATgATggtGTTGCCAACATTGACATCACAGACGAGATGATCACCCTCACTGGCCCCCACTCAATCATTGGCAGAACCATGGTGATCCATGAGAAGGCCGACGACCTGGGAAAAGGAGGCAATGATGAGAGTCTGAAGACGGGCAATGCTGGCGCTCGTCTGGCGTGTGGAGTCATTGGCATCGCCCAGTAA (SEQ ID NO: 5);

[0103] The mutant SOD1 amino acid sequence involved in the example (based on wild type, His at position 68 → Leu, Asn at position 93 → Asp, and Gly inserted between positions 93 and 94):

[0104] MVMKAVCVLKGAGETSGTVYFEQESDAAAVKLTGEIKGLATGEHGFHVHAFGDNTNGCISAGPHFNPLNKTHAGPNDEDRHVGDLGNVTAEDDGVANIDITDEMITLTGPHSIIGRTMVIHEKADDLGKGGNDESLKTGNAGARLACGVIGIAQ (SEQ ID NO: 6).

[0105] This invention provides a novel oil-resistant and low-temperature-adaptable SOD1 mutant and its preparation method. Through rational design or high-throughput screening, a SOD1 variant is obtained that maintains high activity in oily environments while being temperature-sensitive (e.g., inactivated at high temperatures but stable at low temperatures). This enzyme can be widely used in food preservation, skincare, oil antioxidants, and low-temperature biotechnology, addressing key deficiencies of existing SOD1 enzymes in industrial applications. The unique protein sequence of this SOD1 mutant and its functional properties (oil-resistant, low-temperature active, and high-temperature sensitive) are the core protection points of this invention. Any imitation or application based on this sequence or equivalent mutations (conservative substitutions) falls within the scope of protection of this invention.

[0106] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0107] The present invention will be further described below with reference to the embodiments.

[0108] Example 1: Determination of the optimal temperature of SOD1 enzyme

[0109] SOD1 enzyme activity determination method: SOD activity was determined using the nitroblue tetrazolium (NBT) photoreduction method. The specific method is as follows: purified enzyme solution was added to a test tube containing 3 mL of reaction mixture (2 mL each of 3 μmol / L EDTA, 2.25 mmol / L NBT, and 60 μmol / L riboflavin, all prepared with 0.05 mol / L pH 7.8 phosphate buffer, added to 54 mL of 14.5 mmol / L L-methionine, all prepared immediately before use and wrapped in aluminum foil to protect from light). After mixing, the tube was placed on a transparent test tube rack and illuminated in a light incubator for 20 minutes. The test tube was removed and the OD was quickly measured. 560 The value of the enzyme solution was replaced by the buffer solution, and the tube without light was the maximum reduction value. The enzyme activity unit definition: The amount of enzyme required to inhibit the photoreduction of NBT by 50% per unit time is one enzyme activity unit.

[0110] An appropriate amount of enzyme solution was taken and reacted at 4°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, and 70°C for 30 minutes. The reaction system was then prepared according to the above-mentioned assay method, and the enzyme solution treated at different temperatures was added to the reaction solution. The absorbance at 560 nm was measured using a microplate reader. The group without enzyme was set as a blank group, and the highest enzyme activity was set as 100%. The ratio of the enzyme activity at the remaining temperatures to the highest enzyme activity was the relative enzyme activity, and the relative enzyme activity was calculated. The results showed that the wild-type SOD1 had the best activity at 10°C and would be rapidly inactivated if it was stable above 20°C. However, the mutant SOD1 had an optimal temperature of 20°C and still maintained good activity (>60%) at 40°C. Unlike most thermostable SOD enzymes, this SOD1 was rapidly inactivated at high temperatures, with enzyme activity decreasing rapidly after 40°C and reaching 80% activity loss at 50°C (see Figure 1 The relative enzyme activities among the groups were compared, with the mutant SOD1 20℃ group as the reference group).

[0111] Example 2: Determination of the optimal pH of SOD1 enzyme

[0112] The method for determining SOD1 enzyme activity was the same as in Example 1. To determine the optimal pH value of SOD1, buffer solutions of different pH values ​​were prepared: NaAc-HAc buffer solution (pH 4, pH 5), KH2PO4-K2HPO4 buffer solution (pH 6, pH 7, pH 8), and Glycine–NaOH buffer solution (pH 9, pH 10). The buffer solutions of different pH values ​​were mixed with equal amounts of enzyme solution, and the enzyme activity of SOD1 in the buffer solutions of different pH values ​​was determined using the NBT method. The results showed that the optimal pH values ​​of both wild-type SOD1 and mutant SOD1 were between 7.0 and 8.0 (see Figure 2 ), wild-type SOD1 is very sensitive to high pH, ​​with less than 10% activity at pH 10, but still maintains a certain activity at a low pH of 4; unlike wild-type SOD1, mutant SOD1 has a certain activity between 4 and 10, and has a wider pH tolerance.

[0113] Example 3: Effects of oils and emulsions on SOD1 enzyme activity

[0114] The SOD1 enzyme activity assay was the same as in Example 1. Emulsions containing different oil concentrations were prepared according to Table 1, mixed at high speed to form a uniform emulsion, and then mixed with SOD1 enzyme solution at a ratio of 2:1. The SOD1 enzyme activity was measured after the reference group without emulsion was set at 100%. The results showed that the mutant SOD1 activity was still maintained at over 70% in an emulsion containing 15% oil (Formulation 3) (see Figure 3 ), while the wild-type SOD1 also has a certain degree of oil tolerance, but its activity is poorer than that of the mutant type.

[0115] At the same time, a lotion was prepared based on the ingredients of commonly used skin moisturizing products on the market. The formula is shown in Table 2. After refrigeration at 20°C, the residual SOD1 activity was tested. The results showed that the mutant SOD1 could maintain good activity for a long time in the lotion. After 240 hours, it still maintained about 73% of the relative enzyme activity. The mutant SOD1 has very high stability (see Figure 4 ). Therefore, the superoxide dismutase mutant SOD1 of the present invention has a strong tolerance to oil and fat, good stability, and good application prospects.

[0116] Table 1: Emulsion composition table

[0117]

[0118] Table 2: Emulsion ingredients

[0119]

[0120] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A mutant of superoxide dismutase, characterized in that With the following mutations: (i) His at position 68 was mutated to Leu; (ii) Asn 93 mutated to Asp; (iii) Gly is inserted between positions 93 and 94.

2. The mutant according to claim 1, wherein The amino acid sequence is shown in SEQ ID NO:

6.

3. A nucleic acid molecule, characterized in that A gene encoding the mutant according to claim 1 or 2.

4. A recombinant expression vector, characterized in that: Comprising the nucleic acid molecule of claim 3.

5. A host cell, characterized in that Comprising the nucleic acid molecule according to claim 3 or the recombinant expression vector according to claim 4; The host cells do not include totipotent cells.

6. The method for preparing a mutant according to claim 1, wherein: Prepared based on any of the following: (I) the nucleic acid molecule according to claim 3; (II) the recombinant expression vector according to claim 4; (III) The host cell according to claim 5.

7. Use of the mutant according to claim 1 in the preparation of food, medicine or cosmetic products.

8. Food, characterized in that Comprising the mutant according to claim 1.

9. A drug, characterized in that Comprising the mutant according to claim 1.

10. A beauty product, characterized in that Comprising the mutant according to claim 1.

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