Plant lactobacillus expression system based on tyr mutant and its application in black hair agent development

Abstract

The application discloses a Lactobacillus plantarum expression system based on a TYR mutant and application of the Lactobacillus plantarum expression system in development of black hair agents, and belongs to the field of synthetic biology and functional hair dyeing materials. A tyrosinase mutant is screened, and the conversion rate of conversion for preparing 5,6-dihydroxyindole reaches 80%. The mutant is expressed in the Lactobacillus plantarum, and can ferment black boletus extract to prepare a fermentation filtrate rich in 5,6-dihydroxyindole, so that the application has wide application value in the production field of cosmetics and green and safe hair dyes.

Description

Technical Field

[0001] This invention relates to a Lactobacillus plantarum expression system based on TYR mutants and its application in the development of hair dyes, belonging to the fields of synthetic biology and functional hair dyeing materials. Background Technology

[0002] Black truffle extract is widely studied due to its rich content of various active ingredients. In the cosmetics industry, it is used as an antioxidant, moisturizer, and natural colorant. Its antioxidant properties help combat free radicals and slow down the skin aging process, while its excellent moisturizing properties maintain skin hydration and prevent dryness. More importantly, as a natural colorant, black truffle extract offers a safe and harmless alternative to chemically synthesized dyes, meeting current consumer demand for natural and additive-free cosmetics.

[0003] 5,6-Dihydroxyindole, as a natural organic compound, shows great potential in the development of green and safe hair dyes. Traditional chemical hair dyes contain many irritating or harmful chemicals, while 5,6-dihydroxyindole, as a natural ingredient, can not only provide coloring effects comparable to traditional chemical dyes but also reduce irritation to the scalp and hair, and minimize the environmental impact of chemicals. Furthermore, 5,6-dihydroxyindole exhibits good biocompatibility and safety, making it an ideal choice for developing green and safe hair dye products.

[0004] Traditional sources of tyrosinase, especially those derived from basidiomycetes such as shiitake and mushrooms, have natural enzyme activity and substrate affinity that are insufficient to meet the high-yield and high-efficiency requirements of industrial fermentation, becoming a key factor restricting the industrialization of 5,6-DHI.

[0005] Lactobacillus plantarum fermentation technology has unique advantages in enhancing the content of active ingredients in plant extracts. As a probiotic, Lactobacillus plantarum can not only ferment under mild conditions but also transform common components in extracts into substances with higher bioactivity through its metabolic activities. This biotransformation process is highly efficient, safe, and environmentally friendly, achieving ingredient enhancement without the use of external chemical additives, meeting modern consumers' demand for natural and healthy products. Furthermore, the Lactobacillus plantarum fermentation process is easy to control and scale up, suitable for industrial production, and offers good economic benefits. However, existing Lactobacillus plantarum methods cannot be directly used to produce 5,6-dihydroxyindole, and the biotransformation production of 5,6-dihydroxyindole is low, limiting its industrial production. Therefore, there is an urgent need to develop a new method to apply the highly efficient, safe, and environmentally friendly Lactobacillus plantarum to the efficient production of 5,6-dihydroxyindole. Summary of the Invention

[0006] The purpose of this invention is to provide a tyrosinase mutant (TYR-M) derived from horny fungus and its expression system, which solves the problems of low catalytic efficiency of natural tyrosinase and poor yield of 5,6-DHI biosynthesis, thereby achieving efficient production of industrial-grade green hair dye raw materials.

[0007] The present invention provides a tyrosinase mutant TYR-M, whose amino acid sequence has undergone directed mutations at multiple key sites, such as positions 66, 135, 185, 213 and 244, compared with the parent tyrosinase. The amino acid sequence of the parent is shown in SEQ ID NO: 1.

[0008] In one embodiment, the amino acid sequence of the tyrosinase mutant is mutated at the following sites relative to the tyrosinase (SEQ ID NO: 1) derived from horny fungus: glycine (G) at position 66 is mutated to isoleucine (I), alanine (A) at position 135 is mutated to serine (S), serine (S) at position 185 is mutated to arginine (R), proline (P) at position 213 is mutated to lysine (K), and aspartic acid (D) at position 244 is mutated to threonine (T).

[0009] In one embodiment, the amino acid sequence of the tyrosinase mutant is shown in SEQ ID NO:3.

[0010] The present invention also provides a gene encoding the mutant.

[0011] The present invention also provides an expression vector carrying the gene.

[0012] The present invention also provides genetically engineered bacteria that express the mutant, contain the gene, or contain the expression vector.

[0013] In one embodiment, the genetically engineered bacteria uses Lactobacillus plantarum WCFS1 as the host.

[0014] The present invention also provides a method for preparing 5,6-dihydroxyindole, wherein the genetically engineered bacteria are fermented in a fermentation system containing Truffle extract to prepare 5,6-dihydroxyindole.

[0015] In one embodiment, the preparation method of the *Tuber nigricans* extract is as follows: *Tuber nigricans* slices are dried and pulverized, 2-10 times the volume of 60-95% ethanol is added for reflux extraction, the extract is filtered, vacuum concentrated at a temperature not exceeding 55°C, the ethanol is recovered, 1-2 times the volume of water is added to the concentrate, the mixture is allowed to stand for 24-36 hours, filtered, the filtrate is subjected to microfiltration, ultrafiltration, and nanofiltration, the filtrate is vacuum concentrated, freeze-dried, and sterilized by irradiation to obtain the *Tuber nigricans* extract.

[0016] In one embodiment, the method involves fermentation at 36-37°C for at least 48 hours.

[0017] The present invention also provides a fermentation filtrate rich in 5,6-dihydroxyindole prepared by fermenting *Tuber nigricans* extract using the genetically engineered bacteria.

[0018] The present invention also provides the application of the fermentation filtrate in the preparation of cosmetics or hair dyes.

[0019] Beneficial effects: (1) Improved conversion efficiency: The tyrosinase mutant of Auricularia auricula obtained by screening achieved a conversion rate of 80% in the process of converting 5,6-dihydroxyindole in the extract of Auricularia auricula-judae, which significantly increased the content of 5,6-dihydroxyindole.

[0020] (2) Safety and environmental friendliness: Using Lactobacillus plantarum as a bioengineering tool, the safety and environmental friendliness of the process are ensured while increasing the content of 5,6-dihydroxyindole. Green and safe hair dyes are developed using the fermentation filtrate rich in 5,6-dihydroxyindole, which is safe, efficient and convenient.

[0021] (3) Broad application prospects: This invention is not only applicable to increasing the content of 5,6-dihydroxyindole in Truffle extract, but can also be extended to the enhancement of active ingredients in other plant extracts, and has broad market prospects.

[0022] In summary, this invention is not only technically innovative, providing an effective method to increase the content of 5,6-dihydroxyindole, but also PPD-free, ammonia-free, hydrogen peroxide-free, and non-toxic. It is easy to use, has a high coloring rate, produces natural colors, and offers good photoprotection. It shows broad application prospects in practical applications, especially in the production of cosmetics and green and safe hair dyes. Attached Figure Description

[0023] Figure 1 Map of the pNZ8048-TYR-M expression plasmid.

[0024] Figure 2 The ability of wild-type TYR and mutant TYR-M to catalyze the conversion of tyrosine to 5,6-dihydroxyindole.

[0025] Figure 3 The study investigated the changes in the content of tyrosine, dopa, dopamine, and 5,6-dihydroxyindole in *Tuber nigricans* extract before and after fermentation using *Lactobacillus plantarum* expressing the TYR-M mutant protein. Detailed Implementation

[0026] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0027] Test method: Detection of active ingredients (tyrosine, dopa, dopamine, and 5,6-dihydroxyindole): The sample was centrifuged, and the supernatant was filtered through a 0.22 μM filter. Detection conditions: Shimadzu LC-20AT high-performance liquid chromatography system, SPD-M20APAD detector, Waters C18 column (250 x 4.6 mm, 5 μM), column temperature 30℃. The mobile phase consisted of methanol, acetonitrile, and 0.4% formic acid in a volume ratio of 50:15:35, separated at a flow rate of 1 mL / min. The injection volume was 10 μL, and the detection wavelength was 310 nm.

[0028] Enzyme activity (U / mg) is defined as the amount of enzyme that converts 1 micromole of tyrosine to 5,6-dihydroxyindole per minute under standard conditions (37 ℃, reaction pH 6.5).

[0029] The raw materials used in the embodiments of this invention are as follows: 1. The tyrosinase gene used is derived from keratinous fungus (Auricularia auricula-judae). Auricularia cornea The transcriptome of the gene was reverse transcribed to obtain cDNA, which was then used as a template for gene cloning. The expression vector used was the nisin-inducible expression plasmid pNZ8048, which was self-constructed.

[0030] 2. MRS medium for microbial culture: peptone 10.0 g / L, beef extract 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, sodium citrate 2.0 g / L, K₂HPO₄ 2.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 mL / L, final pH adjusted to 6.5, used for the growth and fermentation of *Lactobacillus plantarum*. Nisin was used as an expression inducer, with a final concentration controlled at 10 ng / mL.

[0031] 3. The fermentation substrate, *Tuber nigricans* extract, was obtained in-house. The raw material was dried *Tuber nigricans* powder, extracted by reflux with 75% ethanol for 2 hours, concentrated under reduced pressure to 1 / 5 of the original volume, filtered through a 0.22 μm membrane, ultrafiltered, and freeze-dried for later use. This extract is rich in tyrosine and its derivatives, making it suitable as a substrate for tyrosinase conversion reactions.

[0032] 4. The L-tyrosine used in the in vitro catalytic reaction and activity verification was purchased from Sigma-Aldrich with a purity ≥99%. Copper ions were provided in the form of CuSO4·5H2O as the metal active factor of tyrosinase, and the concentration was controlled within 100 μM.

[0033] 5. The sodium sulfate, ascorbic acid, glycerin, sodium carboxymethyl cellulose, and other ingredients used in the hair dye formula were all purchased from Sinopharm Group, with a purity of ≥98%. Phosphate buffered saline (PBS) was used to adjust the pH of the hair dye to 6.5 to enhance the stability of the reaction system.

[0034] 6. All water used in the experiments was deionized water, all reagents were of analytical grade or higher, and all consumables were treated to be free of DNase and RNase contamination to ensure the accuracy and repeatability of the experiments.

[0035] Example 1: Construction of Lactobacillus plantarum expressing the TYR-M mutant Using the pNZ8048 vector as a template, PCR amplification was performed using 5'-AAGCTTTCTTTGAACCAAAA-3' (upstream primer) and 5'-GGTGAGTGCCTCCTTATAAT-3' (downstream primer). Using the TYR-M gene fragment (SEQ ID NO:2) as a template, the mutant gene tyr-M (SEQ ID NO:4) was amplified using 5'-ATTATAAGGAGGCACTCACCATGGCTGCAGAGCAAGCCGT-3' (upstream primer) and 5'-TTTTGGTTCAAAGAAAGCTTTCACGCCGTTGCCTTCTTCC-3' (downstream primer). PCR system: 0.2 μM primers each, 0.2 mM dNTPs, 10 ng template, 5 μL 2× Buffer, 0.5 μL Hieff, and deionized water to bring the volume to 100 μL. The PCR reaction conditions were: 94 ℃ for 20 s, 94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 2 min, and 4 ℃ for 5 min, for 30 cycles of steps 2-5. The obtained PCR products were ligated using a Gibson Assembly kit (Anhui General Biotechnology). The ligation product was transformed into the commercial host *Lactobacillus plantarum* WCFS1. After verification and sequencing, the correct clone was selected and named *L. plantarum-TYR-M*. The plasmid pNZ8048-TYR-M expression plasmid was extracted from the recombinant bacteria. The plasmid map is shown below. Figure 1 .

[0036] Example 2: Preparation of wild-type TYR and mutant TYR-M The recombinant strains WCFS1-TYR-WT and WCFS1-TYR-M, expressing the wild-type TYR gene and the mutant TYR-M gene respectively, constructed in Example 1, were inoculated into MRS liquid medium containing chloramphenicol (5 μg / mL) and cultured statically at 37°C for 12 hours. After the cell density reached OD600=0.6, nisin (final concentration 10 ng / mL) was added to induce expression of the target enzyme at the same temperature, and the culture was continued for another 24 hours. After expression, the bacterial cells were collected and sonicated with PBS buffer (pH 6.8) to obtain crude enzyme solution. The enzyme solution was collected, and the specific enzyme activity was detected by reacting 1 mL of fermentation broth with 1 mL of aqueous solution containing 10 mM tyrosine at 37 °C for 5 min. The content of 5,6-dihydroxyindole was detected by HPLC. The results showed that the wild-type enzyme activity was 3.2 U / mg, and the mutant enzyme activity was 14 U / mg, which was about 4.4 times higher than that of the wild-type enzyme.

[0037] Example 3: Wild-type TYR and mutant TYR-M catalyze the conversion of tyrosine to 5,6-dihydroxyindole The reaction system was as follows: total volume 1.0 mL; final L-tyrosine concentration: 15 mM; final CuSO4 concentration: 100 μM; enzyme solution: 50 μL (enzyme activity 1.2 U / mL); PBS buffer was added to make up the volume, pH 6.5. The reaction was carried out at 37°C for 60 minutes. After termination, the pH was adjusted to 3.0 with acetic acid. The precipitate was removed by centrifugation, and the 5,6-dihydroxyindole content in the supernatant was detected by HPLC. A Shimadzu LC-20AT high-performance liquid chromatography system was used, with an SPD-M20A PAD detector and a Waters C18 column (250 x 4.6 mm, 5 μM) at 30 °C. The mobile phase consisted of methanol, acetonitrile, and 0.4% formic acid in a volume ratio of 50:15:35, and separation was performed at a flow rate of 1 mL / min. The injection volume was 10 μL, and the detection wavelength was 310 nm. Results are as follows. Figure 2 As shown, the 5,6-dihydroxyindole content in the supernatant of the recombinant bacteria expressing the mutant can reach 0.96 mg / mL, which is 336% higher than that of the recombinant bacteria expressing the wild-type enzyme.

[0038] Example 4: Fermentation of Lactobacillus plantarum expressing the TYR-M mutant increased the content of 5,6-dihydroxyindole. Dry and pulverize *Ichthyophthirius multifiliis* flakes, extract by reflux with 2-10 times the volume of 60-95% ethanol, filter the extract, concentrate under vacuum at a temperature not exceeding 55℃, recover the ethanol, add 1-2 times the volume of water to the concentrate, let stand for 24-36 hours, filter, and then perform microfiltration (membrane pore size: 0.22 μm; operating pressure: 0.1-0.2 MPa; temperature: 4-10 ℃), ultrafiltration (molecular weight cutoff: 10 kDa; operating pressure: 0.2-0.3 MPa; temperature: 4-10 ℃), and nanofiltration (molecular weight cutoff: 200-500 Da; operating pressure: 0.5-1.0 MPa; temperature: below 10 ℃). Concentrate the filtrate under vacuum (vacuum degree: 0.08 MPa; temperature: below 40 ℃), freeze-drying (pre-freezing temperature: -40 ℃; freeze-drying pressure: 10~30 Pa; freeze-drying time: 24~36 h), irradiation sterilization (irradiation source: Co 60 Gamma rays (dose: 5–8 kGy) were used to prepare *Tuber nigricans* extract. The obtained *Tuber nigricans* extract was dissolved in 1 liter of distilled water and mixed thoroughly to prepare a culture medium. This culture medium was pasteurized at 80°C for 30 minutes to ensure complete sterilization. *Lactobacillus plantarum* expressing the TYR-M mutant constructed in Example 1 was inoculated at a 10% inoculum volume (10% of the culture medium volume) and fermented at 37°C for 48 hours. After fermentation, the filtrate was separated by filtration. This filtrate contained *Tuber nigricans* extract transformed by *Lactobacillus plantarum* fermentation and was rich in 5,6-dihydroxyindole. The contents of tyrosine, dopamine, and 5,6-dihydroxyindole before and after fermentation of the *Tuber nigricans* extract by *Lactobacillus plantarum* are as follows: Figure 3 As shown.

[0039] Example 5: Preparation of a green and safe hair dye from the obtained fermentation filtrate rich in 5,6-dihydroxyindole. The green and safe hair dye in this embodiment is composed of Agent A and Agent B, which are packaged separately.

[0040] The preparation method of Agent A is as follows: First, dissolve 0.08g of the fermentation filtrate rich in 5,6-dihydroxyindole prepared in Example 3, 0.055g of sodium sulfite, 0.055g of sodium bicarbonate, 0.035g of ascorbic acid, 0.035g of palmitic acid, and 0.06g of disodium ethylenediaminetetraacetate in 5mL of water at room temperature to form an aqueous phase; separately, add 1.2g of sodium carboxymethyl cellulose and 1.85mL of nonylphenol polyoxyethylene ether to 3.15mL of isopropanol and heat until melted to form an oil phase. Slowly add the aqueous phase to the oil phase while stirring, and after homogenization, Agent A is obtained. The preparation method of Agent B is as follows: Add 1.2g of sodium carboxymethyl cellulose to 4.5mL of water at 80℃. After cooling to room temperature, adjust the pH value to 3.5 with 40% phosphoric acid. After homogenization, Agent B is obtained.

[0041] When using, mix agent A and agent B at a volume ratio of 1:2, then apply evenly to white hair with a small brush. After leaving it at room temperature for 30 minutes, the original white hair will be dyed black with a natural color.

[0042] Hair dyeing effect comparison test: The L'Oréal black hair dye was applied evenly to the white hair with a small brush and left at room temperature for 30 minutes. The original white hair was then dyed black.

[0043] Take equal amounts of hair dyed with the Trichoderma melanin hair dye prepared in this embodiment (1) and hair dyed with L'Oréal black hair dye (2), place them under sunlight at the same time, and wash them with an equal amount of distilled water every other day, and retain the washing solution; repeat the operation seven times in total, and retain the washing solution from each operation.

[0044] The absorbance of the cleaning solution at 510 nm was measured using a 752 UV-Vis spectrophotometer. If the black color is easily washed off, the darker the color of the collected cleaning solution, the greater its absorbance. The measurement results are listed in Table 1 below.

[0045] Table 1. Comparison of hair dyeing effects between Green Safe Hair Dye and L'Oréal Black Hair Dye

[0046] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A tyrosinase mutant TYR-M, characterized in that, Compared with the parent tyrosinase, the amino acid sequence of the parent tyrosinase has undergone directed mutations at multiple key sites, including positions 66, 135, 185, 213, and 244. The amino acid sequence of the parent tyrosinase is shown in SEQ ID NO:

1.

2. The tyrosinase mutant TYR-M according to claim 1, characterized in that, The amino acid sequence of the tyrosinase mutant TYR-M is shown in SEQ ID NO:

3.

3. The gene encoding the tyrosinase mutant TYR-M as described in claim 1 or 2.

4. An expression vector carrying the gene of claim 3.

5. Genetically engineered bacteria that express the mutant of claim 1, or contain the gene, or contain the expression vector.

6. Genetically engineered bacteria, characterized in that, Using Lactobacillus plantarum WCFS1 as a host, the tyrosinase mutant TYR-M described in claim 1 or 2 is expressed.

7. A method for preparing 5,6-dihydroxyindole, characterized in that, The genetically engineered bacteria of claim 6 are fermented in a fermentation system containing Truffle extract to prepare 5,6-dihydroxyindole.

8. The method according to claim 7, characterized in that, Ferment at 36-37°C for at least 48 hours.

9. A fermentation filtrate rich in 5,6-dihydroxyindole prepared by fermenting *Tuber nigricans* extract with the genetically engineered bacteria described in claim 6.

10. The use of the fermentation filtrate according to claim 9 in the preparation of cosmetics or hair dyes.

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