An enoxaparin-carnosine ligase and a preparation method of enoxaparin-carnosine conjugate

Abstract

The application discloses an amino acid sequence of tranexamic acid-carnosine ligase, and a preparation method of tranexamic acid-carnosine conjugate. The amino acid sequence of the tranexamic acid-carnosine ligase is shown as SEQ ID NO: 4. The tranexamic acid-carnosine ligase is fused by an adenylation (A) domain, a thioesterification (T) domain and a condensation (C) domain. The adenylation domain is obtained by mutation and modification of an adenylation domain in non-ribosomal peptide synthetase from Streptomyces coelicolor. After the mutation and modification, the adenylation domain has high specificity for a substrate tranexamic acid, can efficiently recognize and activate the tranexamic acid, so that the tranexamic acid-carnosine ligase can efficiently catalyze the reaction of the tranexamic acid and L-carnosine to generate the tranexamic acid-carnosine conjugate.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing a tranexamic acid-carnosine ligase and a tranexamic acid-carnosine conjugate. Background Technology

[0002] Tranexamic acid (TXA), chemically known as trans-4-(aminomethyl)cyclohexanecarboxylic acid, is a synthetic lysine derivative that has shown good efficacy in treating skin pigmentation disorders such as melasma and post-inflammatory hyperpigmentation. However, due to its small molecular weight and strong hydrophilicity, tranexamic acid diffuses extremely rapidly after penetrating the stratum corneum and entering the aqueous environment of the epidermis. The local concentration around nerve endings spikes from zero to a peak value in a very short time, triggering a stinging reaction.

[0003] L-Carnosine (β-alanyl-L-histidine) is a natural dipeptide composed of β-alanine and L-histidine, and is recognized in the skincare field as a highly effective anti-glycation agent and antioxidant. Despite its remarkable skincare benefits, carnosine's application in the body is greatly limited. Highly active carnosinase exists in human serum, tissues, and even the skin surface, which can rapidly hydrolyze L-carnosine into amino acids. Once L-carnosine is hydrolyzed into amino acids, its anti-glycation and antioxidant effects are significantly weakened. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for preparing tranexamic acid-carnosine ligase and tranexamic acid-carnosine conjugates, which uses tranexamic acid-carnosine ligase to couple tranexamic acid and L-carnosine, overcoming the problems of high skin irritation of tranexamic acid and easy hydrolysis of L-carnosine in the prior art.

[0005] In a first aspect, the present invention provides a tranexamic acid-carnosine ligase, the amino acid sequence of which is shown in SEQ ID NO:4.

[0006] Compared with the prior art, the tranexamic acid-carnosine ligase of the present invention is composed of an adenylation (A) domain, a thioesterification (T) domain, and a condensation (C) domain. The adenylation domain is obtained by mutating the adenylation domain of a non-ribosomal peptide synthase derived from Streptomyces cerevisiae. The mutation sites are D303G, A304V, E307L, R346F, Q367L, F390G, I398V, N399D, and K404G. After the mutation, the adenylation domain has high specificity for the substrate tranexamic acid and can efficiently recognize and activate tranexamic acid. Thus, the tranexamic acid-carnosine ligase of the present invention can efficiently catalyze the reaction of tranexamic acid and L-carnosine to generate tranexamic acid-carnosine conjugates.

[0007] In a second aspect, the present invention provides a nucleic acid molecule that encodes the above-mentioned tranexamic acid-carnosine ligase.

[0008] Thirdly, the present invention provides an expression vector carrying the aforementioned nucleic acid molecules.

[0009] Fourthly, the present invention provides a recombinant strain carrying the above-mentioned nucleic acid molecule or the above-mentioned expression vector.

[0010] Fifthly, the present invention provides the application of the above-mentioned tranexamic acid-carnosine ligase in the preparation of tranexamic acid-carnosine conjugates, the structure of which is as follows: .

[0011] Sixthly, the present invention provides a method for preparing a tranexamic acid-carnosine conjugate, comprising the following steps: The above recombinant strain was fermented and cultured to induce the expression of tranexamic acid-carnosine ligase, resulting in a fermentation broth containing tranexamic acid-carnosine ligase. The substrates tranexamic acid and L-carnosine were added to the fermentation broth, and the reaction was carried out at a temperature of 25℃-37℃ and a pH of 6.0-7.0 to generate tranexamic acid-carnosine conjugates.

[0012] Compared with existing technologies, this invention directly adds tranexamic acid and L-carnosine to a fermentation broth containing tranexamic acid-carnosine ligase, and uses the whole cells of recombinant strains as a biocatalyst for biotransformation. This allows an amide bond to be formed between the carboxyl group of tranexamic acid and the amino group of β-alanine at the N-terminus of L-carnosine, thereby synthesizing the target compound, tranexamic acid-carnosine conjugate. This method is economical, efficient, and simple, and can conveniently and quickly prepare tranexamic acid-carnosine conjugate.

[0013] Furthermore, the above-mentioned recombinant strain was fermented to induce the expression of tranexamic acid-carnosine ligase, including: The recombinant strain was inoculated into a fermenter for fermentation. When the glucose in the fermenter was depleted, a feed solution was added and the dissolved oxygen (DO) value was maintained at 25%-35%. The fermentation was continued until the OD value reached its maximum. 600 When the pH value reaches 80-120, add an inducing agent and induce culture for 22-26 hours to obtain a fermentation broth containing tranexamic acid-carnosine ligase.

[0014] Furthermore, the initial fermentation conditions in the fermenter were: temperature 25℃-35℃ and pH 5.0-6.0.

[0015] Furthermore, the induction culture temperature is 25℃-35℃.

[0016] Furthermore, the feed solution is an aqueous glycerol solution, and the feed rate is 8 mL / h-12 mL / h per liter of fermentation broth.

[0017] In the above technical solution, the concentration of glycerol in the glycerol aqueous solution is 50% (w / v), that is, every 100 mL of glycerol aqueous solution contains 50 g of glycerol.

[0018] Furthermore, the inducer is galactose, and the final concentration of galactose is 1.5g-2.5g of galactose per 100mL of fermentation broth.

[0019] Furthermore, the substrates tranexamic acid and L-carnosine are added in the form of a mixed solution of tranexamic acid and L-carnosine; in the mixed solution of tranexamic acid and L-carnosine, the concentration of tranexamic acid is 0.5 mol / L-1.5 mol / L and the concentration of L-carnosine is 0.5 mol / L-1.5 mol / L.

[0020] Furthermore, the amount of the mixed solution of tranexamic acid and L-carnosine added is 5%-30% of the fermentation broth volume.

[0021] Furthermore, a mixed solution of tranexamic acid and L-carnosine is added per liter of fermentation broth at a rate of 0.05 mL / min to 1.0 mL / min. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the synthesis reaction of tranexamic acid-carnosine conjugate. Detailed Implementation

[0023] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0024] To overcome the problem that the anti-glycation and antioxidant effects are greatly weakened after L-carnosine is hydrolyzed into amino acids in traditional technologies, the technicians of this application have creatively proposed to couple tranexamic acid and carnosine to form a new conjugate, in order to overcome the defects of simply mixing functional molecules physically, and based on this, designed a simple and environmentally friendly biosynthetic route.

[0025] In a first aspect, embodiments of the present invention provide a tranexamic acid-carnosine ligase, the amino acid sequence of which is shown in SEQ ID NO:4.

[0026] The tranexamic acid-carnosine ligase of this invention is composed of an adenylation (A) domain, a thioesterification (T) domain, and a condensation (C) domain. The adenylation domain is obtained by mutating the adenylation domain in the non-ribosomal peptide synthase (NRPS) derived from *Streptomyces cerevisiae*, with mutation sites D303G, A304V, E307L, R346F, Q367L, F390G, I398V, N399D, and K404G. After mutation, the adenylation domain exhibits high specificity for the substrate tranexamic acid, enabling efficient recognition and activation of tranexamic acid. The thioesterification domain is homologous to the adenylation domain and functions to load the activated tranexamic acid. The final condensation domain is derived from the fengycin synthase cluster of *Bacillus* and functions to catalyze the condensation of tranexamic acid with L-carnosine to form an amide bond. This invention utilizes the synergistic action of the three structural domains mentioned above to efficiently catalyze the reaction of tranexamic acid and L-carnosine to generate tranexamic acid-carnosine conjugates.

[0027] Secondly, embodiments of the present invention provide a nucleic acid molecule that encodes the aforementioned tranexamic acid-carnosine ligase.

[0028] Optionally, the gene sequence of the above nucleic acid molecule is shown in SEQ ID NO:3.

[0029] Thirdly, embodiments of the present invention provide an expression vector carrying the aforementioned nucleic acid molecules.

[0030] The expression vectors mentioned above can be selected from conventional expression vectors in the field, such as pKLAC2, pKLAC1, or pYES2.

[0031] Fourthly, embodiments of the present invention provide a recombinant strain carrying the aforementioned nucleic acid molecule or the aforementioned expression vector.

[0032] The above-mentioned recombinant strain can be constructed by: ligating the encoding gene of tranexamic acid-carnosine ligase into an expression vector to obtain a recombinant expression vector, and then transforming the recombinant expression vector into a host strain to obtain a recombinant strain.

[0033] In this invention, *Kluyveromyces lactis* is preferred as the host strain. This strain is not only recognized as a "Generally Recognized As Safe" (GRAS) microorganism by the US FDA, ensuring extremely high safety for its fermentation products in the food and cosmetic fields, but it also possesses the potential for high-density fermentation in simple culture media, with high cell density beneficial for increasing yield per unit volume. Furthermore, *Kluyveromyces lactis* exhibits strong protein expression and secretion capabilities, making it suitable for intracellular enzyme expression and facilitating its subsequent use as a whole-cell catalyst. Finally, *Kluyveromyces lactis* has mature, commercially available expression systems, including strong promoters, integrative expression vectors, and efficient screening methods, facilitating the rapid construction and screening of high-yield engineered strains. For example, *Kluyveromyces lactis* can be specifically selected as *K. lactis* GG799, *K. lactis* Y33, or *K. lactis* CBS2359.

[0034] Fifthly, embodiments of the present invention provide the application of the above-mentioned tranexamic acid-carnosine ligase in the preparation of tranexamic acid-carnosine conjugates, the structure of which is as follows: .

[0035] The tranexamic acid-carnosine ligase of this invention catalyzes the formation of an amide bond between the carboxyl group (-COOH) of tranexamic acid and the amino group (-NH2) of the β-alanine at the N-terminus of L-carnosine, thereby yielding a tranexamic acid-carnosine conjugate. Following the nomenclature rules of the International Union of Pure and Applied Chemistry (IUPAC), this tranexamic acid-carnosine conjugate is named N-{[4-(aminomethyl)cyclohexyl]carbonyl}-β-alanyl-L-histidine, with the molecular formula C2. 17 H 27 N5O4, with an exact molecular weight of 365.2066 g / mol.

[0036] On the one hand, compared to free tranexamic acid, the tranexamic acid-carnosine conjugate has a larger molecular weight, resulting in a slower diffusion rate within the skin after penetrating the stratum corneum. This solves the problem of small tranexamic acid molecules causing localized, high-concentration "impact" and skin irritation. On the other hand, in the tranexamic acid-carnosine conjugate, the amide bond between tranexamic acid and L-carnosine is a non-α-amide bond, exhibiting resistance to common proteases that specifically recognize and hydrolyze α-amide bonds in vivo. Furthermore, compared to natural L-carnosine, this invention couples the carboxyl group of tranexamic acid to the amino group at the N-terminus of carnosine, essentially adding a large molecular "cap" (acylation modification) to the N-terminus of carnosine. This completely disrupts the specific spatial recognition of the substrate by carnosinase, preventing it from binding and initiating hydrolytic catalysis. This solves the problem of the amide bond within natural L-carnosine being easily decomposed by carnosinase, ensuring the tranexamic acid-carnosine conjugate of this invention remains highly stable before reaching the target site on the skin.

[0037] After the tranexamic acid-carnosine conjugate of this invention enters the skin, the amide bond connecting tranexamic acid and L-carnosine can be recognized and slowly hydrolyzed by endogenous enzymes present in the deep epidermis and dermis. This achieves controlled release of the two active ingredients, tranexamic acid and L-carnosine, at targeted sites in the skin (such as melanocytes in the deep epidermis and collagen fibers in the dermis). The released tranexamic acid inhibits melanin production, while the released L-carnosine scavenges reactive carbonyl groups, inhibits AGEs (advanced aging products), and eliminates free radicals, thereby improving skin elasticity and reducing wrinkles. Through this targeted and slow release mechanism, the tranexamic acid-carnosine conjugate not only has a gentle and non-irritating effect but also ensures that the two active ingredients work synergistically to maximize skincare efficacy.

[0038] Sixthly, embodiments of the present invention provide a method for preparing a tranexamic acid-carnosine conjugate, comprising the following steps: S1. The above recombinant strain was fermented and cultured to induce the expression of tranexamic acid-carnosine ligase, and a fermentation broth containing tranexamic acid-carnosine ligase was obtained.

[0039] The specific fermentation method for fermenting the recombinant strain in the above steps can be as follows: The recombinant strain was inoculated into a fermenter for fermentation culture. YPD medium could be used as the initial culture medium in the fermenter. When the glucose in the fermenter was depleted, a feed solution was added, and the dissolved oxygen (DO) value was maintained at 25%-35%. The fermenter was cultured until the OD value reached its maximum. 600 When the pH value reaches 80-120, add an inducing agent and induce culture for 22-26 hours to obtain a fermentation broth containing tranexamic acid-carnosine ligase.

[0040] In some embodiments, the initial fermentation conditions in the fermenter are: a temperature of 25°C-35°C and a pH of 5.0-6.0. For example, the temperature can be 25°C, 30°C, or 35°C; and the pH can be 5.0, 5.5, or 6.0.

[0041] In some embodiments, the induction culture temperature is 25°C-35°C. For example, the induction culture temperature can be 25°C, 30°C, or 35°C.

[0042] In some embodiments, the feed solution is an aqueous glycerol solution, and the feed rate is 8 mL / h to 12 mL / h per liter of fermentation broth. For example, the feed rate can be 8 mL / h, 10 mL / h, or 12 mL / h per liter of fermentation broth. The concentration of glycerol in the aqueous glycerol solution can be 50% (w / v), i.e., 50 g of glycerol per 100 mL of the aqueous glycerol solution.

[0043] In other embodiments, the inducer is galactose, and the final concentration of galactose is 1.5g-2.5g per 100mL of fermentation broth. For example, the final concentration of galactose can be 1.5g, 2.0g, or 2.5g per 100mL of fermentation broth.

[0044] S2. Add the substrates tranexamic acid and L-carnosine to the fermentation broth, and react to generate tranexamic acid-carnosine conjugates at a temperature of 25℃-37℃ and a pH of 6.0-7.0. For example, the reaction temperature can be 25℃, 30℃, or 37℃, and the pH can be 6.0, 6.5, or 7.0.

[0045] In the above steps, the addition of substrates tranexamic acid and L-carnosine involves adding a mixed solution of tranexamic acid and L-carnosine. To prepare this mixed solution, the appropriate masses of tranexamic acid solid and L-carnosine solid can be directly weighed, dissolved in the same volume of solvent, and brought to the target volume to achieve a concentration of 0.5 mol / L-1.5 mol / L for both tranexamic acid and L-carnosine. The solvent can be sterile water, and the pH of the mixed solution should be adjusted to 6.0-7.0 using a 10% sodium hydroxide solution.

[0046] In the above-mentioned mixed solution of tranexamic acid and L-carnosine, the concentration of tranexamic acid is 0.5 mol / L-1.5 mol / L, and the concentration of L-carnosine is 0.5 mol / L-1.5 mol / L. For example, the concentration of tranexamic acid in the mixed solution can be 0.5 mol / L, 1.0 mol / L, or 1.5 mol / L; the concentration of L-carnosine in the mixed solution can be 0.5 mol / L, 1.0 mol / L, or 1.5 mol / L.

[0047] In the above steps, the amount of the mixed solution of tranexamic acid and L-carnosine added is 5%-30% of the fermentation broth volume. For example, the amount of the mixed solution added can be 5%, 10%, 15%, 20%, 25% or 30% of the fermentation broth volume, preferably 10%-20%.

[0048] In the above steps, the mixed solution of tranexamic acid and L-carnosine is added to the fermentation broth by a feeding method at a rate of 0.05 mL / min to 1.0 mL / min per liter of fermentation broth. For example, the feeding rate can be 0.05 mL / min, 0.1 mL / min, 0.2 mL / min, 0.5 mL / min, 0.8 mL / min, or 1.0 mL / min per liter of fermentation broth.

[0049] The above-mentioned flow rate ensures that the substrate concentration is maintained near the enzyme's saturation constant, avoiding feedback inhibition or osmotic damage to the recombinant strain caused by high substrate concentration.

[0050] It should be understood that, unless otherwise specified, all raw materials used in the following examples are commercially available.

[0051] Example 1 Using bioinformatics methods, an adenylated domain of a wild-type nonribosomal peptide synthase (NRPS) derived from *Streptomyces coelicolor* was screened out. This adenylated domain can activate cyclohexyl-related substrates and was named A-domain-ori.

[0052] Site-directed saturation mutagenesis was performed on 10 key amino acid residues in the substrate binding pocket of the aforementioned A-domain-ori to construct a mutant DNA library with a theoretical library capacity of approximately 2010.

[0053] The mutant DNA library was conjugated to a fluorescent reporter system and expressed in a yeast surface display system. Using fluorescently labeled tranexamic acid derivatives as screening substrates, flow cytometry sorting (FACS) was used to screen for mutants capable of binding to these substrates.

[0054] Deep sequencing was performed on the screened positive clone mutants, yielding more than 105 valid sequence-function data pairs.

[0055] The above data were used to train a gradient boosting decision tree model to predict the effects of different mutation combinations on tranexamic acid binding activity.

[0056] Using the trained model, virtual screening was performed to predict multiple multi-point mutation combinations with the highest predictive activity.

[0057] The predicted mutants were synthesized and tested, and an optimized adenosine domain named TCL-A-domain was finally obtained.

[0058] The amino acid sequence of the adenylation domain (A-domain-ori) of the above-mentioned wild-type non-ribosomal peptide synthase is shown in SEQ ID NO:1, and the amino acid sequence of the optimized adenylation domain (TCL-A-domain) is shown in SEQ ID NO:2. Based on SEQ ID NO:1, the optimized adenylation domain (TCL-A-domain) underwent the following site-directed mutations targeting nine key residues in the substrate binding pocket: amino acid position 303 was mutated from D to G, amino acid position 304 from A to V, amino acid position 307 from E to L, amino acid position 346 from R to F, amino acid position 367 from Q to L, amino acid position 390 from F to G, amino acid position 398 from I to V, amino acid position 399 from N to D, and amino acid position 404 from K to G.

[0059] The ATP-PPi exchange assay measures the kinetic parameters of the enzyme. The specific experimental method is as follows: Prepare 6 solutions containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM ATP (adenosine triphosphate), and 1 mM [ 32 The reaction system of [P]-sodium pyrophosphate (radioisotope-labeled PPi) was used. Tranexamic acid was added to each of the six reaction systems at concentrations of 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM, respectively. Then, a purification enzyme with a final concentration of 1 μM was added to each of the six reaction systems to initiate the reaction. After reacting each system at 30°C for 10 minutes, the amount of [P]-sodium pyrophosphate (radioisotope-labeled PPi) generated in each system was measured. 32 The radioactivity intensity of P-ATP was used to calculate the products in each reaction system. 32 The production of P-ATP.

[0060] Through products 32 The yield of P-ATP was calculated, and the initial rate V of the enzyme-catalyzed reaction under different concentrations of tranexamic acid substrate was obtained. The Michaelis-Menten equation was fitted with the initial rate V and the concentration of tranexamic acid to obtain the maximum reaction rate Vmax and Michaelis constant Km of the enzyme. Then, the catalytic constant kcat and the catalytic efficiency constant kcat / Km were obtained.

[0061] The above experiment was repeated twice, with the purification enzymes used in the two experiments being the wild-type adenosylation domain A-domain-ori and the optimized adenosylation domain TCL-A-domain, respectively. The results are shown in Table 1.

[0062] Table 1 The results above show that the catalytic constant kcat of the wild-type adenylation domain A-domain-ori is significantly smaller than that of the optimized adenylation domain TCL-A-domain. The Michaelis constant Km of the wild-type adenylation domain A-domain-ori is significantly larger than that of the optimized adenylation domain TCL-A-domain. The catalytic efficiency constant kcat / Km of the wild-type adenylation domain A-domain-ori is significantly smaller than that of the optimized adenylation domain TCL-A-domain. This indicates that the optimized adenylation domain TCL-A-domain has a stronger affinity for the substrate amino acid cyclic acid, resulting in a faster catalytic rate and higher catalytic efficiency.

[0063] Example 2 The DNA sequence was deduced by reverse engineering the amino acid sequence of the optimized adenylated domain TCL-A-domain to obtain the coding gene for TCL-A-domain. The coding gene for TCL-A-domain, the coding gene for its homologous thioesterification domain (T-domain), and the coding gene for a condensation domain (C-domain) with good catalytic activity (derived from the fengycin synthase cluster of Bacillus) were tandemly to obtain the coding gene for the complete tranexamic acid-carnosine ligase, named TCLigase.

[0064] The above-mentioned coding gene TCLigase was optimized according to the codon preference of Kluyveromyces lactis. The optimized coding gene sequence was cloned into the expression vector pKLAC2 to obtain a recombinant plasmid, named pKLAC2-TCLigase.

[0065] The optimized encoding gene TCLigase has the sequence shown in SEQ ID NO:3, and the amino acid sequence of the tranexamic acid-carnosine ligase it encodes is shown in SEQ ID NO:4.

[0066] The recombinant plasmid pKLAC2-TCLigase was digested with the restriction endonuclease SacII to obtain a linearized recombinant plasmid pKLAC2-TCLigase. The linearized recombinant plasmid pKLAC2-TCLigase was electroporated into K. lactis GG799 competent cells. The transformed strain was plated on YCB-Acetamide (acetamide as the sole nitrogen source) plates and cultured at 30°C for 72 h. The largest single colony on the plate was picked, and genomic DNA was extracted. The copy number of the TCLigase gene was determined by qPCR. Strains with higher copy numbers generally showed higher protein expression levels. The strain with the highest copy number was named K. lactis-TCL and used for subsequent fermentation.

[0067] The above-mentioned YCB-Acetamide medium formula is as follows: 1.17% (w / v) yeast carbon base (YCB), 5 mM acetamide, 2% (w / v) agar powder, with water as the solvent, and used after sterilization.

[0068] Example 3 The above-mentioned strain K. lactis-TCL was inoculated into a 5L fermenter (containing 2.5L of initial medium) for high-density culture. The initial medium in the fermenter was YPD medium. Fermentation was carried out at 30℃ and pH 5.5. When the glucose in the initial medium was depleted and the dissolved oxygen (DO) suddenly increased (approximately 12-14 hours of fermentation), a 50% (w / v) glycerol aqueous solution (containing 50g of glycerol per 100 mL of glycerol aqueous solution) was added as a feed. DO-stat feedback control was used during feeding to maintain DO at 30%, and the average flow rate of the glycerol aqueous solution was 10 mL / h per liter of fermentation broth. Fermentation was continued to increase OD... 600 Once the concentration reaches 100, galactose at a final concentration of 2% (w / v) is added for induction culture (i.e., 2g of galactose is added per 100mL of fermentation broth for induction culture). The strain is induced to express tranexamic acid-carnosine ligase. The culture is then induced at 30℃ for 24 hours to obtain a fermentation broth containing tranexamic acid-carnosine ligase.

[0069] After fermentation, the pH of the fermentation broth was adjusted to 6.5 using a 10% NaOH solution. Then, a mixed aqueous solution of tranexamic acid and L-carnosine (pH 6.5) at a constant rate of 0.2 mL / min per liter of fermentation broth was pumped into the broth. The concentrations of tranexamic acid and L-carnosine in the mixed aqueous solution were both 1 mol / L. Biotransformation was carried out for 48 hours. Using whole cells in the fermentation broth as a catalyst, the reaction of tranexamic acid and L-carnosine was catalyzed to generate tranexamic acid-carnosine conjugates at a temperature of 30℃ and a pH of 6.5.

[0070] Figure 1 This is a schematic diagram of the reaction catalyzed by tranexamic acid-carnosine ligase to synthesize tranexamic acid-carnosine conjugates.

[0071] After the above biotransformation is completed, the tranexamic acid-carnosine conjugate in the transformation solution is purified. The purification steps are as follows: The conversion solution was filtered using a ceramic membrane with a pore size of 0.22 μm to remove bacterial cells, and the clear filtrate was collected. The filtrate was then loaded onto an Amberlite XAD-16 macroporous adsorption resin column at a loading volume of 1 BV and a loading flow rate of 1.5 BV / h. After loading, the sample was first washed with 4 BV of water to remove impurities, and then eluted with 4 BV of 20 vol% ethanol at a flow rate of 1 BV / h. The resin eluent was collected.

[0072] Anion exchange chromatography: The resin eluent was concentrated under reduced pressure at 45°C and -0.09 MPa until no ethanol odor remained to remove ethanol. The concentrate was diluted 2-fold with water, and the pH was adjusted to 8.0 using 10% NaOH solution to obtain the sample solution. The sample solution was loaded onto an anion exchange chromatography column packed with DEAE Sepharose Fast Flow packing material. The column was pre-equilibrated with 20 mM Tris-HCl buffer at pH 8 before loading. The loading volume was 0.5 BV, and the loading flow rate was 1.2 BV / h. After loading, the column was washed with equilibration buffer for 2 column volumes, and then eluted with 20 mM Tris-HCl buffer (pH 8.0) containing 0.2 M NaCl at a flow rate of 1.0 BV / h. The eluent containing the target product was collected.

[0073] The elution product was collected for RP-HPLC: a C18 column was used; mobile phase A was 0.1% (v / v) trifluoroacetic acid aqueous solution, and mobile phase B was 0.1% (v / v) trifluoroacetic acid acetonitrile solution; the flow rate was 10 mL / min, and the detection wavelength was 214 nm. The gradient elution program was as follows: 0 min–5 min: maintain 5% volume of mobile phase B and 95% volume of mobile phase A; 5 min–25 min: the volume percentage of mobile phase B was linearly increased from 5% to 30%, and the volume percentage of mobile phase A was linearly decreased from 95% to 70%; 25 min–30 min: the volume percentage of mobile phase B was linearly increased from 30% to 95%, and the volume percentage of mobile phase A was linearly decreased from 70% to 5% (wash); 30 min–35 min: maintain 95% volume of mobile phase B and 5% volume of mobile phase A. The fraction of the major absorption peak at 15 min–20 min was collected.

[0074] Drying: After collecting the fraction by RP-HPLC, the collected fraction was subjected to vacuum freeze-drying. First, it was pre-frozen at -70°C for 4 hours, then dried under a vacuum of 8 Pa for 36 hours. During the drying process, the cold trap temperature was maintained at -80°C, and the shelf temperature was set as follows: first, the temperature was increased from -30°C to 0°C at a rate of 0.5°C / min and maintained at this temperature for 20 hours (completing the main drying stage); then, the temperature was increased from 0°C to 25°C at a rate of 1.0°C / min and maintained at this temperature for 16 hours (completing the secondary drying stage). After drying, a white powder of tranexamic acid-carnosine conjugate was obtained.

[0075] Finally, 25.5 g of a white powder containing tranexamic acid-carnosine conjugate with a purity of 99.1 wt% (HPLC detection) was obtained from 4 L of conversion buffer, with an overall yield of approximately 70% of the substrate conversion. Mass spectrometry analysis (ESI-MS) showed that the molecular ion peak of the tranexamic acid-carnosine conjugate was at m / z 366.21 [M+H]. + This is consistent with the theoretical value.

[0076] Example 4 In vitro anti-glycation activity assay To establish an in vitro glycation model of bovine serum albumin (BSA)-methylglyoxal (MGO): BSA was dissolved in phosphate buffer (pH 7.4) to prepare a 20 mg / mL BSA solution, and MGO was dissolved in phosphate buffer (pH 7.4) to prepare a 5 mM MGO solution. The BSA and MGO solutions were mixed at a 1:1 volume ratio to obtain the model solution.

[0077] Four model solutions were established according to the above method. One model solution served as a blank control group. The other three model solutions were supplemented with the compound of the present invention (i.e., the tranexamic acid-carnosine conjugate prepared in Example 3 above), L-carnosine, and aminoguanidine (positive control) at a final concentration of 1 mM, respectively. After incubation at 37°C in the dark for 24 hours, the characteristic fluorescence intensity of AGEs (advanced glycosylation end products) was measured using a fluorescence spectrophotometer at an excitation wavelength of 370 nm and an emission wavelength of 440 nm. The inhibition rate of the characteristic fluorescence intensity of AGEs relative to the blank control group was calculated. The results are shown in Table 2.

[0078] Inhibition rate = (characteristic fluorescence intensity of AGEs in blank control group - characteristic fluorescence intensity of target AGEs) ÷ characteristic fluorescence intensity of AGEs in blank control group × 100%.

[0079] Table 2 The results above show that the tranexamic acid-carnosine conjugate of the present invention inhibited the formation of AGEs by 85.2% at 1 mM, which is comparable to the effect of 1 mM L-carnosine (inhibition rate of 89.5%) and significantly better than the blank control group. This indicates that the compound of the present invention completely retains the core anti-glycation efficacy of L-carnosine.

[0080] Example 5 In vitro tyrosinase inhibitory activity assay Preparation of the reaction system: Add the tranexamic acid-carnosine conjugate to be tested and mushroom tyrosinase to a final concentration of 20 U / mL in a sodium phosphate buffer solution with a concentration of 50 mM and a pH of 6.8.

[0081] Five reaction systems were prepared according to the above method, with final concentrations of tranexamic acid-carnosine conjugate in the five reaction systems being 50 µM, 100 µM, 200 µM, 500 µM, and 1000 µM, respectively.

[0082] Preparation of blank control group: Add mushroom tyrosinase to a final concentration of 20 U / mL in sodium phosphate buffer with a concentration of 50 mM and a pH of 6.8.

[0083] After pre-incubating the above five reaction systems and the blank control group at 30℃ for 10 minutes, the substrate L-DOPA was added to each system to initiate the reaction at a final concentration of 0.5 mM. The product, dopachrome, exhibits characteristic absorption at 475 nm. The absorbance change at 475 nm for each reaction system was monitored, and the rate of absorbance change was used to characterize the enzyme activity of mushroom tyrosinase. Using the enzyme activity of mushroom tyrosinase in the blank control group as a baseline, the inhibition rate of different concentrations of tranexamic acid-carnosine conjugate on the enzyme activity of mushroom tyrosinase was calculated. A dose-response curve was constructed with the concentration of tranexamic acid-carnosine conjugate on the x-axis and the enzyme activity inhibition rate on the y-axis, thus obtaining the half-maximal inhibitory concentration (IC50) of tranexamic acid-carnosine conjugate on mushroom tyrosinase. 50 The results are shown in Table 2.

[0084] Using the same test method described above, the half-maximal inhibitory concentrations (IC50) of tranexamic acid and kojic acid against mushroom tyrosinase were calculated respectively. 50 The results are shown in Table 3.

[0085] Table 3 The results above show that the half-maximal inhibitory concentration (IC50) of the tranexamic acid-carnosine conjugate of this invention against mushroom tyrosinase is... 50 The concentration of 150 µM was significantly better than the half-maximal inhibitory concentration (IC50) of free tranexamic acid for the inhibition of mushroom tyrosinase. 50 >1000 µM), indicating that the coupled tranexamic acid-carnosine conjugate significantly enhanced the direct inhibitory effect on tyrosinase.

[0086] Example 6 Cellular level melanin production inhibition detection Experiments were conducted using B16F10 mouse melanoma cells. B16F10 cells were seeded into four wells of a six-well plate and divided into four groups (1-4). After culturing for 24 hours, α-melanocyte-stimulating hormone (α-MSH) at a final concentration of 100 nM was added to groups 1-4 to induce melanin production. Simultaneously, the compound of this invention (i.e., the tranexamic acid-carnosine conjugate prepared in Example 3 above) was added to group 1 of B16F10 cells at a final concentration of 100 µM; tranexamic acid was added to group 2 of B16F10 cells at a final concentration of 100 µM; kojic acid was added to group 3 of B16F10 cells at a final concentration of 100 µM; and group 4 served as a blank control group, receiving only α-MSH without the test sample.

[0087] After culturing for another 72 hours, B16F10 cells from each group were collected and lysed at 80°C using sodium hydroxide solution (1 mol / L)-dimethyl sulfoxide (10%, v / v) to dissolve melanin granules. The absorbance was measured at 405 nm. Total protein content was determined using the BCA method for standardization. With the melanin content of the blank control group as 100%, the relative melanin content and melanin inhibition rate of the other three groups were calculated. The results are shown in Table 4.

[0088] Melanin inhibition rate = (Melanin content of blank control group - Target melanin content) ÷ Melanin content of blank control group × 100%.

[0089] Table 4 The results above show that, compared to the blank control group, at a concentration of 100 µM, the tranexamic acid-carnosine conjugate of this invention reduced melanin production by 45%, tranexamic acid by 30%, and kojic acid by 50%. This indicates that, compared to tranexamic acid, the tranexamic acid-carnosine conjugate of this invention significantly enhances the inhibitory effect on melanin production, demonstrating its excellent skin-whitening and spot-fading potential at the cellular level.

[0090] Example 7 In vitro skin irritation test Following OECD testing guideline 439, irritation testing was conducted using a commercially available reconstructed human epidermal model (EpiDerm™). Four reconstructed human epidermal models were established. The tissues of these four models were exposed to 5% (w / v) aqueous solution of the compound of this invention, tranexamic acid-carnosine conjugate (i.e., 5 g tranexamic acid-carnosine conjugate per 100 mL of solution), 5% (w / v) aqueous solution of tranexamic acid (i.e., 5 g tranexamic acid per 100 mL of solution), 5% (w / v) aqueous solution of SDS (i.e., 5 g SDS per 100 mL of solution), and PBS buffer adjusted to pH 6.5 (close to the physiological pH of skin), respectively. After treatment for 60 minutes, each model was rinsed and cultured for another 42 hours. Finally, cell viability was assessed using the MTT assay. The results are shown in Table 5.

[0091] Table 5 The results above show that the cell viability of the model tissue treated with tranexamic acid decreased to 45% (<50%), and it was classified as an irritant (GHS Category 2) according to OECD standards. In contrast, the cell viability of the model tissue treated with the tranexamic acid-carnosine conjugate of this invention reached 88% (>50%), and it was classified as a non-irritant. This demonstrates that the tranexamic acid-carnosine conjugate of this invention significantly improves skin mildness compared to tranexamic acid.

[0092] Example 8 Metabolic stability and prodrug activation validation in skin homogenates To verify the prodrug mechanism of the tranexamic acid-carnosine conjugate of this invention, an in vitro skin metabolism experiment was conducted. Fresh porcine back skin was homogenized, and the tranexamic acid-carnosine conjugate of this invention was added to a final concentration of 10 µM, followed by incubation at 37°C. Samples were taken at 0, 2, 4, and 8 hours after incubation. After protein precipitation with acetonitrile, the supernatant was collected and analyzed by LC-MS / MS to quantitatively determine the concentration of the tranexamic acid-carnosine conjugate and the concentration of free active substances (free tranexamic acid and free L-carnosine). The results are shown in Table 6.

[0093] Table 6 The results above show that the concentration of the tranexamic acid-carnosine conjugate of this invention decreases in a time-dependent manner with increasing incubation time, and approximately 60% is degraded after 8 hours. Simultaneously, the concentrations of the free active substances, namely free tranexamic acid and free L-carnosine, increase in a corresponding time-dependent manner. This result directly demonstrates that the tranexamic acid-carnosine conjugate of this invention can be hydrolyzed by skin enzymes, releasing free tranexamic acid and free L-carnosine, thus confirming its prodrug design mechanism of action.

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A tranexamic acid-carnosine ligase, characterized in that, The amino acid sequence of the tranexamic acid-carnosine ligase is shown in SEQ ID NO:

4.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the tranexamic acid-carnosine ligase as described in claim 1.

3. An expression carrier, characterized in that, The expression vector carries the nucleic acid molecule as described in claim 2.

4. A recombinant bacterial strain, characterized in that, The recombinant strain carries the nucleic acid molecule of claim 2 or the expression vector of claim 3.

5. The use of the tranexamic acid-carnosine ligase according to claim 1 in the preparation of tranexamic acid-carnosine conjugates, wherein the structure of the tranexamic acid-carnosine conjugates is as follows: 。 6. A method for preparing a tranexamic acid-carnosine conjugate, characterized in that, Includes the following steps: The recombinant strain according to claim 4 is fermented and cultured to induce the expression of the tranexamic acid-carnosine ligase in the recombinant strain, thereby obtaining a fermentation broth containing tranexamic acid-carnosine ligase. The substrates tranexamic acid and L-carnosine are added to the fermentation broth, and the reaction is carried out at a temperature of 25℃-37℃ and a pH of 6.0-7.0 to generate the tranexamic acid-carnosine conjugate.

7. The preparation method according to claim 6, characterized in that, The fermentation culture of the recombinant strain according to claim 4, inducing the recombinant strain to express the tranexamic acid-carnosine ligase, includes: The recombinant strain described in claim 4 was inoculated into a fermenter for fermentation culture. When the glucose in the fermenter was depleted, a feed solution was added and the dissolved oxygen (DO) value was maintained at 25%-35%. The culture was continued until the OD value reached its maximum. 600 When the pH value reaches 80-120, add an inducing agent and induce culture for 22-26 hours to obtain a fermentation broth containing tranexamic acid-carnosine ligase.

8. The preparation method according to claim 7, characterized in that, The initial fermentation conditions in the fermenter are: temperature 25℃-35℃, pH 5.0-6.0; and / or, The induction culture temperature is 25℃-35℃; and / or, The feed solution is an aqueous glycerol solution, and the feed rate is 8 mL / h - 12 mL / h per liter of fermentation broth; and / or, The inducer is galactose, and the final concentration of galactose is 1.5g-2.5g per 100mL of fermentation broth.

9. The preparation method according to any one of claims 6 to 8, characterized in that, The added substrates tranexamic acid and L-carnosine are a mixed solution containing tranexamic acid and L-carnosine; In the mixed solution of tranexamic acid and L-carnosine, the concentration of tranexamic acid is 0.5 mol / L-1.5 mol / L, and the concentration of L-carnosine is 0.5 mol / L-1.5 mol / L.

10. The preparation method according to claim 9, characterized in that, The amount of the mixed solution of tranexamic acid and L-carnosine added is 5%-30% of the volume of the fermentation broth; and / or, The mixed solution of tranexamic acid and L-carnosine is added per liter of fermentation broth at a rate of 0.05 mL / min to 1.0 mL / min.

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