Process for the enzymatic synthesis of ascorbyl phosphate succinyl tripeptide-1

Abstract

This invention discloses a method for the bioenzymatic synthesis of ascorbate phosphate succinyl tripeptide-1, belonging to the field of bioengineering technology. This method involves constructing a fusion protein expression system through genetic engineering, releasing the target tripeptide GHK (glycylhistyllysine) through specific enzymatic cleavage, and then conjugating it to the succinyl group of ascorbate phosphate via an enzymatic catalytic esterification reaction, ultimately obtaining high-purity ascorbate phosphate succinyl tripeptide-1. Compared with traditional chemical synthesis methods, this method has advantages such as shorter synthesis steps, milder conditions, environmental friendliness, and high yield (>85%), making it suitable for large-scale industrial production and showing broad application prospects in the cosmetics and pharmaceutical fields.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for synthesizing ascorbate phosphate succinyl tripeptide-1 via a bioenzymatic process. Background Technology

[0002] Ascorbic acid (vitamin C) and its derivatives are widely used in cosmetics and pharmaceuticals due to their antioxidant, whitening and collagen-promoting effects. However, ascorbic acid is unstable in air and easily oxidized and deactivated, which limits its direct application. To improve stability, a variety of derivatives have been developed, such as ascorbate phosphate and ascorbate palmitate, but these derivatives still have problems such as low skin absorption rate or poor biotransformation efficiency. In recent years, peptide-conjugated ascorbic acid derivatives (such as ascorbate phosphate succinyl tripeptide-1) have attracted attention because they have both anti-wrinkle and whitening effects, but the existing synthesis methods mainly rely on chemical methods (such as liquid phase or solid phase synthesis), which have the following defects: (1) Chemical synthesis defects: expensive condensing agents (such as DCC, HOBt) and toxic solvents (such as DMF) are required, and racemic byproducts are easily generated, making purification difficult. (2) Efficiency and cost issues: chemical methods are complicated, the product yield is low (about 30%-50%), and the cost of resin and protecting reagents is high, making them unsuitable for large-scale production. (3) Environmental burden: The extensive use of organic reagents leads to pollution, which is inconsistent with the trend of green production. To address these issues, enzymatic synthesis of peptides is gradually emerging. For example, by constructing fusion proteins through genetic engineering and releasing target peptides through protease cleavage, efficient preparation of short peptides can be achieved. However, existing biological methods mostly focus on the synthesis of the simple tripeptide GHK, and have not yet addressed the conjugation technology with ascorbic acid derivatives. Therefore, developing an efficient and green enzymatic method for the synthesis of ascorbic acid phosphate succinyl tripeptide-1 is of great significance. Summary of the Invention

[0003] The present invention aims to provide an environmentally friendly, simple, and high-yield bioenzymatic synthesis method for ascorbate phosphate succinyl tripeptide-1, overcoming the shortcomings of chemical synthesis methods and enabling large-scale production.

[0004] The technical solution adopted in this invention is as follows: A method for the bioenzymatic synthesis of ascorbate phosphate succinyl tripeptide-1 includes the following steps: (1) Construct a fusion protein gene containing a GHK tandem repeat sequence, transform it into host bacteria, and induce the expression of the fusion protein; (2) The fusion protein was digested with a specific protease to release the GHK tripeptide monomer; (3) In a buffer solution, the esterification reaction of GHK with ascorbic acid phosphate succinyl group was catalyzed by lipase. (4) Purify and identify the target product.

[0005] The repeat units of the GHK tandem repeat sequence contain specific protease recognition sites. The fusion protein adopts a structure in which a lysosomal tag (such as thioredoxin, Trx) is directly fused with a (GHK)n tandem repeat sequence, where n = 4-10, preferably 6-8.

[0006] Its general amino acid sequence formula is: [Trx] - [flexible linker peptide]- (GHK)n, which is: MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSG - (GHK)n.

[0007] The host bacterium is Escherichia coli BL21(DE3).

[0008] The specific protease is lysyl endopeptidase, enterokinase, or trypsin.

[0009] The lipase described is Candida antarctica lipase B, immobilized on magnetic nanoparticles. CALB exhibits near-specific recognition of the ε-amino group in the acylation of lysine and its derivatives, while acting almost entirely on the α-amino group. This allows the GHK tripeptide to achieve precise conjugation between the ε-amino group and the succinyl group of ascorbate phosphate without any chemical protecting groups, thus preventing the formation of α-substitution or disubstitution byproducts at the source. The serine residue (Ser105) at the active site of CALB first forms an acyl-enzyme intermediate with the succinyl group of ascorbate phosphate. Subsequently, the ε-amino group of GHK (rather than the α-amino group) can effectively enter the hydrophobic channel of the enzyme and attack this intermediate due to its side chain length and spatial conformation, ultimately forming the target amide bond, thus regenerating the enzyme itself.

[0010] The enzymatic digestion reaction is carried out at a pH of 7.5-8.5 and a temperature of 25-37℃; the ratio of enzyme to substrate fusion protein is 1:10-200.

[0011] The esterification reaction is carried out at a pH of 6.0-7.5 and a temperature of 30-45℃.

[0012] Based on the theoretical GHK amount, the molar ratio of ascorbate phosphate succinyl group to the theoretical GHK amount is 1.2-1.5:1, and the amount of immobilized Candida antarcticis lipase B is 5-15% of the theoretical GHK weight.

[0013] Preferably, the molar ratio of ascorbate phosphate succinyl group to the theoretical amount of GHK is 1.2:1, and the amount of immobilized Candida antarcticis lipase B is 10% of the theoretical weight of GHK.

[0014] Purification was performed using preparative HPLC with a C18 reversed-phase column and a water / acetonitrile gradient system containing 0.1% trifluoroacetic acid as the mobile phase.

[0015] This invention lays the foundation for downstream one-pot conjugation processes by optimizing the design of linker peptides and the number of repeating units. A repeating unit count of 6-8 maximizes the yield of GHK per unit of fermentation broth.

[0016] The enzyme digestion product from step (2) is directly mixed with ascorbate phosphate succinyl groups without separation. In a buffer solution, the esterification reaction between GHK and ascorbate phosphate succinyl groups is catalyzed by lipase to generate the target product. The preparation of GHK and the subsequent conjugation reaction are integrated into a "continuous enzymatic process" without the need for intermediate purification. The protease efficiently releases GHK, and the lipase catalyzes in situ without any gaps, achieving synergistic effects at the process level.

[0017] The two enzymes selected in this invention have good compatibility in the reaction system (pH, temperature, and buffer composition). The protease digests the fusion protein to release GHK, and the lipase CALB catalyzes the conjugation of GHK with the succinyl group of ascorbate phosphate, thus constructing a "one-pot" cascade process. The protease digestion reaction solution does not require any intermediate treatment and can be directly used as the substrate source for the lipase-catalyzed reaction, omitting the separation and purification steps of GHK, thereby maximizing the overall conversion efficiency.

[0018] The ascorbate phosphate succinyl tripeptide-1 synthesized by the above method is a compound formed by the amide bond of ascorbate phosphate to the ε-amino group of the lysine side chain of tripeptide GHK (glycyl-histyl-lysine) via a succinyl linker. Its molecular formula is C22H31N6O13P, and its structure is shown below: .

[0019] The product is a white to off-white powder, readily soluble in water and phosphate buffer, slightly soluble in ethanol, and practically insoluble in organic solvents such as acetonitrile and dichloromethane. It exhibits optimal chemical stability in aqueous buffer solutions with a pH of 6.0–7.5. The ascorbic acid moiety, due to phosphorylation, significantly enhances its antioxidant stability while retaining its biotransformation properties, including the release of active ascorbic acid and GHK via enzymatic decomposition in vivo or on the skin. UV spectroscopy reveals a characteristic absorption peak at 245 nm, originating from the conjugated structure of the ascorbic acid phosphate moiety. Infrared spectroscopy shows strong absorption at approximately 1650 cm⁻¹ (amide I band) and 1540 cm⁻¹ (amide II band), confirming the formation of amide bonds.

[0020] Application of ascorbate phosphate succinyl tripeptide-1 in cosmetics or pharmaceuticals.

[0021] This invention employs a tandem repeat gene design: by increasing the number of GHK tandem repeats (n=4-10), the expression level of the fusion protein is significantly improved (reaching over 40% of the total protein), solving the problem of low direct expression efficiency of short peptides. A fully enzymatic process from the fusion protein to the target conjugate was constructed. First, the fusion protein is precisely cleaved using specific proteases (lysyl endopeptidase / enterokinase / trypsin), efficiently releasing the GHK monomer (release rate >90%). Subsequently, without intermediate purification, an immobilized lipase CALB is innovatively introduced to catalyze a regioselective conjugation reaction between the ε-amino group of GHK and the succinyl group of ascorbic acid phosphate in an aqueous buffer. The entire process is carried out in an aqueous buffer, completely eliminating the toxic condensing agents (such as DCC, EDC) and organic solvents (such as DMF, DCM) required by traditional chemical synthesis. The reaction conditions are mild (25-45℃, atmospheric pressure), with few byproducts, conforming to the principles of green chemistry and sustainable development.

[0022] Beneficial effects:

[0023] High efficiency and environmental protection: The bio-enzymatic method avoids the use of toxic condensing agents and solvents, the reaction conditions are mild, and there are few by-products, which meets the requirements of green production; the expression level of fusion protein is high (up to 30%-50% of the total protein), and the GHK release efficiency is >90%.

[0024] Advantages in yield and purity: The yield of enzyme-catalyzed conjugation reaction is >85%, and the purity of the final product is >95%, without the need for complex purification.

[0025] Low cost: No expensive resins or protecting reagents are required, lipases can be reused, and they are suitable for continuous production.

[0026] With broad application prospects, the product combines the whitening effect of ascorbic acid with the collagen-promoting effect of GHK, and can be used in anti-wrinkle skin care products, wound healing agents, etc. Attached Figure Description

[0027] Figure 1 HPLC chromatogram of the product in Example 1; Figure 2 : H-NMR nuclear magnetic resonance of the product in Example 1; Figure 3 C-NMR nuclear magnetic resonance of the product in Example 1; Figure 4 The UV-VIS concentration of the product in Example 1 was c = 5.0 × 10⁻⁶. -5The results of measurements using a standard 1 cm optical path cuvette for a solution of mol / L. Figure 5 The IR of the product in Example 1 was obtained by preparing a solution with c = 0.0003 mol / L and measuring it using a liquid cell with b = 0.1 cm (thickness of the infrared liquid cell). Figure 6 Raman of the product in Example 1. Detailed Implementation

[0028] The lysyl endopeptidase, enterokinase, trypsin, CALB, etc. used in this invention are all obtained through conventional and mature methods that are publicly available in the prior art. Whether purchased commercially or prepared in the laboratory / industrial setting, they do not require new technical means or creative labor. This ensures that those skilled in the art can obtain the enzyme and implement the technical solution of this invention without expending creative effort.

[0029] Lysyl endopeptidase (Lys-C): off-white lyophilized powder, CAS number 72561-05-8, EC number 3.4.21.50.

[0030] Enterokinase (EK): This enzyme specifically recognizes the sequence Asp-Asp-Asp-Asp-Lys (DDDDK) and cleaves it at the C-terminus of a lysine residue. Commercially available recombinant products are usually derived from recombinant bovine enterokinase light chain fragments expressed in genetically engineered E. coli, and there are also GMP-grade products produced using recombinant yeast secretory expression systems (such as Pichia pastoris).

[0031] Trypsin: Traditionally derived from bovine pancreas, it cleaves peptide bonds at the carboxyl terminus of lysine (Lys) or arginine (Arg).

[0032] Candida antarctica lipase B (CALB): This enzyme is derived from the fungus Candida antarctica. Commercially available recombinant products are usually derived from Aspergillus oryzae expression systems. There are also immobilized enzyme preparations made by fermentation, extraction, and purification using genetically engineered bacteria, followed by adsorption onto macroporous acrylic resins or Immobead 150 carriers. Immobilized CALB appears as white or slightly yellow granules, with an enzyme activity typically ≥1800 U / g, and an optimal catalytic temperature of 30-50℃.

[0033] The enzymes used in the following examples are from the following sources: Lysyl endopeptidase (Lys-C), WakoPure Chemical Industries, 125-05061, derived from Achromobacter lyticus, lyophilized powder form, specific activity ≥4.5 AU / mg.

[0034] Enterokinase (EK), New England Biolabs, P8070S, recombinant bovine enterokinase, light chain subunit, purity ≥95%, activity ≥10 U / μL.

[0035] Trypsin, Sigma-Aldrich, T1426, is derived from bovine pancreas and is treated with TPCK to inhibit chymotrypsin activity, with an activity ≥10,000 BAEE U / mg.

[0036] Antarctic Candida lipase B (CALB, free enzyme), Novozymes, Lipozyme® CALB L, liquid enzyme preparation, protein concentration approximately 20-30 mg / mL, activity ≥5,000 U / g.

[0037] Immobilized Candida antarcticis lipase B (CALB, industrial reference, Novozymes, Novozym® 435), immobilized on macroporous acrylic resin, particle size 0.3-0.9 mm, activity ≥10,000 U / g.

[0038] CALB immobilized with magnetic nanoparticles, prepared in-house (according to the method of this invention), Fe3O4@SiO2-NH2@CALB, immobilized on amino-functionalized magnetic nanoparticles, with a particle size of 50-100 nm and an activity ≥3,000 U / g.

[0039] Preparation of enzyme immobilization (CALB immobilized by magnetic nanoparticles, Fe3O4@SiO2): FeCl2·4H2O and FeCl3·6H2O were dissolved in 500 mL of deionized water at a molar ratio of 1:1.75 (11.2 g FeCl2·4H2O and 32.4 g FeCl3·6H2O). Under nitrogen protection, NH3·H2O (25%, w / w) was slowly added in an 80 °C water bath to adjust the pH to 10.0, and the reaction was stirred for 1 hour. The black precipitate was collected by magnetic separation, washed multiple times with deionized water and ethanol, and dried under vacuum at 60 °C overnight to obtain Fe3O4 magnetic nanoparticles. To prevent aggregation and provide a modifiable surface, silica coating was performed using a sol-gel method. 2 g of Fe3O4 nanoparticles were ultrasonically dispersed in an ethanol / water mixture (volume ratio 4:1, total 500 mL), and 10 mL of NH3·H2O and 5 mL of tetraethyl orthosilicate (TEOS) were added. The reaction was stirred at room temperature for 6 hours. The product was collected by magnetic separation, washed three times with ethanol, and dried under vacuum at 60°C to obtain Fe3O4@SiO2 core-shell magnetic nanoparticles.

[0040] Surface functionalization of magnetic nanoparticles: 2 g of Fe3O4@SiO2 nanoparticles were ultrasonically dispersed in 100 mL of anhydrous toluene, and 4 mL of 3-aminopropyltriethoxysilane (APTES) was added. The mixture was refluxed at 110 °C for 24 hours under nitrogen protection. After the reaction, the nanoparticles were magnetically separated, washed three times successively with toluene and ethanol, and dried under vacuum at 60 °C to obtain amino-functionalized magnetic nanoparticles Fe3O4@SiO2-NH2.

[0041] Immobilization of CALB: 1 g of amino-functionalized magnetic nanoparticles were ultrasonically dispersed in 50 mL of phosphate buffer (50 mM, pH 7.0), and glutaraldehyde was added to a final concentration of 2.5% (v / v). The mixture was activated by shaking at room temperature for 2 hours. Excess glutaraldehyde was removed by magnetic separation, and the nanoparticles were washed three times with phosphate buffer.

[0042] The activated magnetic nanoparticles were redispersed in 20 mL of CALB enzyme solution (enzyme protein concentration of approximately 5 mg / mL, dissolved in 50 mM phosphate buffer, pH 7.0) and immobilized at 4°C with shaking for 18 hours. The immobilization ratio was 100 mg CALB protein per gram of carrier (equivalent to 5 times 20 mg CALB / g carrier). After the reaction, magnetic separation was performed, and unbound enzyme protein was removed by washing three times with phosphate buffer to obtain immobilized CALB magnetic nanoparticles (Fe3O4@SiO2-NH2@CALB). The obtained immobilized enzyme can be freeze-dried and stored at 4°C for later use.

[0043] Characterization of the immobilized enzyme: The prepared immobilized CALB particles have a diameter of approximately 50-100 nm, exhibit superparamagnetism, and a saturation magnetization of approximately 15-40 emu / g, facilitating rapid separation and recovery under an external magnetic field. Enzyme activity assays show that the protein loading of the immobilized enzyme can reach 80-100 mg / g of the carrier, with an apparent specific enzyme activity of approximately 60%-80% of that of the free enzyme. The optimal catalytic temperature of the immobilized enzyme is 30-50℃, and the optimal pH is 6.0-8.0. After storage at 4℃ for 30 days, it retains more than 85% of its initial activity, and after repeated use 8-10 times, the relative enzyme activity remains above 70%.

[0044] Purification was performed using preparative HPLC with a C18 reversed-phase column and a water / acetonitrile gradient system containing 0.1% trifluoroacetic acid as the mobile phase.

[0045] Ascorbate phosphate succinyl group: First, ascorbate phosphate is synthesized, and then succinyl group is introduced by esterification with succinic anhydride to obtain an activated carboxyl group for subsequent amino conjugation with GHK.

[0046] The steps are as follows: ① Using L-ascorbic acid as raw material, phosphorylation modification was carried out using the sodium trimetaphosphate method. L-ascorbic acid was dissolved in deionized water to prepare a 20-30% solution. The pH was adjusted to 9.0-10.0 with calcium hydroxide suspension to convert it into calcium salt. Sodium trimetaphosphate (molar ratio of 1.2-1.5:1 with ascorbic acid) was added in batches under stirring. The reaction was carried out at 25-35℃ for 30-60 minutes. During the reaction, the pH was maintained at 9.5-10.0 with calcium hydroxide. The reaction progress was monitored by HPLC. After the reaction was completed, the insoluble matter was removed by filtration. The filtrate was precipitated with ethanol. The precipitate was collected and dried under vacuum to obtain crude ascorbic acid phosphate. ② The dried ascorbic acid phosphate and succinic anhydride (molar ratio 1:1.2-1.5) were reacted in anhydrous DMF solvent with 4-dimethylaminopyridine as a catalyst at room temperature to 40°C for 6-24 hours. Succinyl groups were introduced onto the ascorbic acid phosphate through esterification to form an ascorbic acid phosphate succinyl derivative with an activated carboxyl group. After the reaction was completed, the solvent was removed by vacuum distillation. The residue was dissolved in water and the pH was adjusted to 3-4 with dilute hydrochloric acid. Unreacted succinic anhydride was removed by extraction with ethyl acetate. The aqueous phase was freeze-dried to obtain the target intermediate.

[0047] The method of this invention mainly includes the following steps: (1) Bioenzymatic preparation of GHK tripeptide: GHK tandem repeat fusion protein is constructed by genetic engineering, and GHK monomer is released by enzymatic cleavage using specific proteases (such as enterokinase, trypsin or lysyl endopeptidase).

[0048] (2) Enzyme-catalyzed conjugation reaction: In an aqueous buffer system, lipase or esterase is used to catalyze the esterification reaction between the amino group of GHK and the carboxyl group of ascorbic acid phosphate succinyl group to generate the target product.

[0049] (3) Purification and characterization: The product was purified by preparative high performance liquid chromatography (HPLC) and the structure of the product was verified by LC-MS.

[0050] Example 1

[0051] Gene construction: The (GHK)6 tandem repeat sequence was designed and optimized according to the codon preference of Escherichia coli. The gene fragment 5'-GGCCACAAAGGCCACAAAGGCCACAAAGGCCACAAAGGCCACAAAGGCCACAAA-3' was synthesized and linked downstream of the thioredoxin (Trx) tag in the pET28a-Trxm vector via a flexible linker peptide coding sequence (GGTAGCGGT) to construct the recombinant plasmid pET28a-Trxm-(GHK)6. After sequencing verification, it was transformed into the expression host bacterium Escherichia coli BL21(DE3).

[0052] The amino acid sequence of the fusion protein with repeating units (n=6) is shown in SEQ ID NO.1: GH K

[0053] The corresponding amino acid sequence and the codon-optimized coding DNA sequence for E. coli are shown in SEQ ID NO.2: ATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGTTCTGGTAGCGGT GGCCACAAAGGCCACAAAGGCCACAAAGGCCACAAAGGCCACAAAGGCCACAAAGGCCACAAA.

[0054] Fusion protein expression: Single colonies were picked and inoculated into LB medium containing kanamycin and cultured at 37°C until OD600≈0.6. IPTG was added to a final concentration of 0.5 mM and expression was induced at 30°C for 6 hours. The bacterial cells were collected, sonicated, and centrifuged to obtain the supernatant, which yielded a crude extract of soluble fusion protein. SDS-PAGE analysis showed that the expression level of fusion protein accounted for 45% of the total bacterial protein.

[0055] GHK release by enzymatic digestion: Recombinant lysyl endopeptidase (EC 3.4.21.50, Lysyl Endopeptidase® (model 125-05061) manufactured by Wako Pure Chemical Industries, Ltd., dissolved in 50 mM Tris-HCl buffer (pH 8.0) to prepare a working solution of 0.1 mg / mL) from Achromobacter lyticus was added to the supernatant. The enzyme-to-substrate fusion protein ratio was 1:100. The reaction was carried out at 30°C for 4 hours in Tris-HCl buffer at pH 8.0. HPLC analysis showed that the GHK release rate (calculated based on the theoretical GHK content in the fusion protein) reached over 92%.

[0056] Enzyme-catalyzed conjugation: The pH of the above enzymatic digestion reaction solution (GHK can be used directly without separation) was adjusted to 7.0. Ascorbate phosphate succinyl groups (molar ratio to theoretical GHK was 1.2:1) and immobilized Candida antarcticis lipase B (CALB, immobilized on magnetic nanoparticles, prepared according to the above method, Fe3O4@SiO2-NH2@CALB, enzyme activity was measured to be 3500 U / g (using p-nitrophenol butyrate as substrate, pH 7.0, 40℃)) was added. The reaction was carried out in a water bath shaker (150 rpm) at 40℃ for 18 hours. After the reaction, the immobilized enzyme was recovered by magnetic separation, and the supernatant was used for subsequent purification. The conjugation yield was 95%.

[0057] Purification and Identification: The reaction solution was purified by preparative HPLC, and the HPLC chromatogram of the product is shown below. Figure 1 As shown in Table 1, the peak results were obtained. LC-MS confirmed the molecular weight of the product ([M+H]+ = 594.2), HPLC purity was 96.5%, and yield was 87%.

[0058] Table 1

[0059] Example 2

[0060] The number of repeating units n is 4 and 8 respectively, and the rest is the same as in Example 1.

[0061] When n=4, the GHK release rate is 89%, the conjugation yield is 83%, the purity is 95.2%, and the total yield is 74%. When n=8, the GHK release rate is 94%, the conjugation yield is 89%, the purity is 97.1%, and the total yield is 84%.

[0062] Conjugation yield refers to the yield of the enzyme-catalyzed conjugation step, that is, the conversion efficiency of the reaction between GHK and ascorbate phosphate succinyl group catalyzed by CALB lipase. The total yield is GHK release rate × conjugation yield.

[0063] Compared with Example 1, the conjugation yield decreased when n=4 or 8. This may be related to the lower concentration of GHK per unit volume of reaction solution or the effect of differences in trace components in the enzymatic digestion system on lipase activity. However, it is still significantly better than the 30-50% yield of the chemical synthesis method.

[0064] Example 3

[0065] The fusion protein construction and GHK preparation were the same as in Example 1 (n=6, lysyl endopeptidase digestion). The digestion reaction solution was divided into two groups: the experimental group (one-pot method) directly adjusted the pH and added ascorbate phosphate succinyl groups and immobilized CALB for conjugation reaction; the control group first purified the digestion solution by preparative HPLC to obtain GHK monomer (purity >98%), and then dissolved the purified GHK in fresh buffer for conjugation reaction. The reaction conditions for both groups were exactly the same (pH 7.0, 40℃, 18 hours). The results showed that the conjugation yield of the experimental group was 87%, and the total reaction time was 22 hours (including 4 hours of digestion); the conjugation yield of the control group was 85%, but the total process time was extended to 28 hours (including 6 hours of GHK purification), and the purification step resulted in a loss of about 8% of GHK. This example demonstrates that the "one-pot method" process not only simplifies the operation and shortens the cycle, but also avoids product loss caused by intermediate purification.

[0066] Example 4

[0067] The fusion protein was constructed and prepared as GHK in Example 1 (n=6, lysyl endopeptidase digestion). The GHK released by digestion was divided into two groups: the experimental group was used directly (containing α- and ε-amino groups), and the control group had the α-amino group of GHK protected with Fmoc. Both groups were subjected to CALB-catalyzed conjugation reactions with ascorbic acid phosphate succinyl groups under the same conditions (pH 7.0, 40℃, 18 hours). The results showed that the experimental group had a yield of 87%, and the product was identified by LC-MS as a single target ε-substituted product, with no detected α-substituted or disubstituted byproducts; the control group had a yield of 86%, and the product structure was completely identical to that of the experimental group. There was no significant difference in yield and product structure between the two groups, demonstrating that CALB has absolute selectivity for the ε-amino group, and the α-amino group of GHK can directly participate in the reaction without any protecting group, thus avoiding the generation of byproducts from the source. This example provides direct experimental evidence for the "protecting group-free operation" of this invention.

[0068] Example 5

[0069] GHK was digested with enterokinase (pH 7.8, 25°C) and trypsin (pH 8.0, 37°C), respectively, with the remaining steps the same as in Example 1. When digested with enterokinase, the GHK release rate was 88%, the conjugation yield was 85%, and the purity was 95.8%. When digested with trypsin, the GHK release rate was 90%, the conjugation yield was 84%, and the purity was 96.0%.

[0070] Example 6

[0071] The fusion protein construction and GHK preparation were the same as in Example 1 (n=6, lysyl endopeptidase digestion). The digestion products were divided into three equal portions, and three immobilized lipases from different sources were added to each portion: Group A was CALB (immobilized on magnetic nanoparticles), which is the preferred formulation of this invention; Group B was lipase TL from *Thermomyces lanuginosus* (immobilized on resin); and Group C was lipase PS from *Burkholderia cepacia* (immobilized on diatomaceous earth). The reaction conditions for all three groups were the same (pH 7.0, 40°C, 18 hours, enzyme dosage 10% of GHK weight). The results showed that Group A (CALB) had a conjugation yield of 87%, a product HPLC purity of 96.5%, and no byproducts were detected; Group B had a yield of 45%, and HPLC showed a peak of disubstituted byproducts (approximately 12%); Group C had a yield of 38%, and donor hydrolysis byproducts were also present. This example demonstrates that the superior selectivity of CALB for ε-amino groups is irreplaceable by other common lipases, which is the key enzymatic basis for achieving the protection-free synthesis of this invention.

[0072] Example 7

[0073] The fusion protein was constructed and prepared as in Example 1 (n=6, lysyl endopeptidase digestion). The digestion products were divided into five equal portions, and the pH was adjusted to 5.5, 6.0, 7.0, 8.0, and 8.5, respectively, while other conditions remained the same (CALB immobilization 10% w / w, 40℃, 18 hours). The results showed that CALB activity was stable within the pH range of 6.0-7.5, with conjugation yields all >85% (85% at pH 6.0, 87% at pH 7.0, and 86% at pH 7.5). The yield decreased to 62% at pH 5.5, possibly due to protonation inactivation of the enzyme active site. The yield was 78% at pH 8.0 and decreased to 51% at pH 8.5, possibly due to increased ester bond hydrolysis or enzyme inactivation under alkaline conditions. Therefore, the optimal pH for the esterification reaction is 6.0-7.5, within which efficient conjugation can be achieved.

[0074] Example 8

[0075] The esterification reaction was carried out at pH 6.5 and temperature 35°C, with the rest of the reaction being the same as in Example 1. The conjugation yield was 86% and the purity was 95.5%.

[0076] The esterification reaction was carried out at pH 7.5 and temperature 45°C, with the rest of the reaction being the same as in Example 1. The conjugation yield was 88% and the purity was 96.8%.

[0077] Example 9

[0078] The fusion protein was constructed and prepared as GHK in Example 1 (n=6, lysyl endopeptidase digestion). The pH of the digestion reaction solution was adjusted to 7.0, and ascorbate phosphate succinyl groups (molar ratio to theoretical GHK 1.2:1) and immobilized *Candida antarcticis* lipase B (CALB) equivalent to 10% of the theoretical weight of GHK were added. This enzyme was immobilized on magnetic nanoparticles (Fe3O4@SiO2) via hydrophobic adsorption. After reacting at 40°C for 18 hours, the immobilized CALB was recovered by magnetic separation, washed with phosphate buffer, and directly used in the next batch reaction under the same conditions as the first batch. After five reuses, the activity of the immobilized CALB remained above 90% of its initial activity, and the average conjugation yield across the five batches was 86.5%. The immobilized CALB retained over 84% of its initial activity after 11 reuses in the esterification reaction, and the esterification rate remained above 70% after eight consecutive reactions, demonstrating particularly outstanding reusability in oil / water two-phase systems. CALB immobilized on magnetic nanoparticles not only has high catalytic efficiency but also good operational stability, which can significantly reduce the cost of industrial production.

[0079] Comparative example: Comparative Example 1 (Traditional Chemical Synthesis Method) GHK was synthesized using Fmoc solid-phase synthesis, followed by EDC / HOBt condensation with ascorbate phosphate succinyl groups in DMF. The overall yield was 42%, with a purity of 91%, requiring multiple purification steps.

[0080] Comparative Example 2 (Unoptimized fusion protein repeat unit) The expression of a single GHK fusion protein (n=1) was low (<5% of total protein), and the yield after release was less than 30%.

[0081] Comparative Example 3 (Chemical esterification instead of enzymatic method) GHK was esterified with ascorbic acid phosphate succinyl in DMF using DCC / DMAP, with a yield of 55%, but many byproducts and difficult purification.

[0082] Comparative Example 4 (Enzyme catalysis in organic solvents) The conjugation reaction was carried out in tert-butanol, resulting in a 50% decrease in CALB activity, a yield of 72%, and a purity of 93%.

[0083] Comparative Example 5 (Non-specific protease) GHK digestion with chymotrypsin resulted in nonspecific cleavage sites and a recovery rate of <60%.

[0084] Comparative Example 6 (High-Temperature Enzyme Digestion) Lysyl endopeptidase is rapidly inactivated when digested at 45°C, and the GHK release rate is only 75%.

[0085] Synthesis Yield Test Test method: HPLC chromatograph Operating procedure: A C18 reversed-phase column was used with 0.1% trifluoroacetic acid aqueous solution (phase A) and acetonitrile (phase B) as the mobile phase. Elution was performed according to the following gradient program: maintain 5% phase B for 0-2 minutes, increase phase B linearly to 95% within 2-20 minutes and maintain for 5 minutes, then restore to the initial conditions within 0.5 minutes and equilibrate for 3.5 minutes; flow rate 1.0 mL / min, column temperature 30°C, detection at 245 nm wavelength.

[0086] The bio-enzymatic synthesis method of this invention exhibited excellent and stable high yields (74%-87%) in all examples, significantly surpassing traditional chemical synthesis methods or non-optimized biological methods. Comparative Example 1 (traditional chemical method) showed a yield of only 42%, directly demonstrating the overwhelming advantage of this invention in synthesis efficiency (an improvement of over 100%). Core technological innovations—particularly the tandem repeat gene design (Examples 1-3 vs. Comparative Example 2) and the all-aqueous enzyme catalytic system (Example 1 vs. Comparative Example 4)—were proven to be key to achieving high yields: the optimized fusion protein expression (n=6-8) solved the short peptide expression problem, increasing the yield from less than 30% to nearly 90%; and avoiding the use of organic solvents (tert-butanol) further increased the yield by more than 15 percentage points. Furthermore, the use of specific proteases (each example vs. Comparative Example 5) and mild reaction conditions (Example 1 vs. Comparative Example 6) jointly ensured the efficient and complete release of the precursor GHK, laying the foundation for subsequent high-yield conjugation. In summary, the yield data strongly confirms the scientific validity and advanced nature of the tandem repeat design-specific enzyme digestion-aqueous phase enzyme catalysis strategy adopted in this invention, and fully realizes its invention objectives of being efficient, green, and suitable for large-scale production.

Claims

1. A method for the bioenzymatic synthesis of ascorbate phosphate succinyl tripeptide-1, characterized in that, Includes the following steps: (1) Construct a fusion protein gene containing a GHK tandem repeat sequence, transform it into host bacteria, and induce the expression of the fusion protein; (2) The fusion protein was digested with a specific protease to release the GHK tripeptide monomer; (3) In a buffer solution, the esterification reaction of GHK with ascorbic acid phosphate succinyl group was catalyzed by lipase. (4) The target product, ascorbate phosphate succinyl tripeptide-1, was obtained by purification.

2. The method according to claim 1, characterized in that: The number of repeating units n in the GHK tandem repeating sequence is 4-10.

3. The method according to claim 1, characterized in that: The specific protease is lysyl endopeptidase, enterokinase, or trypsin.

4. The method according to claim 1, characterized in that: The lipase is immobilized Candida antarcticis lipase B.

5. The method according to claim 1, characterized in that: The pH of the enzymatic digestion reaction is 7.5-8.5, and the temperature is 25-37℃.

6. The method according to claim 1, characterized in that: The pH for esterification is 6.0-7.5, and the temperature is 30-45℃.

7. The method according to claim 1, characterized in that: The ratio of specific protease to substrate fusion protein is 1:10~200.

8. The method according to claim 1, characterized in that: The molar ratio of ascorbate phosphate succinyl group to the theoretical amount of GHK is 1.2~1.5:1, and the amount of immobilized Candida antarcticis lipase B is 5~15% of the theoretical weight of GHK.

9. Ascorbate phosphate succinyl tripeptide-1 synthesized by the method of any one of claims 1-8.

10. The use of the ascorbate phosphate succinyl tripeptide-1 according to claim 9 in cosmetics or pharmaceuticals.

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